X-ray Absorption Spectroscopy: a

tool to unveil structural dynamics

of hemeproteins

Prof. Alessandro Arcovito

Istituto di Biochimica e Biochimica Clinica

Università Cattolica del sacro Cuore

XCVII Congresso Nazionale della Società Italiana di Fisica

L’Aquila 26-30 settembre 2011

Sructural dynamics of proteins

• A protein structure is always the

compromise between stability and function

and it may be not sufficient alone to fully

understand a biological process to the

molecular level.

• A general approach aimed to identify the

key structural determinants driving the

entire biological process, is called

structural dynamics.

Structural changes in YQR-Mb 316 ns after photolysis.

Complex landscape of protein structural dynamics unveiled by nanosecond Laue crystallography

Dominique Bourgeois, Beatrice Vallone, Friedrich Schotte, Alessandro Arcovito, Adriana E. Miele,

Giuliano Sciara, Michael Wulff, Philip Anfinrud, and Maurizio Brunorii PNAS 2003;100:8704-870

©2003 by National Academy of Sciences

Structural changes in the heme vicinity of YQR-Mb detected at 0.1–316 ns

after CO photolysis; the time frames at 316 ps and 1 ns are not shown.

Extended subnanosecond structural dynamics of myoglobin revealed by Laue crystallography

Dominique Bourgeois, Beatrice Vallone, Alessandro Arcovito, Giuliano Sciara, Friedrich Schotte, Philip A.

Anfinrud and Maurizio Brunori PNAS 2006;103:4924-4929

©2006 by National Academy of Sciences

energy

absorb

ance

XANES EXAFS

Pre-edge edge Post-edge

100 eV 800 eV

μ = absorption coefficient, or

absorption cross section

X-ray Absorption Spectroscopy

IFluo

Io

IT (E) Io exp((E )x)

I fluo(E)(E)

IT

x

XRD vs. XAS

Pro’s Con’s

XRD • long range order information • coordinates error depends on

resolution, completeness and

refinement.

• Need of crystalline material

• at low resolution metal clusters

cannot be resolved unambiguously.

XAS • works with solutions, powder,

crystals

• Very good coordinates error

• short range order information

• Need for a metal center

X-rays

Fluo

detector

CCD

XRD (1.4Å resolution)

Polarised XANES

Daresbury, CCLRC MAD-10

Out of work !!

XANES and XRD on the

same myoglobin crystal

P21 crystal of

myoglobin

MXAN results as restraining

factor in XRD refinement

A B C D (PDB code 1EBC)

e || heme_normal

Neuroglobin (Ngb)

• Able to bind O2, CO, NO

• Ngb concentration in neurons is low (micromolar) and O2

storage or transport functions are ruled out

• Function proposed: 1) O2 sensing 2) enzymatic reductase

activity 3) NO detoxification

• Ngb: monomeric hemeprotein expressed in vertebrates brain.

• Increased hypoxic stress damage in neurons upon repressing

Ngb biosynthesis AND improved recovery following

overexpression.

1. Burmester et al. (2000) Nature 407, 520–523.

Ngb 3D structure

• Classical globin fold.

• X-ray structure of Ngb solved at 1.4 Å.

• Bis-His ligation at the Fe-heme

• Ligand-linked conformational change observed in NgbCO (1.7 A res.), involving sliding of the heme in a different position

Ngb

NgbCO

Vallone et al. Proteins 2004 56:85.

Vallone et al., PNAS 2004, 101: 17351

Light Blue: Mb

Blue: Ngb

Blue: Mb

XRD

XANES

fingerprints

EXAFS fit

Electron

density

map

• The X-ray structure of the dithionite reduced species, is almost identical to ferric (photoreduced?) Ngb

• transition from ferric to ferrous Ngb seems not associated to structural changes, like in electron transfer proteins

joint XRD/EXAFS/XANES on

protein crystals/solution

R32 single crystals

of Ngb contain 18

proteins per unit

cell.

- The Fe-heme structure in

solution and in the cryo-

trapped single crystal (which

cracks at room temperature)

are the same.

- Accordingly, the residual

energy involved in the

protein relaxation

responsible of crystal

cracking after CO binding

does not reside in the heme

pocket

Inorg Chem. 2011 Oct 3;50(19):9423-9

Conclusions

• X-ray Diffraction and polarized X-ray Absorption are complementary

techniques, that could be easily coupled (in proper equipped beamlines

and for metal containing proteins) to both enhance resolution and derive

information on the oxidation state of the metal centre.

• X-ray-induced bond lysis of the Fe-sixth ligand bond seems to be

common in hemeproteins. It could be deliberately used allowing novel X-

ray experiments to be planned (diffraction or absorption time resolved

setup ?, FEL ?) aimed at a dynamical characterization of

pentacoordinate ferrous states in hemeproteins, that is the species

involved in binding external ligands.

Acknowledgements

Stefano Della Longa

Dipartimento di Medicina Sperimentale, Universita` dell’ Aquila, Italy

Paola D’Angelo

Dipartimento di Chimica Università di Roma La Sapienza, Italy

Maurizio Brunori, Beatrice Vallone

Dipartimento di Scienze Biochimiche Università di Roma La Sapienza, Italy

Jean Louis Hazemann, Olivier Proux, Denis Testemale, Vincent Ranieri

FAME beam line CRG BM 30B Grenoble, France

Michael Wulff

ID09 ESRF Grenoble, France

Dominique Bourgeois

Laboratoire de Cristallographie et de Cristallogene se des Proteines, Unite´Mixte de Recherche 9015, Institut de Biologie Structurale Grenoble, France

Philip A. Anfinrud

National Institutes of Health, Bethesda, MD, USA

Samar Hasnain, Richard Strange, Michele Cianci

Molecular Biophysics Group, Daresbury Laboratory, United Kingdom

Beamline 10 at SRS Daresbury, (UK):

MAD/XANES/XRF

b

a

c Photo

n b

eam

a

b

c

e = POLARIZATION VECTOR

E = ELECTRICAL FIELD VECTOR ac plane

e // E photon beam

I0fluo(E) Ie //c* 0.85normal(E) 0.15heme(E)

)(995.0)(005.0)( //90 EEIEI hemenormala

fluo e