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UNIVERSIDAD POLITÉCNICA DE MADRID

ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS

DEPARTAMENTO DE BIOTECNOLOGÍA

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TESIS DOCTORAL

Azotobacter vinelandii nitrogenase: “Kinetics of nif gene

expression and insights into the roles of FdxN and NifQ in

FeMo-co biosynthesis”

Autor: Emilio Jiménez Vicente

Director: Dr Luis Rubio Herrero

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UNIVERSIDAD POLITÉCNICA DE MADRID

ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS

DEPARTAMENTO DE BIOTECNOLOGÍA

Memoria Presentada por

D. Emilio Jiménez Vicente

para optar al grado de Doctor

Director

Dr. Luis Rubio Herrero

Profesor Titular UPM

Madrid, 2014

! This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognize that its copyright rest with the author and that no quotation from the thesis, or any information derived therefrom may be published without the author’s prior, written consent.

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Abstract !

The Molybdenum-nitrogenase is responsible for most biological nitrogen

fixation activity (BNF) in the biosphere. Due to its great agronomical importance,

it has been the subject of profound genetic and biochemical studies. The Mo

nitrogenase carries at its active site a unique iron-molybdenum cofactor (FeMo-

co) that consists of an inorganic 7 Fe, 1 Mo, 1 C, 9 S core coordinated to the

organic acid homocitrate. Biosynthesis of FeMo-co occurs outside nitrogenase

through a complex and highly regulated pathway involving proteins acting as

molecular scaffolds, metallocluster carriers or enzymes that provide substrates

in appropriate chemical forms. Specific expression regulatory factors tightly

control the accumulation levels of all these other components. Insertion of

FeMo-co into a P-cluster containing apo-NifDK polypeptide results in

nitrogenase reconstitution. Investigation of FeMo-co biosynthesis has

uncovered new radical chemistry reactions and new roles for Fe-S clusters in

biology.

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Abbreviations !

2-OG 2-oxoglutarate ADP adenosine diphosphate ATP Adenosine triphosphate Blast Basic Local Alignment Search Tool BNF Biological nitrogen fixation CFU Colony-Forming Units

cGMP Ciclic guanosine monophosphate DNA Desoxiribonucleic acid DTH Sodium dithionite EBP Enhancer Binding Protein EPR Electronic Paramagnetic Resonance ESE Electro Epin Echo

EXAFS Extended X-ray Absorption Fine Structure FAD Flavin-adenine-dinucleotide

FeFe-co Iron-iron cofactor FeMo-co Iron-molybdenum cofactor FeV-co Iron-anadium cofactor

GAF cGMPphosphodiesterase adenylate cyclase FhlA GOGAT Glutamine oxoglutarate aminotransferase

HPK Histidine Protein Kinase IHF Integration Host Factor

Mo-co Molybdenum cofactor MoFe protein Dinitrogenase

MoSto Molybdenum Storage protein mRNA Messenger Ribonucleic acid NAS Normalized Absolute Signal nif gene designation for molybdenum-dependent nitrogen

fixation NifB-co NifB cofactor

NMF N-Methyl-Formamide NMR Nuclear Magnetic Resonance NRVS Nuclear Resonance Vibrational Spectroscopy OD600 Optical Density at 600nm ORF Open Reading Frame

PAS Per (period circadian protein) Arnt (aryl hydrocarbon receptor nuclear translocator protein) Sim (single-minded protein)

PLP Pyridoxal phosphate qRT Quantitative Reverse Transcriptase RNA Ribonucleic acid rnf Genes homologous to Rhodobacter capsulatus nif

genes

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RPM rounds per minute SAM S-Adenosyl Methionine UAS Upstream Activation Sequence UMP Uridine MonoPhosphate

UR uridylyl-removing UTase uridylyltransferase

UV Ultraviolet XAS X-ray Absorption Spectroscopy

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Chapter 1: Introduction

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The nif regulon is defined as nifA and those genes under the control of

NifA that are responsible for the production of a functional Mo nitrogenase. In

Azotobacter vinelandii, nif genes are clustered in two chromosomal regions

adjacent and equidistant from the replication origin [1] comprising at least eight

operons [2-5] (Fig.1). From a genetic standpoint, such a position suggests a

higher gene dosage during active cell growth [1] or just a critical role of these

genes in A. vinelandii life style. Interestingly, all genes known to be required for

the Mo, V and Fe-only nitrogenases (e.g. nifU, nifS, nifV, nifM, and nifB) are

located in Mo nitrogenase regions [4-7]. The so-called major nif cluster contains

at least six transcriptional units, namely orf12orf13, nifHDKTY, nifENX, orf5,

iscAnifnifUSVcysE1nifnifWZMclpX2 and nifF, in which nif genes are interspersed

with a number of open reading frames (ORFs) of unknown function [4]. The

minor nif cluster contains three operons, namely rnfABCDGEH, nifLA and nifB

fdxN nifOQ rhdN grx5nif, and the nafY gene [5, 8-10].

Genes in nif operons are transcribed by #54-containing RNA polymerase,

that recognizes promoter DNA with boxes centered at around -12 and -24 from

the transcription start point with a consensus sequence of 5'-YTGGCACGR-N3-

TTGCW-3' [11, 12]. Transcription by #54-containing polymerases is dependent

of additional transcriptional factors denominated Enhancer Binding Proteins

(EBPs), bind to DNA regions known as Upstream Activator Sequences (UAS) to

initiate RNA synthesis [13]. The two EBPs NtrC and NifA are responsible for

regulating the transcription of genes involved in the assimilation of N2. NtrC is

well characterized in diazotrophic enteric bacteria as involved in nif genes

transcription, meanwhile, nif gene expression in A.vinelandii is NifA-dependent

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but NtrC-independent, where NtrC has been shown to be responsible of

activation of genes involved in assimilation of other N2 sources such as nitrate

[14].

There are cases in which additional DNA binding proteins are needed,

including the integration host factor [15]. IHF is an asymmetric histone-like

protein that binds and bends DNA in specific locations (IHF binding consensus

sequence 5'-WATCAANNNNTTR-3') [16].

Despite of the fact that only 11 gene products form the core of Mo

nitrogenase dependent N2 fixation system (nifH, nifD, nifK, nifE, nifN, nifB, nifU,

nifS, nifV, nifQ, and nifM) a genome-wide transcription profile analysis

(comparing steady-state gene expression levels in N2-fixing A. vinelandii cells

versus NH3 assimilating cells) demonstrated that ca. 400 genes were

differentially expressed under Mo-dependent diazotrophic growth conditions

[17, 18]. Recently, a synthetic biology approach to engineer nitrogenase in

Klebsiella pneumonae was constructed by using 20 genes [19]. The low level of

nitrogenase activity obtained suggest that synthetic N2 fixation operons need

not only to balance gene expression but also to adjust it overtime.

Uncoordinated expression or inadequate Nif protein concentration might be

deleterious to N2 fixation.

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gene clusters. Predicted "54–dependent promoter regions are depicted by

nafY H E G D C B A rnf

L A B fdxN

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major nif cluster

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A. vinelandii chromosome

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arrows. Black arrows represent regions additionally containing NifA-UAS and

IHF motifs; blue arrows represent regions containing NifA-UAS motifs but

lacking IHF motifs; orange arrows represent regions lacking both NifA-UAS and

IHF motifs. Adapted from [20].

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N2 fixation is a very high energy demanding process. Thus, a strictly

regulated mechanism that control nitrogenase synthesis and activity is

mandatory in order to minimize waste of energy.

The NifA-NifL-GlnK complex is key in regulating nif gene expression. In

A. vinelandii this system integrates different environmental and intracellular

signals including cell energy levels (ATP/ADP ratio), N2/carbon balance, and

redox state, to determine if diazotrophic growth is feasible. Different

diazotrophic proteobacteria, such as A. vinelandii, K. pneumoniae and

Pseudomonas stutzeri, display significant differences on their signal integration

mechanisms [21-24].

The biochemical basis of the NifA-NifL-GlnK complex relies on a series

of sensing and interaction motifs that have been studied in detail (Fig. 2A) [25-

27].

NifL is an evolutionary relative of Histidine Protein Kinases (HPK) that

acts as anti-activator of NifA. NifL domain architecture is similar to cytoplasmic

HPKs. It contains two N-terminal PAS motifs (Per-ARNT-Sim) [28, 29] and a C-

terminal transmitter region containing a conserved H domain that acts as signal

integrator and a nucleotide binding domain belonging to GHKL (Gly-His-Lys-

Leu) superfamily of ATPases [30, 31]. PAS1 carries a flavin-adenine-

dinucleotide (FAD) cofactor. FAD cofactor oxidation results in activation of NifL

and consequent inhibition of NifA activity, whereas reduction of the FAD moiety

deactivates NifL [28]. The H domain of NifL is located between the PAS2 and

GHKL domains. Although the conserved His is not essential, other amino acids

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from the H domain play an important role in signal transduction associated with

conformational changes that modulate the interaction of NifL with the 2-

oxoglutarate(2-OG)-bound form of NifA [32-34].

The NifL GHKL ATP-binding domain, does not hydrolyze ATP,

however it seems to perform two different functions: (i) it senses the energy

status of the cell by binding ADP, and (ii) it senses the cellular N2 level through

an interaction with GlnK, a PII-like protein [25, 35, 36].

As mentioned before, NifA is an EBP that activates transcription of "54-

dependent promoters [12]. The N-terminal part of NifA presents a GAF domain

(cGMPphosphodiesterase adenylate cyclase FhlA domain) that binds 2-OG for

allosteric control of NifA. Interaction of 2-OG with the GAF domain of NifA

prevents NifL interaction in the absence of unmodified GlnK [34, 37]. 2-OG is a

Krebs cycle intermediate that provides, not just a direct measure of cellular

carbon status, but also an indirect measure of cellular N2 status. This is

because 2-OG is a substrate of the Glutamine oxoglutarate aminotransferase

(GOGAT) enzyme and a main source of carbon skeletons for amino acid

biosynthesis [38]. Additionally, NifA presents a central catalytic AAA+ domain

that can interact with the "54 RNA polymerase subunit [39]. Binding of NifL

inhibits the ATPase activity of this domain disabling nif gene expression [34,

40]. The C-terminal part of NifA includes a helix-turn-helix DNA-binding domain

that recognizes NifA-UAS located 5' of nif genes.

When A. vinelandii cells are growing under ideal diazotrophic conditions

(N2 starvation and high respiratory rates) the level of oxygen is low and the

levels of 2-OG and ATP are high. Allosteric binding of 2-OG to the GAF domain

produces a conformational change in NifA that impairs NifL binding. Free NifA is

able to activate nif gene expression through DNA binding and activation of the

"54 factor. On the other hand, at relatively low 2-OG levels or, most importantly,

when NifL FAD group is oxidized by excess oxygen, NifL is competent enough

to bind NifA and inhibit its activity. However, additional fine-tuning applies that

will be described below [22].

!

!

! ")!

A. vinelandii relies on GlnK and GlnD to determine its cellular carbon/N2

balance [41]. The glnK gene product is a member of the PII family that

stabilizes NifL-inhibition of NifA by forming a GlnK-NifL-NifA ternary complex in

response to NH4+ excess. Contrary to the well-studied case of enteric bacteria,

including the diazotrophic enterobacterium K. pneumoniae, GlnK expression in

A.vinelandii appears to be independent of NtrC [41].

GlnK is susceptible to be urydilated or de-urydilated by GlnD (an

uridylyltransferase/uridylyl-removing enzyme). The activity of GlnD depends of

glutamine concentration, at high concentration, binding of glutamine to the

enzyme switches activity in favor of uridyl-removing activity, whereas low

glutamine concentration switches activity toward an uridylyltransferase function

[42, 43].

Uridylylation of GlnK by GlnD plays a key role in nif gene activation of

expression [25]. Under conditions of N2 excess (high N2/carbon ratio) resulting

in high concentration of glutamine, non-uridylylated GlnK blocks the ammonium

transporter AmtB [44]. It also interacts with the C-terminal domain of NifL

increasing its inhibitory effect over NifA and forming a ternary NifL-NifA-GnlK

complex that is more stable to dissociation by binding of 2-OG than the NifL-

NifA complex (Fig. 2B). Thus, the N2 signal overrides the carbon source signal.

On the other hand, under conditions of N2 limitation (low N2/carbon balance)

GlnD uridylylates GlnK (GlnK-UMP3), which cannot interact with NifL. In

addition, 2-OG binding to NifA prevents its inhibition by NifL (Fig. 2B).

Interestingly, there is no evidence of NifL interaction with cytoplasmic

membranes in A. vinelandii as it has been shown to be the case in K.

pneumonia, where NifL could be immobilized in the membrane, and therefore,

hijacked from NifA, resulting in activation of nif transcription [45].

!

!

! ".!

&

NH4+

Amtb Amtb GlnK

GlnK-UMP3

2NH3+H2

N2+8e-+8H+

[2-OG]

[Gln]

Nitrogen excess Nitrogen starvation

Nitrogenase

!"#!$$#

P

UR UTase

GlnD

2-OG

NifA

!54

NifA

"%&###"'&#(#&)#

GlnK

nif

ADP P P

FAD NifL

FADH2 NifL

!"#$!nif

redox sensor Nitrogen and Energy status

GAF AAA+ HTH

2-OG

N/C status

Interacts with !54"ATPase

activity DNA-binding

domain"

Signal integrator

GlnK

P P

ADP FAD/H2 PAS1 PAS2 H GHKL NifL

NifA

#!

%!

!

!

! #/!

&&

&(478/9& F2& !@9& *46"[*46$[P;>\& /978;0.5/L& -L-.9M& 56& $1- ."!+/)!0""1 (A)

Schematic domain architecture of A. vinelandii NifL and NifA proteins. (B) GlnK-

NifL-NifA response to environmental and metabolic conditions in A. vinelandii.

Left panel: conditions of N2 excess result in high concentration of glutamine that

leads to deuridylylation of GlnK by the uridylyl-removing (UR) activity of GlnD.

The unmodified form of GlnK can interact with (1) AmtB to block active transport

of NH3, and (2) with NifL in a GlnK-NifL-NifA ternary complex to block activation

of nif gene transcription. Right panel: conditions of N2 limitation result in low

glutamine and high 2-OG concentrations that lead to uridylylation of GlnK

(GlnK-UMP3) by the uridylyltransferase (UTase) activity of GlnD. The modified

form of GlnK is unable to interact with either AmtB or NifL. In addition, high 2-

OG levels induce a conformational change in NifA that prevents NifL inhibition

and allows binding to specific UAS activating nif gene transcription. Adapted

from [20].

!

!

!

!

!

!

!

!

!

! #"!

!

"#7!D+,3?-*9%3+3!,.!@%*)(!5('3*%/!,.!*9%!@,(?EB%-'@!-+*/,&%-)3%!!

!

"#7#"!4%-%/)(!5,-5%C*3!

!

Two different major strategies for metal cofactor biosynthesis can be

found in nature. In some cases, the cofactor is assembled directly in the final

target protein. The nitrogenase [8Fe-7S] P-cluster is an example of proteins

where in situ cofactor assembly takes place [46-50]. In the case of more

complex prosthetic groups, the opposite approach is used. The iron-

molybdenum cofactor (FeMo-co) of nitrogenase, the molybdenum cofactor (Mo-

co) of nitrate reductase, and the H-cluster of [FeFe]-hydrogenase are examples

of cofactors where ex situ assembly occurs [51-53]. FeMo-co synthesis is

completed outside the target enzyme in a biosynthetic pathway completely

independent of the production of the structural polypeptides. Thus, FeMo-co

needs to be inserted into an apo-enzyme in order to render the mature, active

nitrogenase enzyme.

!

"#7#7!F+-+*/,&%-)3%!)-B!)C,<B+-+*/,&%-)3%!

!

NifDK (also referred to as dinitrogenase or MoFe protein or nitrogenase

component I) is a 230-kDa $2%2 tetramer of the nifD and nifK gene products.

The $ and % subunits arrange as a pair of $% dimers that are related by a two-

fold rotation axis. Both $ and % subunits (NifD and NifK respectively) are

phylogenetically related and display a similar tertiary structure consisting of

three domains each. NifDK contains two unique metal clusters per $%-dimer:

the P-cluster and the FeMo-co [54].

!

!

! ##!

The P-cluster is a [8Fe-7S] cluster in which two [4Fe-4S] cubanes share

a µ6-S atom. The P-clusters are located at the interface between the $ and %

subunits at around 12 Å below the protein surface. In the DTH-reduced state of

NifDK, residues $-Cys88 and %-Cys95 provide thiol groups bridging the two

cubanes, whereas residues $-Cys62, $-Cys154, %-Cys70 and %-Cys153 coordinate

the remaining Fe sites in the P-cluster (residue numbers correspond to the A.

vinelandii NifDK).

NifU and NifS are needed for the initial formation of two pairs of [4Fe-4S]

clusters that serve as precursors to the P-clusters. The concerted action of both

NifZ and NifH is required for the biosynthesis of the complete set of P-clusters,

which will be carried out in situ (directly on the NifDK protein). A NifDK protein

carrying only one P-cluster and one pair of [4Fe-4S] cluster or with two pairs of

[4Fe-4S] clusters (but no P-clusters) is obtained from deletion mutants lacking

NifZ or NifH, respectively [55].

The FeMo-co comprises a [Mo-7Fe-9S] cluster with a single carbide

atom residing in the cavity formed by the six central Fe atoms [56-58]. In

addition, the Mo atom is coordinated by the C-2 carbonyl and hydroxyl groups

of the organic acid homocitrate (Fig. 3). FeMo-co is almost completely buried

within the $-subunit of NifDK, 10 Å below the protein surface and 14 Å away

from the P-cluster. Hydrophilic residues form the majority of the protein

environment around FeMo-co, although a number of hydrophobic residues are

required for cofactor positioning. Unlike the P-cluster, FeMo-co is ligated by only

two NifD amino acid residues: $-His442 (which binds to the Mo atom) and $-

Cys275 (which binds to the Fe atom located at the opposite end of the cluster)

[54]. Several other residues surrounding the cofactor binding site are selected

to create a protein environment tailored for FeMo-co binding, such as $-Gly356

and $-Gly357 (which are needed to prevent steric hindrance with the metal

cluster), $-Arg96 and $-Arg359 (which hydrogen bond to and stabilize the

cofactor), or $-Gln191, $-Glu440 and $-Glu427 (which interact directly or through

water molecules with the homocitrate moiety). As expected, residues $-His442,

!

!

! #$!

$-Cys275 and some other residues in the vicinity of FeMo-co are highly

conserved across species.

&

&

&

!

!

! #%!

&

(478/9&K2&".5M41&-./81.8/9&56&(9R5Q152 Adapted from [51]

Apo-NifDK (also referred to as apo-dinitrogenase or apo-MoFe protein) is

a cofactor-less NifDK protein. Several forms of apo-NifDK have been reported

to accumulate in the cell depending on the genetic background [47]. For

example, a "nifH mutation renders apo-NifDK lacking both the P-clusters and

FeMo-co whereas a "nifB mutation renders apo-NifDK that contains P-clusters

but lacks FeMo-co. Here we will use the term apo-NifDK when referring to the

FeMo-co less form found in "nifB strains [56], otherwise a more detailed

definition will be included. This form of apo-NifDK can be readily activated by

the simple addition of FeMo-co. In fact, apo-NifDK activation was used as assay

to isolate FeMo-co from pure preparations of NifDK protein [59, 60].

In general, minor differences are observed in apo-NifDK structure upon

FeMo-co insertion. However, the $III domain undergoes major structural

rearrangements. A comparison between the apo- and holo-NifDK structures

revealed a His triad ($-His274, $-His442 and $-His451) possibly involved in the

formation of an insertion funnel in the structure of "nifB NifDK. The

rearrangement of the $III domain is hypothesized to generate an opening for

FeMo-co insertion and to provide a positively charged path to drive FeMo-co

entrance down to the cofactor binding site [56] (Fig. 4). Site-directed

mutagenesis studies on NifDK are consistent with the important role of the

histidine residues along the insertion funnel to facilitate FeMo-co insertion [55].

!

!

! #&!

(478/9& 32& +./81.8/9-& 56& *46]\& 0>E& 0A5Q*46]\2 (A) Structure of one NifDK #$

pair. (B) Structure of one apo-NifDK #$ pair. Color code: # subunits; #I domain

in cyan, #II domain in purple and #III domain in orange. % subunits; $I domain

in grey, $II domain in green and $III domain in wheat. The #III domain

estructure shows diferent $ sheet orientation in NifDK and apo-NifDK forms.

The cavity formed between domains #I, #II and #III is occupied by FeMo-co in

NifDK, whereas this cavity is empty in apo-NifDK.

!"#$%&'()*

+(,-"#-*

!" #"

!

!

! #'!

"#7#G!H%I,<5,!D+,3?-*9%3+3!

!

The isolation of FeMo-co from pure preparations of NifDK protein is one

of the seminal contributions to the field of nitrogenase biochemistry and to our

understanding of complex metalloproteins assembly in general. FeMo-co

isolation set the basis for the in vitro FeMo-co insertion and FeMo-co synthesis

and insertion assays developed by Vinod Shah and now widely used in the field

[60]. Purified FeMo-co was found to be extremely sensitive to oxygen and

unstable in protic solvents. Thus, FeMo-co extraction must be carried out into

anaerobic N-methyl formamide (NMF) after denaturing and precipitating pure

NifDK in a series of low and neutral pH solutions. FeMo-co isolated in this

manner is stable indefinitely when stored as an anaerobic NMF solution under

liquid nitrogen.

Combined genetic and biochemical studies have determine that FeMo-co

biosynthesis requires (1) enzymes to provide substrates in the appropriate

chemical forms and to catalyze certain critical reactions such as carbide

insertion, (2) molecular scaffolds to aid in the step-wise assembly of FeMo-co,

and (3) metallocluster carrier proteins that escort FeMo-co biosynthetic

intermediates in their transit between scaffolds (Fig 5) (Table 1) [51]. Once fully

assembled, FeMo-co is transferred from the “FeMo-co biosynthetic factory” into

apo-NifDK, either by a hypothetical protein-protein interaction between NifEN

and apo-NifDK [55] or mediated by the FeMo-co binding protein NafY [51]. The

insertion of FeMo-co into apo-NifDK generates mature, functional holo-NifDK.

The specific roles of number of Nif proteins in FeMo-co biosynthesis are

described below:

!

!

! #(!

!0,;9B2&G5;9&56&>46&79>9&A/5E81.-&4>&R5Q>4./579>0-92 Genes from the N2-fixing

bacterium A. vinelandii are listed. The color code is as follows: green, genes

!"#"$ %"&'()*+,#$

!"#$% !"#!"#$#!%&'$()&*+,-$'.!'),/'%-&!

!"#&% 0$,1'),/'%-&!-2!!"#!"#$#!%&'$()&*+,-$!!

!"#'% 3$!$%&'!()*+!",(&)'&&*#(!-4%!('5#!24$),-$(!'(!!,#-6!7$8$-9$!24$),-$!*$!.%&/"!(0,!1""&

!"#(% :)';-.<!2-&!=>#1:?!).4(%#&!@*-(A$%B#(*(6!>-&5(!)-5+.#C!9*%B!D*2:!

!"#)% EA(%#*$#!<#(4.24&'(#F!(#&/#(!'(!:!<-$-&!2-&!=>#1:?!).4(%#&!@*-(A$%B#(*(6!>-&5(!)-5+.#C!9*%B!D*27!

!"#*% G-4@.#!&-.#!)'&&A*$"!D*2H1)-!'$<!(#&/*$"!'(!(%-&'"#!2-&!IJ1).4(%#&!K'$<!+&-@'@.A!-%B#&!>#L-1)-!+&#)4&(-&(M!

#+,-% G-$'%#(!#.#)%&-$(!2-&!>#L-1)-!@*-(A$%B#(*(!

!"#.% :0L1&'<*)'.!+&-%#*$6!N#$#&'%#(!%B#!)-5+.#C!>#:!)-&#!-2!>#L-1)-!K*$).4<*$"!%B#!)#$%&'.!E!'%-5M!

!"#/% O-5-)*%&'%#!(A$%B'(#!

!"#0% G-$'%#(!5-.A@<#$45!%-!%B#!D*2PDQD*2O!)-5+.#C!2-&!>#L-1)-!@*-(A$%B#(*(!

!"#1% 3$!)-5@*$',-$!9*%B!D*2DF!2-&5(!'!()';-.<!+&-%#*$!*$/-./#<!*$!.'%#&!(%#+(!-2!>#L-1)-!'((#5@.A!

!"#-% 3$!)-5@*$',-$!9*%B!D*2PF!2-&5(!'!()';-.<!+&-%#*$!*$/-./#<!*$!.'%#&!(%#+(!-2!>#L-1)-!'((#5@.A!

!2#'% G-4@.#!&-.#!(%'@*.*R*$"!'+-1D*2GJ!'$<!*$!>#L-1)-!*$(#&,-$!

!"#3% 3$/-./#<!*$!S1).4(%#&!2-&5',-$!

!"#4% G*$*%&-"#$'(#!KL->#!+&-%#*$M!T1(4@4$*%6!>#L-1)-!*(!@4&*#<!9*%B*$!%B*(!(4@4$*%!

!"#5% G*$*%&-"#$'(#!KL->#!+&-%#*$M!U1(4@4$*%!-26!S1).4(%#&!*(!.-)'%#<!'%!%B#!*$%#&2')#!-2!T!'$<!U!(4@4$*%(!

!"#6% V#W4*&#<!2-&!5'%4&',-$!-2!D*2O!K>#!+&-%#*$M6!:*5*.'&!%-!+&-.A.!*(-5#&'(#(!

!"#7%G*$*%&-"#$'(#!&#<4)%'(#!K>#!+&-%#*$M6!X@.*"'%#!#.#)%&-$!<-$-&!%-!D*2GJ6!0.(-!&#W4*&#<!2-&!S1).4(%#&!'$<!>#L-1

)-!@*-(A$%B#(#(6!

!

!

! #)!

required in vitro for Mo-nitrogenase synthesis or activity; red, genes required in

vivo for the Mo-nitrogenase and also for the alternative V and Fe-only

nitrogenases. Adapted from [61].

"#7#J!:+.K!)-B!:+.L!

!

Many of the proteins involved in N2 fixation, including nitrogenase itself,

are iron-sulfur (Fe-S) proteins. Given the large amount of Nif proteins expressed

in N2-fixing conditions, a specific [Fe-S] cluster biosynthetic system is found in

diazotrophic microorganisms in addition to the general [Fe-S] cluster

biosynthetic machinery. This redundancy presents at least two major

advantages: (i) it satisfies the high demand of [Fe-S] clusters needed for N2

fixation and (ii) it ensures that a deleterious mutation disturbing this specialized

system only affects cell survival under diazotrophic growth conditions [46].

NifU and NifS are required for the maturation of both NifH and NifDK.

The critical observation to identify a role for these proteins was that whereas

many mutations in nif genes affected either NifH or NifDK, mutations in either

nifU or nifS resulted in a large decrease of activity in both nitrogenase

components [4]. Since the presence of [Fe-S] clusters is common to both

components it was proposed that NifU and NifS proteins had a role in the

assembly of nitrogenase-specific [Fe-S] clusters. An additional key observation

was the fact that, although nitrogenase activity was severely affected in nifU

and nifS mutants, it was not completely lost. This led to the identification of

additional housekeeping NifU and NifS homologues, referred to as IscU and

IscS, which were involved in supplying [Fe-S] clusters for general cellular

functions [62].

NifS is a 87-kDa pyridoxal phosphate-containing (PLP) homodimer [63].

NifS is a cysteine desulfurase that catalyzes a desulfurization reaction of L-

cysteine, rendering L-alanine and a protein-bound persulfide as products. A

highly conserved Cys325 residue located in the active site of the enzyme was

found critical for NifS activity [64]. Based on amino acid sequences, cysteine

!

!

! #.!

desulfurases are classified in two major groups; NifS has a SSGSAC(T/S)S

conserved consensus sequence and falls within group I [50]. Interestingly, a

cysE homologue, encoding an O-acetyl serine synthase, the rate-limiting step

for cysteine biosynthesis, is co-transcribed together with nifS [65].

NifU is a 66-kDa homodimer containing a stable [2Fe-2S] cluster per

subunit [66]. Amino acid sequence conservation analysis, site-directed

mutagenesis experiments and activity assays with separate purified domains

confirmed the presence of three conserved domains in NifU [67]. The central

domain contains a permanent, redox-active, [2Fe-2S] cluster coordinated by

four conserved cysteine residues, whereas the N-terminal and C-terminal

domains present three and two conserved cysteine residues, respectively, for

the assembly of transient [Fe-S] clusters [68]. Spectroscopic and genetic

analyses provided further evidence of formation of labile [2Fe-2S] clusters

within both terminal domains of NifU in reactions containing L-cysteine, Fe2+

and NifS [69].

A series of elegant experiments using apo-NifH as [Fe-S] cluster

acceptor provided further details on the mechanism of NifS/NifU [64, 70]. NifS

activity directs the assembly of transient [4Fe-4S] clusters on NifU, which are

subsequently transferred to apo-NifH endowing protein activity. A NifUS

complex formed during cluster assembly has been reported [69], but NifS was

not required for [Fe-S] cluster transfer from NifU to the target apo-protein.

Although in vitro loading of apo-NifH with [4Fe-4S] clusters was possible simply

by incubating apo-NifH with Fe2+ and S2-, the reaction was significantly faster (at

physiologically significant rates), specific and more efficient (requiring only

equimolar amounts of NifU) when using NifS, Fe2+ and L-cysteine. All together,

these results confirmed the role of NifS as donor of S2- in order to sequentially

load the scaffold NifU for the synthesis of simple [2Fe-2S] and [4Fe-4S] clusters

required for maturation of nitrogenase components.

NifU and NifS are also involved in FeMo-co synthesis (Fig. 5).

Participation of these proteins as providers of [Fe-S] cluster substrates for

FeMo-co biosynthesis was difficult to demonstrate because many FeMo-co

!

!

! $/!

biosynthetic proteins are Fe-S proteins themselves and mutations in nifU or nifS

would have pleiotropic effects on the pathway. This puzzle was solved by

investigating the capability of nifUS double mutants to synthesize NifB-co, an

early precursor to FeMo-co (see below). NifB-co biosynthesis was practically

abolished in nifUS mutants [71]. Because NifU and NifS were shown not to be

essential to render active NifB, the lack of NifB-co was attributed to a lack of

[Fe-S] cluster precursors to assemble NifB-co (and hence FeMo-co).

It is important to note that NifB-co is a biosynthetic intermediate not only

of FeMo-co but also of the FeV-co and the FeFe-co of alternative vanadium and

iron-only nitrogenases. Consistently, nifU and nifS, mutants were shown to be

defective in Mo-nitrogenase, V-nitrogenase and Fe-only nitrogenase activities

[7].

!

!

! $"!

&

&

(478/9& Y2& *4./579>0-9& M9.0;;51;8-.9/& ,45-L>.@9-4-2& This schematic model

illustrates the enzymatic machinery (shown above) involved in different steps of

nitrogenase metallocluster assembly (shown below). Early steps involve NifS

and NifU for the assembly of NifH [4Fe-4S] cluster and the precursors of P-

cluster and FeMo-co of NifDK. P-cluster biosynthesis ocurrs in situ by the NifH-

dependent condensation of the [4Fe-4S] pairs into the P-cluster. On the other

hand, FeMo-co biosynthesis occurs outside NifDK. NifB catalyzes the first

committed step of FeMo-co biosynthesis by assembling NifB-co, the central [Fe-

S-C] core of FeMo-co. NifB-co is then transferred to the NifEN scaffold via NifX.

!!

!!

"##

$"

%!

&

'

%!

$

!"#$%&'()*+,-&./'0(&,&*

*1(2-"#-*+,-&./'0(&,&*

3)-/*4)-'(,/**#$%&'()*&./'0(&,&*

!()*$+,-!.)-/01234/)/! !546-+,-!/01234/)/!

!!78+,9:/24;! 546-+,-!()*$+,-!<+,9:/24;!=;4,:;/-;! <+,9:/24;!

!!

!78+,9:/24;!/01234/)/!

>54+>#!

?!8!

8!?! @! @!

%!?!8!

8!?! %!

?!8!

8!?!@! @!@! @!

@! @!A!(

(A!

@! @!

B!5CD(!

7!7

%!%!

%!

5,')-6(/7&(*8('7$$-#$%&'()*+,-&./'0(9#*47'0:7.&*

6-!)1/4;E-1!

@-F-,)2;G24!

4+!HI<!

H?<!

""

!#FG99!J54+#K!,9:/24;!.)-/01234/)/!

<)!

@! @!?!8!

8!?!

@! @!

(L!

!@M!

(@N!

@L!

!

!

! $#!

Maturation to FeMo-co occurs within a putative NifEN/NifH complex by

sequential addition of Fe, Mo and homocitrate. Finally, FeMo-co is transfered by

NafY to apo-NifDK to generate active NifDK protein.

"#7#M!:+.DA!./,@!3+@C(%!NH%<LO!5('3*%/3!*,!*9%!5,/%!,.!H%I,<5,!

!

The nifB gene product encodes a protein having a S-Adenosyl

Methionine (SAM) radical motif CX3CX2C at its N-terminal region [72]. The C-

terminal end of the protein comprises a NifX-like domain that is conserved in

proteins with ability to bind FeMo-co and its biosynthetic precursors [10, 73]. As

mentioned above, NifB participates in an early biosynthetic step that is common

to FeMo-co, FeV-co and FeFe-co biosyntheses. Therefore, mutants lacking nifB

were incapable of diazotrophic growth under all conditions tested [5, 9].

Consistently, regulation of nifB expression by the transcriptional activators of all

three nitrogenases, NifA, VnfA and AnfA, has been reported [6].

NifB catalyzes the conversion of simple [2Fe-2S] or [4Fe-4S] clusters,

donated by NifU, into a complex [Fe-S] cluster denominated as NifB-co in a

reaction that involves radical chemistry [74]. Interestingly, the metabolic product

of NifB, termed NifB-co, was purified and studied before purification of active

NifB had been accomplished [75]. NifB-co comprises the central [(6-8)Fe-9S-C]

core of FeMo-co but does neither contain a heterometal (e.g. Mo) nor

homocitrate [75, 76]. Early experiments showed that NifB-co served as

precursor to FeMo-co in the in vitro FeMo-co synthesis assay [75], and that it

was the source of most (if not all) Fe and S present in FeMo-co [77].

The NifB protein was first purified from A. vinelandii cells [74]. Isolated

NifB was a 110-kDa homodimer containing ca. 12 Fe atoms and exhibiting an

UV-visible spectrum typical of [Fe-S] proteins. Changes in the NifB redox state

and incubation with SAM altered the properties of its UV-visible spectrum, as

expected for a redox-responsive SAM radical protein. Isolated NifB did neither

carry NifB-co nor was readily active in supporting in vitro FeMo-co synthesis.

!

!

! $$!

However, after incubation with Fe2+ and S2-, Fe content increased to ca. 18

atoms and NifB became active [74] providing the first demonstration of

complete in vitro FeMo-co synthesis from its atomic components. The work of

Curatti also laid the groundwork for further mechanistic experiments by showing

that radical chemistry was absolutely required for NifB activity. Later on, it was

shown that the carbide atom at the center of FeMo-co had its origin in the

methyl group of SAM [78].

An interesting observation is that NifB-co could be inserted in place of

FeMo-co into nitrogenase in vitro. This artificial NifDK/NifB-co complex was

capable of H+ and C2H4 reduction but not N2 fixation [79]. This observation

emphasizes the importance in vivo of carrier proteins in redirecting metal

precursors to the appropriate target proteins (see below).

"#7#P!HB0:A!)-!'-Q-,R-!/,(%!+-!:7!.+0)*+,-!!

!

Electron donation processes are essential not only in the N2 reduction

but also in many steps of FeMo-co biosynthesis. Electron donation involved in

Mo-nitrogenase have been identified, in K. pneumonia, Azoarcus sp, and

Rhodobacter capsulatus. In K. pneumoneiae NifF flavodoxin donates electrons

to NifH, which in turn is the obligate electron donor to NifDK [80]. In R.

capsulatus, an FdxN homolog is involved both in electron transfer to NifDK for

N2 reduction, and in the biosynthesis of nitrogenase components [81, 82]. In

Azoarcus sp. strain BH72, deletion of the fdxN homolog affected switch-off of

nitrogenase activity [83] but no effect on nitrogenase activity nor biosynthesis

was observed.

Among the fifteen ferredoxins encoded in the A. vinelandii chromosome,

FdxN can be classified as a type 2[4Fe-4S]. Thus class type includes

ferredoxins with two metal cluster binding domains containing a

CysX2CysX2CysX3Cys and a CysX2CysX7~9CysX3CysX3~5Cys motifs, although

the cysteine labeled in bold does not appear in the sequence of the A. vinelandii

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FdxN [1]. The fdxN gene is located into the minor nif cluster region downstream

of nifB. Co-expression of nifB and fdxN occurs at similar levels under

diazotrophic conditions that allow for the expression of either Mo or alternative

nitrogenases [84]. Mutation in fdxN partially impaired in vivo nitrogenase activity

and diazotrophic growth, indicating that although not essential, FdxN is involved

in some aspect of nitrogenase activity, biosynthesis or regulation [9].

"#7#S!:+.TA!F+/%5*+-&!I,!*,!H%I,<5,!3?-*9%3+3!

!

NifDK represents up to 5% of the total cellular protein accumulated under

diazotrophic growth. Thus, N2-fixing A. vinelandii cells must cope with a large

demand for Mo, a low abundance transition metal (1-2 ppm in soils). A.

vinelandii produces siderophores, low-molecular-weight molecules with high-

affinity for cation metals, to aid in Mo (and Fe) acquisition [85]. Unfortunately,

siderophores can bind to other metals, such as W, which can eventually be

incorporated into FeMo-co rendering inactive nitrogenase [86]. To discriminate

against tungstate, A. vinelandii carries ATP Binding Cassette transport systems

that are highly-specific for molybdate [87]. Three copies of the modABC operon

are found in the A. vinelandii genome [1], as opposed to a single copy in the

closely related bacterium Pseudomonas stutzeri.

A. vinelandii has a unique Mo-accumulation system based on a Mo

storage (MoSto) protein. MoSto is a $3%3 heterohexamer of the mosA and mosB

gene products with capacity to store up to 100 Mo atoms [88] in the form of

complexes of polynuclear oxoanions [89]. In addition, cellular systems are in

place to keep Mo homeostasis and to direct molybdate to the corresponding

Mo-dependent enzymes (Fig. 6). The molbindin ModG appears to be

responsible for directing Mo to N2 assimilation pathways, such as nitrate

reductase or nitrogenase [87]. NifO has been related to Mo balance between

nitrate reductase and nitrogenase. It was suggested that NifO would direct Mo

towards FeMo-co biosynthesis, thus impairing development of nitrate reductase

activity [90].

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The nifQ gene was identified by screening Nif- mutants, the phenotype of

which could be reverted by a large excess of molybdate or cysteine in the

growth medium [91, 92]. Although nifQ mutants did not accumulate molybdate

they were neither impaired in molybdate transport nor in the activity of

alternative nitrogenases [5, 9] nor Mo-co-containing enzymes such as nitrate

reductase [59].

NifQ proteins are found in all diazotrophic species of the Proteobacteria

phylum, with the exception of some Rhizobia. NifQ proteins do neither contain

molbindin domains nor show sequence similarity to MosA or MosB. They do

contain a highly conserved C-terminal putative metal-binding motif

CX4CX2CX5C.

As isolated from A. vinelandii, NifQ was a monomeric 20-kDa oxygen-

sensitive protein containing ca. 3 Fe atoms and 0.30 Mo atoms per monomer.

NifQ displayed a UV-visible spectrum typical of [Fe-S] proteins. Electronic

Paramagnetic Resonance (EPR) and Electron spin echo (ESE)-EPR analyses

revealed that NifQ carried a novel redox-responsive [Mo-3Fe-4S] cluster [93].

In vitro FeMo-co synthesis assays with purified components

demonstrated that NifQ serves as unique Mo source for FeMo-co synthesis.

Comparison of Mo-content in Nif proteins before and after the FeMo-co

synthesis reaction revealed that, only in the presence of NifH, Mo was

effectively mobilized from NifQ to NifEN, demonstrating that all three proteins

were required for Mo transfer [93].

The exact reaction(s) carried out by NifQ are not known. The complete

processing of Mo from molybdate (MoVI) to the state found in FeMo-co (MoIV)

requires at least three chemical transformations: (i) replacement of O ligands by

S ligands, (ii) reduction of Mo from MoVI to MoIV, and (iii) insertion into an [Fe-S]

environment. It has been suggested that the role of NifQ could be related to

some (or all) of these changes [94].

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;90-.& 64=9& A/519--9-2 (i) molybdate harvesting by siderophores, (ii) molybdate

transport and discrimination against tungstate, (iii) Mo accumulation and

homeostasis, (iv) Mo sorting to the appropriate pathway, and (v) Mo insertion

into FeMo-co. &

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The nifV gene product is a homocitrate synthase that catalyzes the

condensation of acetyl coenzyme A and 2-OG to render R-homocitrate [95].

nifV mutants exhibited slow diazotrophic growth rates [96], a phenotype that

could be reverted in vivo by supplementing the growth medium with homocitrate

[97]. V- and Fe-only nitrogenase dependent growth was also impaired in these

mutants [7], indicating that homocitrate was part of FeV-co and FeFe-co as well.

K. pneumoniae nifV mutants have been shown to incorporate citrate into

a non-functional form of FeMo-co in vivo [98]. The situation was more complex

in A. vinelandii where a mixture of organic acids replacing homocitrate in the

cofactor was found [99]. In vitro FeMo-co synthesis assays carried out with

analogous organic acids in place of homocitrate resulted in syntheses of

cofactors with altered catalytic properties [100, 101]. It is not clear how the

nitrogen-fixing cell manages to discriminate between homocitrate and other

analogous organic acids during FeMo-co biosynthesis. Homocitrate

incorporation occurs within NifEN, presumably after Mo incorporation has taken

place [102]. It is possible that discrimination occurs within the NifEN/NifH

complex. It is also possible that homocitrate concentration in the cell was so

high that it would preclude incorporation of other organic acids.

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NifEN is a 200-kDa $2%2 heterotetramer of the nifE and nifN gene

products that carries two identical [4Fe-4S] clusters at the interface of both

subunits [103]. If isolated from the appropriate genetic backgrounds, NifEN

preparations exhibit trapped FeMo-co biosynthetic intermediates [104, 105].

NifEN is absolutely required for FeMo-co synthesis in vivo [3] and in vitro [106].

NifEN displays high similarity with NifDK at several levels, including

amino acid sequence similarity, [107], position of their metal clusters within the

protein [108] and the ability to catalyze C2H2 and azide reduction albeit at very

low rates [109]. It was the amino acid sequence similarity of NifEN to NifDK that

led to the proposal of NifEN acting as molecular scaffold for FeMo-co

biosynthesis [107].

NifEN is the central node of the FeMo-co biosynthetic pathway, where

Mo, homocitrate (and possibly additional Fe) are incorporated into NifB-co (Fig.

5) [51]. Briefly, NifB-co, is transferred from NifX to NifEN, where it is converted

into the VK-cluster (named after Dr. Vinod K. Shah) [110]. Although both NifB-

co and the VK-cluster lack Mo and homocitrate and serve as precursors to

FeMo-co, there are some differential properties that indicate they are not the

same precursor. First, while NifB-co is EPR silent, the VK-cluster shows EPR

signals both in reduced and oxidized states [110]. Second, EXAFS and NRVS

analysis suggest that NifB-co is no larger than the central [6Fe-9S-C] core of

FeMo-co [76], whereas the VK-cluster was proposed to be a larger [8Fe-9S]

cluster [111]. The recently solved NifEN crystal structure confirmed the

assignment of 8 Fe atoms for the VK-cluster. Third, NifEN-mediated Fe

incorporation into NifB-co at capping positions external to the [6Fe-9S-C] core

was achieved in vitro (Rubio laboratory unpublished results).

In addition to the VK-cluster, NifEN purified from a "nifH background has

been shown to contain Mo in a separate [Mo-3Fe-4S] cluster environment [112].

Occupancy levels for this cluster were low and dependent on the purification

method used, probably due to cluster instability [105]. Nevertheless, it was

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shown to serve as Mo source during FeMo-co synthesis in vitro. The

composition of this cluster resembles the one found in NifQ preparations and,

since NifQ has been shown to be able to transfer Mo to NifEN in vitro [93], a

logical proposal is that the [Mo-3Fe-4S] cluster within NifEN derives from the

NifQ cluster. Another possibility is that this cluster represents a NifQ-

independent Mo insertion pathway that would operate with lower efficiency. This

pathway would be responsible for the reversion of the nifQ mutant phenotype

by the presence of 1000-fold molybdate into the growth medium [91, 113]. Interestingly, NifEN is able to substitute for the homologous VnfEN

protein of the V-nitrogenase [114]. This finding raises questions regarding the

specificity of NifEN in Mo insertion into FeMo-co and opens the possibility of

other elements providing this specificity [94].

Finally, NifEN appears to be the site where homocitrate is

incorporated into the cofactor in a reaction that requires NifH [102] .

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NifH (also referred to as dinitrogenase reductase, Fe protein or

nitrogenase component II) is the obligate electron donor to NifDK. NifH is a 60-

kDa homodimer of the nifH gene product. The NifH structure revealed a twofold

symmetric enzyme with Mg2+!ATP binding sites located at the dimer interface

within each monomer. A single [4Fe-4S] cluster is coordinated at the dimer

interface through Cys97 and Cys132 of each NifH polipedtide chain [115]. NifH

undergoes conformational changes during Mg2+!ATP binding and hydrolysis in

a process following electron transfer from the [4Fe-4S] cluster of NifH to the P-

cluster of the NifDK component [116].

Three accessory proteins are necessary to synthesize active NifH,

namely NifU, NifS and NifM [4]. NifM is similar to prolyl isomerases and has

been proposed to induce a conformational change on NifH that precedes

incorporation of its [4Fe-4S] cluster [117]. NifU and NifS are involved in the

assembly and delivery of the [4Fe-4S] cluster of NifH [70] (Fig. 5).

NifH is a moonlighting protein with at least three essential roles in the

nitrogenase system: (i) it is required for electron transfer to the NifDK

component during catalysis, (ii) it is required to assemble P-clusters from pairs

of [4Fe-4S] cluster precursors, and (iii) it is essential to FeMo-co biosynthesis,

in which process it probably plays multiple roles.

Not all of NifH capabilities are required for the performance of all its

functions. Many lines of evidence show that Mg2+!ATP hydrolysis and electron

transfer are required for catalysis but not for P-cluster nor FeMo-co

biosynthesis. First, nifM mutants were shown unable to fix N2 but able to

support FeMo-co biosynthesis [118]. Second, [4Fe-4S] cluster-deficient apo-

NifH (generated by chemical treatment of NifH to remove the metal clusters)

was able to participate both in P-cluster synthesis and in FeMo-co synthesis

[119]. Third, NifH variants with altered properties of Mg2+!ATP binding and/or

hydrolysis could carry out FeMo-co biosynthesis [120, 121]. On the other hand,

more recent experiments indicate that NifH must be able to hydrolyze

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Mg2+!ATP and to transfer electrons in order to be active in FeMo-co

biosynthesis [111].

NifH is absolutely required for FeMo-co biosynthesis. Neither Mo nor

homocitrate are incorporated into the cofactor in the absence of NifH. However,

the exact mechanism by which NifH exerts its role remains unclear. NifEN and

NifH are able to interact transiently with each other [121] and, in fact, Mo

transfer from NifQ to NifEN occurs only in the presence of NifH [93]. It has been

proposed that NifH would play its role of facilitating Mo insertion into the VK-

cluster simply by docking with NifEN and exerting some sort of conformational

change on it [51].

The proposal of NifH being the element that selectively incorporates Mo

into FeMo-co discriminating against other heterometals has long been

discussed. Several observations do not support a role for NifH and other

dinitrogenases reductases in specifying the heterometal to be inserted into the

cofactor and point out to other proteins (e.g. NifQ) being potentially responsible

for heterometal discrimination. VnfH, the equivalent protein in the V-

nitrogenase, could replace NifH in FeMo-co biosynthesis [122]. Similarly, AnfH

of the Fe-only nitrogenase supported FeMo-co synthesis in vivo [123]. Bishop

and collaborators proved that NifH was able to support V-dependent

diazotrophic growth in the absence of VnfH [124].

Finally, it is well known that NifH must be present to render homocitrate-

containing FeMo-co [102, 111]. However, incorporation of homocitrate into an

isolated Mo-containing FeMo-co precursor has not yet been reported. Thus, a

direct role for NifH in homocitrate incorporation into FeMo-co precursor remains

hypothetical.

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Once a cofactor has been synthesized on a scaffold, it needs to be

transferred to its target protein. When prosthetic groups are very labile and

oxygen-sensitive, direct diffusion is unlikely and the metal clusters are expected

to be always protein-bound within the cell [10]. Additionally, there is a rapid

demand for nitrogenase synthesis during diazotrophic growth, which taken

together, might explain the existence of proteins involved in metallocluster

delivery.

The nifX gene is clustered into a single operon together with nifEN. The

nifENX gene cluster is in fact widespread among bacteria, suggesting the three

gene products have a related role. NifX is a 17-kDa single-domain protein.

Although unable to bind 55Fe or 99Mo [102, 125] or assemble an [Fe-S] cluster,

the product of the nifX gene has been shown able to bind FeMo-co and FeMo-

co precursors [110]. Early studies speculated with a role of NifX in the

incorporation of homocitrate into a FeMo-co precursor [102], or as a negative

regulator of nif-gene expression in response to NH4+ concentration and O2

[126]. However, recent in vitro experiments have demonstrated another roles

for NifX [110]. First, it would work as donor of at least two FeMo-co precursors

(NifB-co and VK-cluster) to NifEN. NifX and NifEN do not form a stable protein

complex, but a transient interaction occurs for the metal cluster exchange to

happen. Second, NifX would function as storage of FeMo-co precursors,

redirecting labile metal clusters to NifEN. This might be especially relevant to

buffer the flux of FeMo-co precursors under stress conditions, hence minimizing

metal cluster losses.

NifX-like domains are present in a group of nitrogenase-related proteins,

and thus serves to define a family of nitrogenase cofactor binding proteins,

including VnfX and the C-terminal domains of NifB, NifY, NafY and VnfY. NafY

is probably the best characterized among them [10, 127, 128].

NafY is a 26-kDa protein with a double role in apo-NifDK stabilization and

in FeMo-co insertion into apo-NifDK. Two functional domains can be defined in

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NafY, with each role mostly assigned to each domain. The 12-kDa N-terminal

domain is sufficient to bind to apo-NifDK in the absence of the rest of the

protein. NMR solution structure of the N-terminal domain of NafY revealed that

it contained a sterile alpha motif domain, a structure frequently involved in

protein-protein interactions [129]. This domain represented the first apo-NifDK

binding structure known, other than NifH, and it exhibited a novel fold for apo-

NifDK binding, different from what is observed in the NifH structure [115].

Interestingly, excess of N-terminal NafY domain or full-length NafY had a

negative effect on apo-NifDK reconstitution in vitro.

The 14-kDa C-terminal domain was shown to bind FeMo-co

autonomously. The crystal structure of the core domain of NafY (defined as the

C-terminal domain missing the last 13 amino acid residues) represents the only

known FeMo-co binding fold different from that of NifDK [130]. Mutational

analyses indicated direct implication of the His121 residue in FeMo-co binding

[128]. These results suggest a model with a series of histidine residues involved

in FeMo-co insertion into apo-NifDK. FeMo-co-bound to NafY via His121 would

be donated to $-His362 (at the entrance of the insertion funnel in apo-NifDK),

followed by the entry into the positively charged environment created by the His

triad ($-His274, $-His442 and $-His451) and finally donation to $-His442, as one of

the ligating residues of FeMo-co in NifDK [56].

Given the low affinity of NafY for NifB-co and the ability of apo-NifDK to

bind NifB-co [79], this might be a physiological mechanism to couple FeMo-co

biosynthesis to apo-NifDK activation, while preventing insertion of biosynthetic

intermediates into the nitrogenase active site.

NifX and NafY are not essential for in vitro FeMo-co synthesis or in vivo

diazotrophic growth under standard laboratory conditions [10, 106]. However,

several caveats need to be considered in order to appreciate their relevance.

First, functional overlap among members of this family complicates the finding

of a mutant phenotype in deletion mutants. A BLAST search for NifX-like

sequences reveals two additional homologues in the genome of A. vinelandii

[1], in addition to the above-mentioned members of this family. Thus, functional

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redundancy might obscure the observation of a phenotype in single mutant

strains. Second, diazotrophic growth experiments of deletion mutant strains are

typically carried out under optimal laboratory conditions. This might preclude the

observation of phenotypes present under nutrient-limited environmental growth

conditions. For instance, double "nifX "nafY mutant or triple "nafY "nifY

nifX::kan mutant were impaired in diazotrophic growth under Mo starvation

conditions [10]. Similar observations indicating the requirement of NifX under

Fe-depleted conditions were reported in Herbaspirillum seropedicae [131].

Third, in vitro experiments proved the additive stimulatory effect of NifX and

NafY on FeMo-co biosynthesis when present in the reaction mixture [106]. NifX

and NafY were not required for NifB-co synthesis, but were able to

independently increase apo-NifDK activation. Fourth, proteins involved in metal

cluster storage and delivery have been described in other cofactor biosynthetic

pathways, including the Mo-co carrier protein in Chlamydomonas reinhardtii

[132], the IscA and ErpA carriers in E. coli [133], and the mammalian MMS19

protein for [Fe-S] cluster assembly [134], to name a few. Similarly, enzyme-

specific chaperones relevant to metal cofactor insertion into multi-subunit

metalloenzymes have been reported in other systems, such as the NarJ

chaperone from E. coli [135] and the copper superoxide dismutase from

Saccharomyces cerevisiae [136]. Hence, it is not surprising to find proteins with

similar roles in the FeMo-co biosynthetic pathway.

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Chapter 2: Objectives

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The possibility to replace chemically-synthesized nitrogen fertilizers by a

process of endogenous fixation of atmospheric N2 by major crop plants is a holy

grail in plant biotechnology. Attempting to advance in such direction, our

laboratory is trying to genetically transfer nitrogenase into non N2-fixing

eukaryotic organisms. In order to implement the complex machinery required for

nitrogenase biosynthesis in heterotopous hosts, it is important to acquire good

knowledge about the roles of gene products involved, the intensity of gene

expression, the timing of intervention and the in vivo stoichiometry of proteins

involved in this process.

In addition, some aspects of the biosynthesis of nitrogenase metal clusters,

FeMo-co and the P-cluster, are still unclear. Elucidation of new roles and

reaction mechanisms is important to determine whether or not their function

could be replaced by housekeeping gene proteins in heterotopous hosts. The

specific objectives of this thesis are:

" To determine Intracellular concentration of Nif proteins and nif gene

expression levels in cells of Azotobacter vinelandii along its adaptation to

diazotrophic growth.

" To elucidate the role of FdxN in the molybdenum nitrogenase.

" To determinate the structure and function of molybdenum groups in

NifQ and establish which of them are involved in molybdenum donation to

FeMo-co biosynthesis.

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Chapter 3: Results.

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3.1 Time-course analysis of nif mRNA and Nif protein accumulation

upon derepression of nitrogenase in A. vinelandii

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Activation of N2 fixation is a tightly regulated process, mainly because of

the high-energy demand it imposes. Switching from N2-fixation repressing

conditions to the N2 fixing state is a complex process that in the model aerobic

free-living bacterium A. vinelandii affects expression of up to 400 genes [17].

In furtherance of providing a framework to engineer N2 fixation in non-

diazotrophic organisms, we are interested in obtaining information about:

- The intensity of nif gene expression along the time, defining at what

moment expression starts, reaches a maximum and fades down to a new basal

level, or starts another wave of expression.

- The precise order in which each Nif protein intervenes during transition

to diazotrophic growth and the stoichiometric balance amount different nif

proteins, including the stage dominated by nitrogenase metal cofactor

biosyntheses and the stage dominated by activity of mature nitrogenase.

- The effect of mutations in regulatory, structural and biosynthetic genes

on the balance of nif gene expression.

- The connection of these data with nitrogenase activity registered in the

cell.

Following the methodology described in Fig. 7, we have quantified the

accumulation of mRNA for 17 genes involved in N2 fixation, including 10

essential Mo-dependent N2 fixation genes (nifH, nifD, nifK, nifE, nifN, nifU, nifS,

nifM, nifB and nifQ), 4 genes that code for non-essential proteins with assigned

roles on N2 fixation (fdxN, clpX2, nifX, nafY), elements responsible of the

regulation of nif gene expression (nifA, nifL), and a nif gene of unknown function

in A. vinelandii (nifY). The research strategy includes analyzing the wild-type

strain (DJ), a strain partially impaired in N2 fixation (!fdxN) [9], a strain

incapable of nif gene expression (!nifA) [137], a strain fully defective in Mo-

nitrogenase activity because it lacks the MoFe protein structural genes ("nifDK)

[138], and a strain unable to synthesize active site FeMo-cofactor (!nifB) [74].

In addition, the intracellular molar concentrations of NifH, NifD, NifK,

NifE, NifN, NifU, NifS, NifX, NifY, and NafY were established by using

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quantitative immunoblot analysis of whole cells and estimating the total cell

volume in the samples (Fig. 8).

A version of this section has been published: Poza-Carrion, C., Jimenez-

Vicente, E., Navarro-Rodriguez, M., Echavarri-Erasun, C. & Rubio, L. M. (2014)

Kinetics of Nif gene expression in a nitrogen-fixing bacterium, J. Bacteriol. 196,

595-603.

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-.9AQE5U>2&NH4+-grown cells (green) were collected by centrifugation, washed

with NH4-free (yellow) or NH4+-containing (green) medium (control cultures),

and resuspended in the same medium at a final OD600 of 0.5. Cells were then

incubated at 30°C with shaking (200 rpm) in 9 independent Erlenmeyer flask. At

0, 10, 30, 60, 120, 180, 240 and 420 minutes each one of these flasks was

collected and subjected to the following analyses:

- Determination of in vivo C2H2 reduction activity

- Cell volume determination

- OD600 measurement

- nif mRNA quantification by qPCR

- Quantitative immunoblot analysis

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measuring average cell volume and multiplying this value by the number of

CFU per ml of culture.

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Time (h)

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3.1.1 Diazotrophic growth and in vivo C2H2 reduction activities of A.

vinelandii wild-type, !fdxn, !nifA, !nifB and !nifDK strains

!

As a result of their inability to fix N2 using the Mo-nitrogenase, !nifA,

!nifB and !nifDK strains were unable of grow under standard diazotrophic

conditions (data not shown). On the other hand, the "fdxN strain exhibited a

Nif+ phenotype, although it diazotrophic growth rate was severely affected (see

section 3.2.1).

In vivo C2H2 reduction activities of A. vinelandii wild type, !fdxn, !nifA,

!nifB and !nifDK mutants under diazotrophic growth conditions were

determined (Fig. 9). Cultures were growth in Burk media lacking NH4+ in order

to initiate Mo-nitrogenase synthesis. C2H2 reduction activity by nitrogenase

starts one hour after induction, and in the wild-type strain reaches maximum

values four hours after derepression. C2H2 reducing activities of the !nifA, !nifB

and !nifDK mutant strains were unable to reduced C2H2 under Mo-nitrogenase

derepressing conditions (as expected), whereas the !fdxN mutant strain was

severely affected in nitrogenase activity, showing 30% of wild type levels.

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Figure 9. In vivo C2H2 reduction activities of A. vinelandii wild type and the

!fdxN, !nifB, !nifA and !nifDK mutants.

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3.1.2 Time-course of nif gene expression and Nif protein

accumulation in wild-type cells upon nitrogen step-down.

-

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!

Absolute levels of nif-specific mRNAs accumulation were estimated by

quantitative real-time PCR (qRT-PCR) against the results using known amounts

of synthetic DNA amplicons as template. nif gene mRNA levels were then

compared to 16S rRNA levels present in each sample to yield normalized

absolute signals (NAS). Fig. 9 and Fig. 10 show that A. vinelandii responded

rapidly to nitrogen step-down by expressing nif genes and developing

nitrogenase activity. The nifLA operon was the fastest to respond with nifA

exhibiting a narrow pulse of expression that peaked 30 min after NH4+ removal

from the medium. Expression of genes within the iscAnifnifUSVcysE1nifnifWZM

clpX2 cluster also peaked after 30 minutes exhibiting NAS values for nifU of

(10), nifS (7.3), nifV (3.6), and clpX2 (1.8). Most genes involved in FeMo-co

biosynthesis (nifENX and nifB fdxN nifOQ operons) showed maximum mRNA

levels 1 h after derepression with NAS values ranging from 2 to 7. Similarly,

nitrogenase structural genes nifH, nifD and nifK showed maximum mRNA levels

1 h after derepression but NAS values ranged from 30 to 50. In contrast, nifY

showed the slowest response with accumulation peak at 2 h and a NAS of 4.3.

Two main patterns of mRNA accumulation occurred when nitrogenase activity

was at steady state: while nifH, nifD, nifK, nifE, nifN, nifX and nafY levels were

at least 25% of their maximum NAS 7 h after derepression, all other genes

returned to basal NAS levels.

!

!

! &'!

!

!

! &(!

Figure 10. Time-dependent profile of nif gene induction in the wild-type

strain of A. vinelandii. (A to C) Expression levels of genes within the major nif

cluster, which contains nifHDKTYENX iscAnif nifUSV cysE1nif nifWZM clpX2

nifF. (D to F) Expression levels of genes within the minor nif cluster, which

contains rnfABCDGEH nafY in one DNA orientation and nifLAB fdxN nifOQ

rhdN grx5nif in the opposite DNA orientation. Black arrows in ORF maps indicate

predicted "54-dependent promoter regions. The gray arrow indicates a

hypothetical constitutive promoter region. Dot lines represent unstudied

interspersed genes within each operon. Data is the average of at least three

biological replicates ± SE.

&

!

!

!

!

!

!

!

! &)!

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!4M9Q158/-9&56&*46&A/5.94>&0118M8;0.45>&4>&$1."!+/)!0""&8A5>&9>.9/4>7&E40Z5./5A@41&7/5U.@&&

Intracellular concentration of each selected Nif protein was estimated by

quantifying A. vinelandii whole-cell immunoblot signals against a standard curve

generated by immunoblotting known amounts of the corresponding purified Nif

protein (Fig. 11) and dividing by total cell volume in the sample (Fig. 8).

Consistently with its role in general synthesis of [Fe-S] clusters for both

nitrogenase structural and biosynthetic proteins, NifU accumulated fast,

showing 90% of its maximum content of 23.7 µM only 1 h after derepression

(Fig. 12A). FeMo-co biosynthetic proteins presented low accumulation levels

ranging from 0.4 &M NifEN to 3.2 µM NifB, 5.7 &M NifQ, and 5.8 &M NifX (Fig.

12B). On the other hand, nitrogenase structural proteins accumulated at very

high levels, up to 108.5 µM NifH, 54.8 µM NifD, and 48.1 µM NifK 4 h after

derepression (Fig. 12C). The NafY chaperone, involved in stabilizing FeMo-co-

deficient apo-NifDK, showed significant basal levels (8 &M) at the start of

derepression and moderate increase over time (up to 19,9 &M). At the time of

maximum in vivo nitrogenase activity (4 h after derepression, see Fig. 9), three

main protein groups could be observed according to their intracellular levels: (i)

low-concentration proteins involved in FeMo-co biosynthesis, which included

NifE (0.4 µM), NifN (0.3 µM), NifB (2.7 µM), and NifQ (2.5 µM); (ii) mid-

concentration proteins dedicated to the transfer or storage of FeMo-co and its

precursors, such as NifX (5.8 µM) and NafY (17.7 µM); and (iii) high-

concentration proteins, which included NifD (54.8 µM), NifK (48.1 µM), and NifH

(107.2 µM). The high levels of NifU (23.7 µM) observed were surprising but in

agreement with its role in providing [Fe-S] clusters to many different proteins.

!

!

! &.!

Figure 11. Immunoblots of whole A. vinelandii cells derepressed for

nitrogenase developed with antibodies against Nif proteins. (A)

Immunoblots to detect cellular levels of NifDK, NifH, NifB, NifEN, NifU, NifQ,

NifX and NafY in wild type A. vinelandii cells. Time elapsed after NH4+ removal

from the medium is indicated at the top of the panel. (B) Immunoblots of known

amounts of purified proteins used to generate quantification standard curves.

Squared correlation coefficients R2 are shown to the right of the panel.

!

!

! '/!

Figure 12. Time-course of Nif protein accumulation in A. vinelandii upon

nitrogen step-down. (A) NifU protein, required for the biosynthesis of [Fe-S]

clusters for the nitrogenase components. (B) FeMo-co biosynthetic proteins. (C)

Nitrogenase structural proteins including the NafY chaperone.

!

!

! '"!

3.1.3 Effect of nifA mutation in the expression of selected nif genes

The !nifA mutant exhibited a Nif-minus phenotype and lacked in vivo

nitrogenase activity, as expected [139] the nifH, nifD, nifK, and nifY mRNA

levels present in the !nifA strain were negligible: 390, 170, 120 and 130 fold

lower than in the wild-type strain, respectively (Fig. 13)0

Similarly, the nifENX and the nifB fdxN nifOQ operons showed massive

drops of expression compared to wild type: 43-fold lower mRNA for nifE, 75 for

nifN, 20 for nifX, 270 for nifB, 44 for fdxN, and 17 for nifQ. Consistently, NifH,

NifDK, NifEN, NifX, and NifB proteins were barely detected by immunoblot

analysis (Fig. 14). Genes within the iscAnif nifUSV cysE1nif nifWZM clpX2 cluster

and also nifL revealed not to be as drastically dependent on NifA since nifU,

nifS, nifV, and clpX2 mRNA levels were 3, 4, 9, 4, and 8 times lower than in wild

type, respectively. However, their time-course expression profile showed a clear

response to nitrogen step-down (Fig. 13). This response was also observed in

immunoblot analysis of NifU accumulation in "nifA strain grown under

diazotrophic conditions (Fig. 14).

Finally, nafY mRNA levels were reduced 10 fold with respect to wild type,

a value lower than previously reported [140], and nifY mRNA was almost

undetectable in the mutant. Surprisingly, despite it differences in nafY and nifY

expression profiles between "nifA and the wild type, the NafY and NifY proteins

accumulation levels were very similar (Fig. 14).

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Figure 13. Time-dependent profile of nif gene expression upon nitrogen

step-down in wild-type A. vinelandii (closed symbols) and the "nifA

mutant (open symbols). Genes are organized according to chromosomal

clustering. A NAS value of 2 means twice the amount of 16 rRNA present in the

sample. Data are the average of three biological replicates (±SE).

!

!

! '%!

Figure 14. Comparison of Nif protein accumulation between "nifA and the

wild-type strain. Immunoblots to detect cellular levels of NifDK, NifH, NifB,

NifEN, NifU, NifY and NafY in whole cells of the wild type and the 'nifA strains.

Time elapsed after NH4+ removal from the medium is indicated at the top of the

panels.

DJ !nifA

0 10

30

45

60

90

120

180

240

420

0 10

30

45

60

90

120

180

240

420

NifB

NifDK

NifEN

NifH

NafY

NifX

NifY

NifU

Time (min)

!

!

! '&!

3.1.4 nif gene expression in !nifB and !nifDK mutants.

To determine the extent of feedback regulation of nif gene expression by

NifDK activity, we investigated the response of A. vinelandii to a situation of

nitrogen step-down when it was unable to fix N2. Two different Nif-minus strains

were selected: a !nifB mutant impaired at the early steps of FeMo-co

biosynthesis and thus unable to accumulate cofactor biosynthetic intermediates,

and a !nifDK mutant that has no defect on FeMo-co biosynthesis but is unable

to fix N2.

The !nifB and !nifDK mutants exhibited similar nifA and nifL mRNA

levels and expression profiles than wild type (Fig. 15 and Fig. 16). However, this

was not the case for other nif genes studied, which mRNAs accumulated at very

high levels in the mutants. Seven hours after switching to diazotrophic growth

conditions, the !nifB strain accumulated 10-times more nifH mRNA and 4-times

more nifD mRNA than wild type (Fig. 15), whereas the !nifDK mutant

accumulated 9-times more nifH mRNA than wild type (Fig. 16). The nifB, nifE,

nifU and nafY genes, whose products are involved in biosynthesis and insertion

of FeMo-co, were also largely overexpressed in both mutants. Interestingly,

clpX2 showed unusual peaks of expression overtime with maximum 18-fold

overexpression in !nifDK and 8-fold in !nifB compared to wild type.

!

!

! ''!

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Figure 15. Time-dependent profiles of nif gene expression upon nitrogen

step-down in "nifDK strain. Wild-type A. vinelandii (closed symbols) and the

!nifDK mutant (open symbols). Data are the average of three biological

replicates (±SE).

!

!

! ')!

Figure 16. Time-dependent profiles of nif gene expression upon nitrogen

step-down in "nifB strain. Wild-type A. vinelandii (closed symbols) and the

!nifB mutant (open symbols). Data are the average of three biological

replicates (±SE).

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3.1.5 Deletion of the fdxN gene alters nif gene expression and Nif

protein accumulation/!

The fdxN gene, located between nifB and nifQ in the minor nif cluster,

encodes a ferredoxin with similarity to 2 x [4Fe-4S] ferredoxins that has been

suggested to have a role in FeMo-co biosynthesis 1.2. The !fdxN strain

consistently accumulated higher levels of nif specific transcripts compared to

wild type (up to 7 times higher), except for lower nifY and nifQ mRNA levels and

the absence of fdxN expression (Fig. 17). Seven hours after derepression, all

genes tested presented higher mRNA levels in the !fdxN mutant than in wild

type (Fig. 17). However, it is important to note that the !fdxN mutant is only

partially impaired in N2 fixation (see section 3.2) and that its phenotype is

therefore very different from that of a !nifB mutant.

Although the expression profiles of most nif genes were similar in the

!fdxN strain and the wild type, a few fundamental differences were observed.

First, nifA and nifL mRNA levels did not decreased rapidly as in the wild type,

but rather stayed at maximum levels during a few hours after derepression (Fig.

17). Second, the response to nitrogen step-down was generally delayed with

most mRNAs showing maximum accumulation levels 2-3 h after derepression

and descending slowly over the 7 h period studied here.

The time-course of Nif protein accumulation in !fdxN was significantly

different from wild type (Fig. 18). NifH rate of accumulation and maximum

intracellular concentration was 1.4-fold higher in the !fdxN (144.5 µM) than in

the wild type (107.2 µM). NifD and NifK followed the opposite trend with lower

accumulation rates and maximum intracellular concentrations almost 2-fold

lower than in wild type (33.2 µM and 29.4 µM, respectively). Importantly, the

!fdxN strain accumulated as much NafY chaperone as NifD and NifK,

suggesting the need to stabilize apo-NifDK probably because of its defect in

FeMo-co biosynthesis. It also accumulated higher concentrations of NifU (2.6-

fold) and the FeMo-co biosynthetic proteins NifB (3.1-fold), NifEN (5.3-fold) and

NifQ (2.3-fold) than wild type. The levels of NifX, which has been proposed to

!

!

! (/!

function as reservoir for FeMo-co precursors [110], were similar in wild type and

!fdxN strains.

The [mRNA]max / [protein]max ratio was used as index to understand

correlations between mRNA accumulation and protein accumulation (Table 2).

Low ratios can be interpreted either as high efficiency in mRNA translation or as

high protein stability. On the other hand high ratios could indicate either low

mRNA translation efficiency or low protein stability. Although this index varied

between 0.3 and 0.6 in most cases, a few significant exceptions were found.

The nafY/NafY index was 0.1, which suggested high stability of the NafY

chaperone. On the other hand, nifN/NifN, nifE/NifN, and nifB/NifB indexes were

4.7, 4.16, and 2.12 respectively, suggesting low protein stability, which is

consistent with the reported ClpX2-mediated degradation of NifB and NifEN

during diazotrophic growth [141].

!

!

! ("!

Figure 17. Time-dependent profile of nif gene expression upon nitrogen

step-down in "fdxN mutant. Wild-type A. vinelandii (closed symbols) and the

'fdxN mutant (open symbols). Data are the average of three biological

replicates (±SE).

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Fig

ure 18. Time-dependent profile of Nif protein accumulation upon nitrogen

step-down in the "fdxN mutant. Wild-type A. vinelandii (closed symbols) and

the "fdxN mutant (open symbols). Proteins are organized by role in nitrogenase

biogenesis: proteins involved in early steps of FeMo-co synthesis (A), late steps

of FeMo-co synthesis (B), FeMo-co insertion into apo-NifDK (C), and

nitrogenase structural proteins (D). Total cell volume was used to calculate

protein concentration (&M). Data are the average of three biological replicates

(±SE).

0

25

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0 1 2 3 4 5 6 7

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Time (h)

0

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50

75

0 1 2 3 4 5 6 7

µM

Time (h)

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50

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100

125

150

175

0 1 2 3 4 5 6 7

µM

Time (h)

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0

10

20

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40

50

0 1 2 3 4 5 6 7

µM

Time (h)

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0

5

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15

0 1 2 3 4 5 6 7

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Time (h)

0

2

4

0 1 2 3 4 5 6 7

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Time (h)

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2

4

0 1 2 3 4 5 6 7

µM

Time (h)

NifE NifN NifQ

0

2

4

6

8

10

0 1 2 3 4 5 6 7

µM

Time (h)

0

5

10

15

0 1 2 3 4 5 6 7

µM

Time (h)

0

25

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0 1 2 3 4 5 6 7

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Time (h)

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"! ?+@4ABA! C-D-CE! F5! AG3H! I3HJK! +LM! NOFP-4L! IQ?K! R-O-! BE-M! PF! S+CSBC+P-! PT-E-!

O+P4FE0!

!0,;9&F2&)5MA0/4-5>&56&`MG*"aM0H[`b/5.94>aM0H&/0.45-&65/&0&>8M,9/&56&!"#&79>9-&4>&U4;E&.LA9&0>E&2#034-$1-."!+/)!0""&-./04>-&!

!

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3.2 Research of FdxN role in FeMo-co biosynthesis.

Biosynthesis of metal clusters for the nitrogenase component proteins

NifH and NifDK involves electron donation events. Yet, electron donors specific

to the biosynthetic pathways of the [4Fe–4S] cluster of NifH, or the P-cluster

and the FeMo-co of NifDK, have not been identified. Previous results

established that an A. vinelandii mutant lacking fdxN was partially impaired in

nitrogenase activity and diazotrophic growth, indicating that, while fdxN is not

essential to nitrogenase activity, it is involved in some aspect of nitrogenase

activity, biosynthesis or regulation. Here we generated a "fdxN mutant strain by

carrying out an in-frame deletion of the entire fdxN gene and investigated the

mutant phenotype in detail to ascertain the specific role(s) of FdxN in the Mo-

nitrogenase. A version of this study has been published: Jimenez-Vicente, E.,

Navarro-Rodriguez, M., Poza-Carrion, C. & Rubio, L. M. (2014) Role of

Azotobacter vinelandii FdxN in FeMo-co biosynthesis, FEBS Lett. 588, 512-6.

!

G#7#"!4/,R*9!,.!"$,5/!@'*)-*!'-B%/!B+)6,*/,C9+5!5,-B+*+,-3!

Under diazotrophic conditions the "fdxN strain exhibited a long lag

phase and a 50% increase in doubling time during exponential growth phase

compare to wild type (4.6 h compared to 3 h in the wild-type strain). On the

other hand, no growth defect was observed in "fdxN strain using NH4+ as N2

source. (Fig. 19). These results are in agreement with a previous report [9].

The !fdxN mutant was severely affected in nitrogenase activity.

Detection of nitrogenase activity in vivo was delayed for two hours, exhibiting a

longer lag phase compared to wild type. Maximum activity was detected seven

hours after derepression, but only reached 35% of maximum wild type activity.

In a genetic complementation experiment, an extra copy of fdxN under the

control of the nifH promoter (PnifH) was integrated in a non-nif region of the

chromosome in wild type and the fdxN mutant strains. No difference in C2H2

!

!

! (&!

reducing activity or diazotrophic growth were found between the wild type and

the wild type harboring PnifH::fdxN. However the adition of PnifH::fdxN was

able to revert the "fdxN mutant phenotype to wild type levels of diazotrophic

growth C2H2 reduction activity, demonstrating that the phenotype of UW344

was due to the !fdxN mutation and not to polar effects over nifOQ rhdN grx5nif

genes (Fig 20).

!

!

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and

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. vin

elan

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J (w

ild ty

pe, b

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ed li

nes)

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ant s

train

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ng N

2 (sq

uare

s) o

r NH

4+

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les)

as

sole

nitr

ogen

sou

rce.

!

!

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Figure 20. Genetic complementation of the "fdxN mutation by PnifH::fdxN

in trans. (A) In vivo nitrogenase activity as determined by whole-cell C2H2

reduction assays in A. vinelandii DJ (wild type), UW344 (!fdxN), and derivative

strains UW369 (PnifH::fdxN) and UW371 (!fdxN PnifH::fdxN). (B) growth of A.

vinelandii DJ, UW344, UW369 and UW371 strains at the initial stages after

nitrogenase derepression.

Wild type

UW345

UW369

UW371

!"

#"

nmol

C2H

4 for

med

·min

·OD-

1!OD

600!

!

!

! ()!

3.2.2 !fdxN cells accumulate a mixture of NifDK and apo-NifDK

The "fdxN strain accumulated substantially less NifDK protein than the

wild type while NifH levels were found to be 1.2-fold higher in "fdxN than in wild

type (Fig. 21). Since nifH and nifDK genes are co-transcribed from the same

promoter, the specific decrease in NifDK accumulation suggests either

increased degradation or a defect in the rate of NifDK synthesis

While immunoblot analysis provided an estimate of total NifDK

accumulated in cells, it did not distinguish between active NifDK and inactive

cofactor-less apo-NifDK. In vitro activity assays were performed to determine

the amount of active NifH and NifDK proteins in cell-free extracts obtained from

wild type and "fdxN cells. The levels of active NifDK in cell-free extracts were

determined by the C2H2 reduction assay after addition of excess purified NifH

(Table 3). Considering that NifDK specific activity is 2000 nmol C2H4 formed per

min per mg of protein, it was estimated that wild type and "fdxN cell-free

extracts contained ca. 75 and 20 µg active NifDK per mg of protein in the

extract, respectively.

!

!

! (.!

Figure 21. Comparison of NifDK and NifH protein accumulation between

"fdxN and the wild-type strain. Immunoblots to detect cellular levels of NifDK

and NifH in whole cells of the wild type and the 'fdxN strains. Time elapsed

after NH4+ removal from the medium is indicated at the top of the panels.

!

!

! )/!

To compare the accumulation of NifH in wild type and mutant cell-free

extracts, a titration curve with purified NifDK was necessary; this is because low

NifH/NifDK ratios are detrimental to C2H2 reducing activity of nitrogenase. The

result of this experiment revealed that wild type and "fdxN strains exhibited

almost identical NifH activity levels (Table 3 and Fig. 22).

On the other hand, an excess of NifH protein does not disturb NifDK

specific activity determination and can be used to estimate the concentration of

active NifDK.

In other experiments, cell-free extracts were supplemented with 0.27

nmol of isolated FeMo-co in N-methyl formamide (NMF) solution plus excess

purified NifH. Control reactions contained the same amounts of NMF but no

FeMo-co. Addition of FeMo-co had no significant effect on wild-type cell-free

extracts; an activity of 86.0 ± 3.7 moles of C2H4 formed per min per mg of

protein was obtained compared to 82.5 ± 4.4 in the control reaction. In contrast,

FeMo-co addition increased "fdxN NifDK activity by 32% (40.3 ± 4.1 moles of

C2H4 formed per min per mg of protein compared to 30.6 ± 2.9 in the control

reaction), showing the presence of FeMo-co-activatable apo-NifDK in the

mutant extracts. Thus, "fdxN mutation caused, not only a decrease in total

NifDK levels, but also the accumulation of apo-NifDK probably by a defect in

NifDK biosynthesis.

!

!

! )"!

!

!

!

!

Figure 22. Titration of wild type and !fdxN NifH activities in cell-free

extracts by addition of purified NifDK component. NifDK and NifH were

used to supplement cell-free extracts of wild type and !fdxN strains prior to

determination of C2H2 reduction activities. An excess of pure NifH (240 µg) or

specific amounts of pure NifDK (0, 35, 105, 210, 350 µg) were added per 1 mg

of protein in the extract. Results are the average of 4 independent experiments.

!"

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()"*+,-." ()"*+,/"

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0%1+#2,/!3

4%

!

!

! )#!

!

!

Reaction mixture Activitya

Wild type extract 13.9 ± 2.1

+NifDK 21.6 ± 0.7

+NifH 75.3 ± 5.9

!fdxN extract 5.5 ± 1.9

+NifDK 19.2 ± 5.6

+NifH 19.5 ± 4.7

+!U+CB-E!+O-!PT-!+D-O+,-E!F5!+P!C-+EP!5FBO!+EE+VE!N-O5FOA-M!

E-N+O+P-CV0!JN-S454S!+SP4D4P4-E!+O-!-@NO-EE-M!+E!LAFC!W#>%!

5FOA-M!N-O!A4L!N-O!A,!NOFP-4L!4L!PT-!-@PO+SP0!

!

Table 3. Nitrogenase component activities in cell-free extracts of wild type

and "fdxN strains. Activities were measured by using the C2H2 reduction

assay. The amount of NifH in cell-free extracts was determined after addition of

105 µg NifDK. The amount of NifDK in cell-free extracts was determined after

addition of excess of NifH.

!

!

! )$!

3.2.3 Purification and characterization of "fdxN NifH and NifDK

nitrogenase components

Pure NifH and NifDK preparations were obtained from wild-type and the

"fdxN strains (Fig. 23 A and B). These preparations were used to investigate

activity, metal content, and associated EPR signals. No difference was

observed between "fdxN and wild-type NifH in their capacity to titrate NifDK

activity (Fig. 23C). Maximum activities around 2000 nmol of C2H4 formed per

min per mg of NifDK were obtained at NifH:NifDK molar ratios of 40, as

expected. On the other hand, purified "fdxN NifDK showed 2.5-fold lower

activity than wild type NifDK, since titration with NifH rendered a maximum

activity of 700 nmol of C2H4 formed per min per mg protein (Fig. 23D). !fdxN

NifH and NifDK metal contents and EPR signals were determined and

compared to those of wild-type proteins. Similar to wild type, the !fdxN NifH

was shown to contain 4.2 ± 0.1 mol Fe per mol NifH dimer (Fig. 23E) and to

exhibit the same intensity of the NifH [4Fe–4S] cluster EPR signal (Fig. 24A). Fe

and Mo content in purified !fdxN NifDK were 18.8 ± 1.9 mol and 1.0 ± 0.1 mol

per mol of NifDK tetramer (Fig. 23F), respectively, a ratio that is consistent with

a complement of 2 P-clusters and only 1.2 FeMo-co in the mutant protein. The

DTH-reduced "fdxN NifDK exhibited the characteristic S = 3⁄2 EPR signal arising

from FeMo-co although with an intensity quantified as 40% relative to wild-type

NifDK (Fig. 24F). Furthermore, the slight differences appreciated between the g-

values of NifDK and !fdxN NifDK suggest that the FeMo-co environment was

not identical in the two proteins. No S = ( EPR signal indicative of the presence

of P-cluster precursors [142] was observed in !fdxN NifDK. These results

indicate that !fdxN NifDK was partially deficient in FeMo-co consistent with a

role of FdxN on FeMo-co biosynthesis.

!

!

! )%!

Figure 23. Properties of purified !fdxN NifH and !fdxN NifDK proteins. (A)

SDS-PAGE analysis of purified NifH and !fdxN NifH preparations. (B) SDS-

PAGE analysis of purified NifDK and !fdxN NifDK preparations. (C) tritation of

NifH and !fdxN NifH activities. (D) determination of NifDK and !fdxN NifDK

specific activities. (E) Fe content of purified NifH and !fdxN NifH preparations.

(F) Fe and Mo contents of purified NifDK and !fdxN NifDK preparations.

!

!

! )&!

Figure 24. Derivative EPR spectra of NifH and NifDK proteins purified from

wild type and the !fdxN mutant. (A) spectral conditions for NifH were 9.65

GHz microwave frequency, 10 G modulation amplitude, 6,3 mW microwave

power, and 12 K. (B) spectral conditions for NifDK were 9.65 GHz microwave

frequency, 10 G modulation amplitude, 0.6 mW microwave power, and 5 K.

Apo-NifDK spectrum is added as control lacking the S = 3⁄2 EPR signal from

FeMo-co.

!"""# !$""# %"""# %$""# &"""# &$""# '"""# '$""#

!"#$%

!"#$%%!"#$%

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,'-.%

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!""# $""# !!""# !$""# %!""# %$""# &!""# &$""# '!""# '$""# (!""#

!"#/0%

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1234!"#/0%

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3.2.4 FdxN is involved in NifB-co biosynthesis

UW235 (!nifENX) strain is unable to process NifB-co into FeMo-co

because it lacks the NifEN protein, a mutation that results in NifB-co

accumulation [75]. The UW396 double mutant ("fdxN "nifENX) strain was

generated to investigate whether the absence of fdxN affected NifB-co

accumulation. NifB-co activity levels were estimated by using the NifB-co

dependent FeMo-co synthesis and insertion assay, a biochemical

complementation in which UW45 (nifB) cell-free extracts are incubated with

extracts of the strains being analyzed under conditions that allow FeMo-co

synthesis and insertion into apo-NifDK present in the UW45 extract (see

chapter 5 materials and methods). The activity of reconstituted NifDK is then

determined by standard procedures. NifB-co present in UW235 extracts

reconstituted NifDK activity to a value of 5.40 ± 0.79 moles of C2H4 formed per

min per mg of protein. In comparison, NifB-co present in UW396 extracts only

activated NifDK to 1.13 ± 0.07 moles of C2H4 formed per min per mg of protein,

demonstrating that FdxN activity was required for NifB-co biosynthesis.

!

!

! )(!

3.3 Study of Mo environment(s) in NifQ.

!

We have used Mo and Fe K-edge X-ray absorption spectroscopy (XAS)

to study the ligand environment and the oxidation state of the Mo and Fe atoms

of NifQ. Interpretation of these data should allow a better understanding of the

speciation of Mo in NifQ, and thus the role of NifQ in the Mo biochemistry of

FeMo-co synthesis. This work is a collaborative effort with Dr. Simon George at

UC-Davis and Dr. José A. Hernandez at Midwestern University. A version of this

section is being prepared for submission to the Journal of American Chemical

Society.

Mo and Fe K-edge EXAFS analysis suggested that NifQ contains two

different [Fe-S] clusters, a [Mo-3Fe-4S] cluster, which likely includes Mo-O at

2.12 Å, and the [3Fe-4S] Mo-free version of this cluster. This observation

agrees with metal analysis of pure NifQ preparations showing only 0.3 Mo

atoms per NifQ monomer, and also with EPR experiments, which suggested

that each NifQ molecule carries either a [3Fe-4S] cluster or a [Mo-3Fe-4S]

cluster [93]. The best Mo EXAFS fits suggest that a second Mo environment

that accommodates about half of the bound Mo is also present and includes

short 1.73 Å Mo-O and possible Mo-S bonds at 2.23 Å.

NifQ was incubated with either $, $'-bipyridyl or CuCl2, to alter the ratio of

its Mo environments. Mo K-edge EXAFS of the resulting NifQ proteins is shown

in Fig. 25 while fitting parameters are presented in Table 4. A control reaction in

which NifQ was incubated with assay buffer showed slightly different ratios of

Mo=O content than as-isolated NifQ (compare panels A and B in Fig. 25). Mo

EXAFS was consistent with the $, $'-bipyridyl chelating a fraction of the Fe

present in the [Mo-3Fe-4S] cluster without largely altering this Mo environment

(Fig. 25C). On the other hand, treatment with CuCl2 practically eliminated the

second Mo environment, as indicated by the increase in the number of 2.26 Å

Mo-S and 2.69 Å Mo-Fe bonds and the decrease of the 1.73 Å Mo-O bonds

(Fig. 25D). Interestingly, Cu(II) treatment abolished NifQ ability to serve as Mo

source in the in vitro FeMo-co synthesis and insertion assay (Table 5) while $,

$'-bipyridyl treatment decreased it only by 34%.

!

!

! ))!

Figure 25. Mo K-edge EXAFS of NifQ. (A) As-isolated NifQ. (B) NifQ after

buffer exchange treatment. (C) NifQ after $, $'-bipyridyl treatment. (D) NifQ

after CuCl2 treatment. Data and fits are presented as broken and solid lines,

respectively. Left panels, k3 weighted Mo EXAFS spectra. Right panels, Mo-S

phase corrected Fourier transforms from left panels.

!

!

! ).!

NifQ – As Isolated NifQ – Buffer control + Bipyridyl + Cu(II)

N R (Å) #2 (Å2) N R (Å) #2 (Å2) N R (Å) #2 (Å2) N R (Å) #2 (Å2)

Mo-S 1.5 2.336 0.00400 1.39 2.325 0.00454 1.53 2.25 0.00454 2.89 2.263 0.00454

Mo-Fe 1.5 2.713 0.00343 1.44 2.713 0.00345 0.95 2.701 0.00345 2.07 2.690 0.00345

Mo-O (short) 0.88 1.734 0.00186 0.76 1.734 0.00200 0.57 1.736 0.00200 0.60 1.730 0.00200

Mo-O (long) 1.05 2.124 0.00500 1.52 2.095 0.00514 1.24 2.088 0.00514 2.43 2.186 0.00514

"E0 (eV) -9.70 -9.29 -9.23 -7.92

Scale Factor 1.05 1.05 1.05 1.05

Table 4. EXAFS curve fitting parameters for the spectra of NifQ samples

treated with reagents to selectively eliminate each Mo environment. The

corresponding spectra are illustrated in Fig. 25. N is the number of

backscattering atoms. R is the distance. #2 is the Debye-Waller factor. "E0 is

the threshold energy offset. Threshold energies ("E0) were constrained to be

the same for all components. The estimated uncertainties in N, R, #2 and "E0

were in general less than ± 0.25, ± 0.006 Å, ± 0.0008 Å2, and ± 0.6 eV,

respectively.

!

!

! ./!

Table 5. Effect of altering Mo environments in NifQ on its ability to support

in vitro FeMo-co synthesis. FeMo-co synthesis and nitrogenase activity

assays were performed with purified components. Reaction mixtures included a

large excess of apo-NifDK. After FeMo-co synthesis, the amount of

reconstituted NifDK is determined by the acetylene reduction assay.

Nitrogenase specific activity is given in nmol C2H4)min-1)mg-1 protein. Values

are the average of at least two independent determinations ± SD.

!

Activity

NifQ

treated with assay buffer 50.2 ± 2.8

treated with #, #’-bipyridyl 32.9 ± 8.1

treated with CuCl2 1.9 ± 0.1

MoO42- 36.4 ± 5.3

No Mo source 2.0 ± 0.1

!

!

! ."!

!

!

!

!

!

!

!

!

!

!

!

!

!

! .#!

!

!

!

!

!

!

!

!

!

!

!

!

!

4. General discussion

!

!

! .$!

J#"!8+@%<5,'/3%!)-)(?3+3!,.!"#$!@$:;!)-B!:+.!C/,*%+-!)55'@'()*+,-!+-!:7<.+0+-&!'63437+84)2!(#")*+",##!!!

A. vinelandii responds in an agile and highly coordinated manner to

nitrogen step-down in preparation for diazotrophic growth. The earliest

expression was detected in the nifLA operon encoding the regulatory proteins

NifL and NifA [143, 144]. Maximum nifA mRNA accumulation occurred 10 to 30

minutes after NH4+ removal from the medium and triggered a series of events

that led to nitrogenase biosynthesis and diazotrophic growth. However, 1 h after

derepression nifA mRNA levels collapsed to barely detectable (Fig. 10 and Fig.

26). Expression of nifL followed an identical pattern, but nifL mRNA levels were

considerably lower despite being located closer to the promoter of the nifLA

operon. Since there are no apparent additional promoters upstream nifA, a

plausible scenario could be that nifL has faster mRNA degradation rate than

nifA in a mechanism involving specific non-coding RNAs capable of selectively

labeling nifL mRNA degradation, such as the one described in [145]. The DNA

region upstream nifLA contains one RpoN (#54)-binding motif and two NifA-

binding UAS suggesting that NifA self regulates expression of the nifLA operon.

Consistently, very low nifL mRNA levels were observed in the "nifA mutant (Fig.

13 and Fig. 26).

Rapid accumulation of nifU, nifS, nifV and clpX2 mRNAs was also

observed but their pattern of disappearance was not as drastic as for nifL and

nifA (Fig. 10 and Fig. 26). The products of nifU and nifS are involved in [Fe-S]

cluster biosynthesis for both nitrogenase components [4] including serving

precursors for the early stages of FeMo-co biosynthesis [71]. The product of

clpX2 is part of a protease system that specifically targets NifB and NifEN

proteins for degradation [146]. Expression of nifU, nifS and nifV genes, which

products are required for all three nitrogenases in A. vinelandii [147] was not

completely eliminated in the "nifA mutant (Fig. 13 and Fig. 26).

!

!

! .%!

&

(478/9& F:2& _0/40.45>& 4>& !"#& 79>9& 9HA/9--45>& .@/587@& 0E0A.0.45>& .5&

E40Z5./5A@41& 7/5U.@2&Different responses to the change towards diazotrophic

growing conditions in A. vinelandii wild type, a mutant lacking the transcriptional

activator NifA, a mutant lacking the nitrogenase structural genes NifDK, and

mutants partially ("fdxN) or completely ("nifB) impaired in FeMo-co

biosynthesis. (A) Wild-type vs. "fdxN mutant; (B) Wild-type vs. "nifA mutant;

(C) Wild type vs. "nifB mutant; (D) Wild-type vs. "nifDK mutant. mRNA

expression data were visualized with the Multi Experimental Viewer program

(http:://www.tm4.org/). Scale color bars show mRNA normalized absolute

signals; black indicates lack of mRNA whereas intensive green indicates the

nifH nifD nifK

nifY nifE nifN nifX nifU nifS nifV nifA nifB fdxN nafY

nifL nifQ clpX2

0 10m

30

m

1h

2h

3h

4h

7h

Wild type !fdxN

0 10m

30

m

1h

2h

3h

4h

7h

100 0 100 0

10 0 10 0

1 0 1 0

nifH nifD nifK

nifY nifE nifN nifX nifU nifS nifV nifA nifB fdxN nifQ

nifL nafY

0 10m

30

m

1h

2h

3h

4h

Wild type !nifA

0 10m

30

m

1h

2h

3h

4h

50 0 50 0

5 0 5 0

1 0 1 0 clpX2

nifH nifD

0 10m

30

m

1h

2h

3h

4h

7h

Wild type !nifB

0 10m

30

m

1h

2h

3h

4h

7h

100 0 100 0

10 0 10 0

1 0 1 0

nifE nifU

nifY

nifA

nafY

nifL

clpX2

nifB

nifH nifD

0 10m

30

m

1h

2h

3h

4h

7h

Wild type !nifDK

0 10m

30

m

1h

2h

3h

4h

7h

100 0 100 0

10 0 10 0

1 0 1 0

nifE nifU

nifY

nifA

nafY

nifL

clpX2

nifB

A B

C D

7h

7h

!

!

! .&!

maximum mRNA level in each experiment. Each data point represents the

mean of at least three biological replicates. !

One RpoN-binding motif and two putative AnfA-binding UAS are located

upstream iscAnif and also between iscAnif and nifU suggesting that expression of

the iscAnif nifUSV cysE1nif nifWZM clpX2 operon is also under the control of

AnfA. It has been previously shown that A. vinelandii nifA mutant was able to

express V- or Fe-only nitrogenases when molybdate was not present in the

growth medium [147], and that VnfA and AnfA transcriptional activators were

able to modulate expression of nifB [148] and nifM [149]. No regulation of nifU

and nifS expression by either VnfA or AnfA has been reported.

This study provides accurate quantification of expression strength in

terms of mRNA and Nif protein accumulation as well as timing of expression

required for N2 fixation. Expression of most genes studied here peaked 1-2 h

after derepression. Importantly, expression of the nifB fdxN nifOQ rhdN grx5nif

operon appears to stop earlier than that of the nifHDKTY operon (Fig. 10 and

Fig. 26), evidencing correlation between expression timing and protein function.

We found that, in general, genes required for early stages of nitrogenase

biosynthesis, i.e. nifU, nifS, fdxN and nifB were expressed and turned-off earlier

than genes required in later stages of the pathway.

Although each nif operon yielded similar mRNA levels for the genes it

contains, clear exceptions were found in the cases of nifL (described above),

nifY and nifQ. The nifY mRNA levels were 10-fold lower than those of nifH, nifD

and nifK, while nifQ had 4-fold lower mRNA levels than nifB and fdxN. The

presence of intergenic secondary structures within the nifHDKTY operon

affecting mRNA levels has been described [18]. Likewise, the lower expression

of nifQ with respect to nifB and fdxN was observed before [150]. We suggest

that mRNA stability within nif operons may be modulated by the presence of

uncharacterized non-coding RNAs.

The accumulation pattern of Nif proteins indicates that the diazotrophic

cell is capable of maximizing its resources by logically arranging protein

synthesis based on their functions. NifU was the first protein to reach maximum

!

!

! .'!

accumulation (Fig. 27) because of its essential role in [Fe-S] cluster

biosynthesis for nitrogenase components including early steps in FeMo-co

biosynthesis. However, NifH was the most prevalent protein 1 h after

derepression and remained as such during the next 6 h.

!

!

! .(!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

U

X

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0.1 0.6 2 5 6 10 15 30 60 100 150

1 h 1 h

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7 h 7 h

Wild type fdxN

!

!

! .)!

Figure 27. Concentration of Nif proteins on both wild type and !fdxN

strains. Left Panels A, C, E, G and I correspond with wild type cells after 1, 2,

3, 4, and 7 hours of induction time respectively. Right panels B, D, F, H, and J

correspond with Nif protein concentration after 1, 2, 3, 4, and 7 hours of

induction in !fdxN cells. Violet spheres represents µM concentration values.

It is possible that early NifH abundance is required to support P-cluster

[151] and FeMo-co syntheses [152]. NifH achieved its maximum concentration

4 h after derepression coinciding with maximum NifDK concentration and

maximum in vivo nitrogenase activity, in line with NifH structural role in the

nitrogenase complex. At the time when maximum nitrogenase activity occurs,

the in vivo NifH/NifDK molar ratio was 2. This is in contrast to the optimum ratio

in the in vitro assays reported to be between 40 NifH to 1 NifDK [153].

!

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Once nitrogenase was fully operative, the demand for biosynthetic

components such NifB and NifEN decreased. NifB catalyzes the biosynthesis of

NifB-co, an early intermediate in the FeMo-co biosynthetic pathway [154].

Similarly to NifU, NifB concentration was highest 1 h after derepression (3&M)

but it slowly decreased down to 1 µM after 7 h of derepression. NifE and NifN

polypeptide concentration was similar throughout the experiment, consistent

with its stoichiometric requirement to form the NifE2N2 heterotetramer in which

FeMo-co is assembled [51]. The NifEN/NifDK molar ratio also decreased

overtime from 0,02 one hour after derepression to 0,007 after 7 h. The fact that

NifB and NifEN are actively degraded during diazotrophic growth [146] may

explain their very low concentrations. It is however likely that NifB and NifEN

are able to perform multiple turnovers in vivo providing enough FeMo-co for an

excess of NifDK molecules.

In contrast to other biosynthetic proteins, NifQ and NafY concentration

steadily raised, suggesting that they are needed along and further the initial

diazotrophic growth stages (Fig. 12 and Fig. 27). The roles of NafY in apo-

NifDK stabilization and in FeMo-co insertion to form holo-NifDK [155] might be

still required at later stages. NifQ accumulation pattern is difficult to interpret

because its role is to donate the Mo atom to the NifEN/NifH complex for its

incorporation into FeMo-co [156] and, therefore, a pattern of accumulation

similar to those of NifB or NifEN was expected.

New insights about timing and regulation of nif expression were obtained

from the study of the 'fdxN mutant. The 'fdxN strain is partially impaired in

FeMo-co biosynthesis and accumulates as much apo-NifDK as holo-NifDK

[157]. The pattern of nif gene expression in this mutant was characterized by a

lag in nif gene expression and increased nif mRNA accumulation (Fig 15 and

Fig. 26). Importantly, nifA mRNA levels were increased but this is not a general

effect of mutants impaired in nitrogenase activity because 'nifB and 'nifDK

mutants showed wild-type profiles for nifA expression. Thus, it is possible that

FdxN has an additional regulatory role besides its role in FeMo-co biosynthesis.

!

!

! "//!

Although nifD and nifK mRNA levels remain high during derepression in

the 'fdxN strain, NifDK protein was much less abundant and therefore the

corresponding [mRNA]max/[Protein]max index was significantly higher than in wild

type. In addition, NafY was more abundant in the 'fdxN mutant reaching

equimolecular concentration to NifDK. It is known that NafY associates to the

FeMo-co deficient apo-NifDK providing stabilization [155, 158]. Altogether,

these observations indicate that the 'fdxN mutant accumulates significant

amounts of apo-NifDK. It also accumulated 3-fold the amount of FeMo-co

biosynthetic proteins, including NifU, NifS, NifB, NifE, NifN, and NifQ compared

to wild type (NifH concentration increased 1.4 fold), probably to compensate its

impairment in FeMo-co biosynthesis. Stoichiometry among biosynthetic proteins

remained almost identical as if the fine-tuned regulation coordinating FeMo-co

biosynthesis was not affected.

Finally, the 'nifB and 'nifDK mutants, who are unable to fix N2 and

therefore remain under nitrogen starvation, responded to derepression by

accumulating enormous amounts of nif mRNAs. The mechanism by which A.

vinelandii detects a defect in nitrogenase biosynthesis or activity is not well

understood. Interestingly, nifA mRNA accumulation was unaltered in the 'nifB

and 'nifDK mutants, evidencing the importance that postranslational

modulation of NifA activity has in nif gene expression [143].

Previous studies had shown that A. vinelandii cultures derepressed

under N2-free atmosphere rendered uncontrolled accumulation of active

nitrogenase in response to the inability to produce ammonia [159]. This

phenomenon, known as hyperderepression, is similar to the responses of 'nifB

and 'nifDK mutants and would explain the higher accumulation of mRNAs in

these mutants. The overenhanced responses to derepression were more

intense in !nifB and !nifDK strains (Nif- mutants) than in "fdxN (a partially

nitrogenase activity impaired strain), consistent with the existence of at least

two negative feedback regulatory mechanisms. The first such mechanism

responded to the levels of fixed nitrogen (hyperderepression), whereas the

second mechanism appeared to respond to the levels of the mature NifDK

component.

!

!

! "/"!

J#7!$%3%)/59!,.!HB0:!/,(%!+-!H%I,<5,!E+,3?-*9%3+3#!!

A polar fdxN::lacZ-Km insertional mutant was previously reported to be

impaired in NifDK activity [9]. V- and Fe-only nitrogenase activities were also

affected in this mutant and, consistently, fdxN expression was observed under

all three diazotrophic growth conditions [9]. To narrow down the role of FdxN in

nitrogen fixation, we generated a mutant strain carrying a non-polar in-frame

fdxN deletion and sequentially asked a series questions. The "fdxN strain did

not exhibit a strict Nif minus phenotype but was clearly impaired in nitrogenase

activity and diazotrophic growth.

The lack of a complete Nif minus phenotype is likely due to some degree

of functional overlap with other ferredoxins. The A. vinelandii genome sequence

reveals the presence of 14 additional ferredoxin encoding genes annotated as:

fdxA, vnfF, iscfdx, fixX, asfB, xylT, lapQ, Shethna protein, Rieske-type

ferredoxin, Avin01510, Avin03470, Avin10510, Avin25850, and Avin39700 [1].

Some of these genes and their ferredoxin products have been studied. The

fdxA gene encodes A. vinelandii ferredoxin I, which was suggested to have

some functional overlap with the NifF flavodoxin. The role of ferredoxin I was

not clear: while fdxA mutants grew at wild-type rates, fdxA nifF double mutants

grew slower both under diazotrophic and non-diazotrophic conditions [160].

Ferredoxin I contains one [3Fe–4S] cluster and one [4Fe–4S] cluster and its

atomic structure has been solved [161]. The vnfF gene is located immediately

downstream of vnfH [124] and it has been suggested to participate in electron

donation to the V-nitrogenase. Indeed, vnfF transcript levels increased 100-fold

under Mo-independent diazotrophic growth conditions [17]. The iscfdx gene

belongs to an operon involved in [Fe–S] cluster assembly for general purposes

[62]. Its role is to supply electrons to the cysteine desulfurase IscS aiding in the

reduction of S0 to S-2 [162]. The Shethna protein is part of the so-called

nitrogenase conformational protection mechanism as it has been shown to

associate with nitrogenase under conditions of O2 stress and protect it from

oxidative damage [163]. The roles of many other A. vinelandii ferredoxins

remain unassigned.

!

!

! "/#!

We first asked whether wild-type nitrogenase activity levels could be

obtained in "fdxN cell-free extracts by using the in vitro nitrogenase assay, in

which electron donation to nitrogenase is carried out by DTH, and the answer

was negative. The fact that "fdxN cell-free extracts were specifically defective in

NifDK activity did not support a role for FdxN in electron donation during

nitrogenase catalysis but rather in NifDK expression, biosynthesis or stability.

We then asked whether NifDK expression was affected and found that

the "fdxN mutant accumulated 2-fold lower levels of NifDK than wild type.

However, NifH levels were slightly higher in the "fdxN mutant, and it is known

that nifHDK gene expression is driven by a common promoter upstream nifH.

Apart from NifH, the "fdxN mutant accumulated higher levels of other proteins

involved in FeMo-co synthesis, such as NifU, NifB, NifQ and NifEN (Fig. 27).

This phenotype can be interpreted as a compensatory mechanism to increase

nitrogenase activity. Thus, low NifDK levels were probably due to a defect in

either biosynthesis or stability, or both. Lower apo-NifDK levels have been

reported for mutants affected either in P-cluster assembly or in FeMo-co

biosynthesis or insertion [10, 164, 165]. These effects have been interpreted as

decreased apo-NifDK stability. However, these results do not completely rule

out an additional role for FdxN in modulating nif gene expression. We note that

an Azoarcus sp. BH72 fdxN mutant strain exhibited a complex phenotype that

affected switch off regulation of nitrogenase activity [32].

Detection of FeMo-co activatable apo-NifDK [138, 164] in "fdxN cell-free

extracts further suggested that (i) FdxN was involved in FeMo-co biosynthesis,

and (ii) nitrogenase P-cluster synthesis was not altered. P-cluster synthesis is a

NifH-and reductant dependent process that occurs by the in situ fusion of two

[4Fe–4S] cluster subunits within apo-NifDK [55]. It is important to note that P-

cluster synthesis is prerequisite for FeMo-co insertion. Therefore, mutants

unable to synthesize the P-clusters would accumulate a form of apo-NifDK that

cannot be activated by the simple addition of FeMo-co [47].

Finally, purified "fdxN NifDK showed properties that were consistent with

the absence of part of its FeMo-co complement: lower specific activity, lower Mo

!

!

! "/$!

and Fe content, and lower intensity of FeMo-co EPR signal. The presence of

FeMo-co-deficient apo-NifDK in the "fdxN mutant could explain its lower levels

overall, since this form of the enzyme has been reported to be less stable than

NifDK [164].

Being the fdxN gene part of the nifB fdxN nifOQ rhdN grx5nif operon, it

was reasonable to hypothesize that FdxN could function in concert with either

NifB or NifQ. Both NifB and NifQ require a source of reductant to synthesize

their products, namely NifB-co [74] and a [3Fe–4S–Mo] cluster [94],

respectively. Indeed we found that the "fdxN strain was severely impaired in

NifB-co biosynthesis. On the other hand, FdxN participation in the same

reaction as NifQ is unlikely because the phenotype of fdxN mutation could not

be reverted by growing the cells diazotrophically in the presence of 1 mM

sodium molybdate, conditions known to revert nifQ mutant phenotype [9]. NifU

and NifS have been shown to provide NifB with [Fe–S] cluster precursors for

NifB-co synthesis [71]. They are also required for the synthesis of [Fe–S]

clusters for other nitrogenase proteins, including NifH [70] and the P-clusters of

NifDK [55]. Assembly of [4Fe–4S] clusters in NifU requires reductant [68], as it

does the transfer of these clusters to target apo-proteins [67]. The results of this

study do not support the participation of FdxN in either these processes

because no defect was observed in "fdxN NifH with regard to its activity or

[4Fe–4S] cluster content. In conclusion, this study establishes the involvement

of FdxN in NifB-co biosynthesis by NifB. We suggest that the role of FdxN is to

serve as electron donor to NifB during catalysis, probably by reducing the SAM-

bound [4Fe–4S] cluster and driving the formation of the 5’-deoxyadenosyl

radical.

!

!

! "/%!

J#G!]-!^+*/,!H%I,<5,!3?-*9%3+3!)33)?!'3+-&!:+.T!*/%)*%B!R+*9!_`!_a<E+C?/+B?(!,/!b'b(7!)3!,-(?!I,!3,'/5%#!!

NifQ is known to donate Mo to the NifEN/NifH biosynthetic machinery to

complete FeMo-co [93]. The presence of two apparent Mo binding

environments in NifQ is relevant to the Mo chemistry for FeMo-co biosynthesis.

The existence of this second Mo form in NifQ may explain the observation that

only 50% of the Mo is typically transferred from native NifQ to NifEN/NifH under

FeMo-co synthesis conditions [93]. It must be noted that it is not clear whether

or not the two Mo sites are coexisting in the same NifQ monomer.

Mo is initially imported into the cell as MoO42- and the subsequent conversion of

this Mo(VI) oxyanion into the reduced Mo(IV) present in FeMo-co requires three

chemical events: replacement of oxo-ligands by S ligands, reduction of Mo and

insertion of Mo into an Fe-S environment. The [3Fe-4S] cluster observed in

isolated NifQ molecules is an obvious site for Mo binding, reduction and

subsequent transfer. This is not just because a fraction of the [Fe-S] clusters in

NifQ were found to be [Mo-3Fe-4S] centers, but also because the reduced [3Fe-

4S]0 state is known to be able to coordinate heterometals to complete [M-3Fe-

4S] cubanes in reversible equilibrium that can be gated by the cluster oxidation

state [166]. This means that the [3Fe-4S] cluster could be capable of binding

and reducing Mo, releasing it in response to an oxidative trigger. However, a

necessary preliminary step would be the partial or total replacement of MoO42-

oxo-ligands by S. The observation in NifQ of a separate Mo environment with

an apparent mixture of Mo=O and short Mo-S bonds suggests this second site

may be the locus for this Mo binding and subsequent S substitution in a

mechanism that is unclear.

An alternative hypothesis for the role of the second environment is that it

represents the form of Mo that is delivered to NifEN for FeMo-cofactor

biosynthesis. When the second Mo environment of NifQ was eliminated by a

CuCl2 treatment, NifQ was unable to donate Mo in the in vitro FeMo-co

synthesis assay. The fact that the treatment with #, #'-bipyridyl to attack NifQ

[Mo-3Fe-4S] cluster only decreased NifQ ability to serve as Mo source by one

!

!

! "/&!

third, points to a role of the second Mo enviroment in direct transfer of Mo to

NifEN (Fig. 28).

!

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!

!

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(478/9&F<2&R5E9;&65/&.@9&/5;9&56&*46T&4>&R5&4>15/A5/0.45>&4>.5&(9R5Q152!Brown

and blue arrows represent events carried out by NifQ and NifEN, respectively.

Two Mo environments are present in NifQ: a [Mo-3Fe-4S] cluster that

incorporates Mo from molybdate, and an Fe-free Mo environment that could be

involved in Mo transfer to NifEN.

!

!

!

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5. Conclusions !

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1. There is a temporal coordination in the transcription of the nif genes

according to their role in nitrogenase biosynthesis.

2. A logical organization of Nif protein accumulation according to their function

is defined in Azotobacter vinelandii cells during their adaptation to diazotrophic

growth.

3. RNAm levels of nifL are surprisingly lower than those of nifA. An unknown

regulation mechanism must be involved in this event.

4. Expression of nifUSV is not strictly dependent on nifA under diazotrophic

conditions.

5. The !fdxN mutant shows higher accumulation of Nif proteins involved in

FeMo-co synthesis without altering their stoichiometry ratios.

6. The !fdxN mutant is impaired in the activity of the NifDK component of

nitrogenase and, as a result of this, it shows low in vivo nitrogenase activity as

well as low diazotrophic growth rate.

7. NifDK protein purified from !fdxN cells contains half complement of FeMo-co

compared to the wild type NifDK. EPR analysis suggests that the environment

around FeMo-co is altered in the !fdxN-NifDK protein.

8. FdxN has a specific role in biosynthesis. In particular, FeMo-co.

9. FdxN is required for the biosynthesis of the NifB cofactor. This is the first role

unequivocally assigned to any ferredoxin in Azotobacter vinelandii nitrogenase

biosynthesis.

10. NifQ ability to transfer Mo directly to NifEN depends on the presence of an

envioroment that includes Mo-O and Mo-S bondE0!

!

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! ""/!

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1.3 References !

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