More Siderophore Stuff

Steven “Babyface” Backues

Donnie “Big D” Berkholz

Brooks “Mad Dog” Maki

Overview of Iron Uptake

Two basic strategies:

- Reduction before uptake

- Reduction after uptake

Reduction Before Uptake

• Release of “reductants” into environment, or reducing enzyme bound to cell surface

• Advantages:• No need for permeases, which can be used by

pathogens such as phages

• Disadvantages• Less specific, and can lead to toxicity from

other metals (Cu(II), Cd(II), Co(II), Ni(II)

Reduction After Uptake

• Uses siderophores to bind Fe(III), which is released inside the cell, usually via reduction of iron from Fe(III) to Fe(II)

• Highly specific, but requires more energy to form the siderophores and uptake system

Is Reduction Difficult?

For various fungal siderophores, reduction potential of Fe(III) is around –400mV

The reduction potential of NAD+/NADH or NADP+/NADPH is around –320mV

- So, there is a positive G°’ but not any more positive than in many other NADH or NADPH driven reactions

- Below pH of 7.9, decreasing pH favors reduction

Other Release Mechanisms?

• Degradation of the siderophore?

• Release without reduction?• For Fe(III) only partially coordinated by a

siderophore, Cl- ions can increase dissociation rates 100-1000 fold.

Use and Storage of Iron

• After reduction, Fe(II) is always bound to carrier proteins until used

• Iron is always stored as Fe(III)

Ferritin

Ferritin is found in mostanimals, plants, and some bacteria.

It can store up to 5,000 atoms of Fe(III) as [FeO(OH)]8[FeO(H2PO4)].

Siderophores as Iron Storage

• Mössbauer spectroscopy shows that reduction is not rate-limiting for siderophore uptake.

• Experiments with 55Fe and a fluorescent ferrichrome analogue showed that while loaded siderophores were taken up within minutes, the iron was not fully released for up to 16 hours after uptake.

In some fungi, one type of siderophore is used for uptake and another for storage

- in N. crassa, coprogen shuttles, while ferricrocin stores- in R. minuta, Rhodotorulic acid used only for storage, not for uptake

Amphiphilic Siderophores

• Prior to binding, these siderophores are micelles with hydrophobic centers

• With the addition of Fe(III) they form vesicles.

• Vesicles are approx. 100 nm across with hydrophobic ring lined with hydrophilic heads

• This structure is important in photoreactivity

Photoreactivity

• Light mediated decarboxylation of an alpha-hydroxy acid complexed to a transition metal ion is well known.

• It has been found that this reaction also occurs in Fe-siderophore complexes.

• Fe(III) petrobactin was readily photolyzed in this way under ocean surface conditions.

Photoreactivity, the sequel

• Photolysis is mediated by light in the ultraviolet spectrum

• Therefore these reactions occur deep into the euphotic zone (80 m)

• Fe-siderophore complexes are structurally stable in sterile sea water.

Photoreactivity, the final chapter

Two main products of photochemical reaction:

hydrophobic

(fatty acid tail)

hydrophilic

(head group - peptide)

Fe (III) is reduced to Fe(II)

Fe Cycling

• What happens to Fe(II)?• Direct biological uptake• Oxidation back to Fe (III) (possibly complexed by

another siderophore)• Possible chelation by organic ligands?

• The photo-oxidized ligand continues to bind Fe(III)

• Iron bound by these ligands may be more available for uptake, as stability is reduced from original siderophoreReferences

Iron Scavenging by Pathogens

• Within animals, all of the iron is generally complexed and being used, so bacteria must steal it, often by use of siderophores.

Exochelins

• Exochelins are released by M. tuberculosis.

• They scavenge metal primarily from transferrin and lactoferrin, human iron binding proteins; less effectively from ferritin

• They transfer their iron to mycobactins in the M. tuberculosis cell wall

Heme Acquisition System A

• This is a protein, not a siderophore

• It or similar proteins are produced from many gram negative bacteria

• It binds an entire heme molecule, extracting it from hemoglobin, then releasing it to the bacterial membrane receptor HasR.

                                            

      “The heme binding site is made up of some hydrophobic residues and is held by the two ligands: residue His32 lies on one side while Tyr75 completes the coordination of the heme iron.”

References

Ardon, Orly et. al. Microbiology, 1997, 143 3625-3631 Boukhalfa, Hakim; Crumbliss, Alvin, Inorganic Chemistry 2001, 40 4183-4190 Czjzek, Mirjam et. al. AFMB Activity Report 1996-1999 (http://afmb.cnrs-mrs.fr/subjects/pdf/21.pdf) De Luca, Nicoala; Wood, Paul Advances in Microbial Physiology, 2000, 43 39-74 Gobin, Jovana; Horwitz, Marcus Journal of Experimental Medicine, 1996, 183 1527-1532 Matzanke, Berthold; Winkelmann, Günther FEBS Letters, 1981, 130 50-53

More references

• Photochemical cycling of iron in the surface ocean mediated by microbial iron(iii) binding ligands. K. Barbeau, E.L. Rue,

K.W. Bruland, A. Butler. Letters to Nature 27 Sep. 2001

• Scientists Chart Iron Cycle in Ocean. National Science Foundation 27 Sep. 2001

• Sunlight Affects Iron Cycles. Pamela Zurer Biogeochemisty 1 Oct. 2001

• Marine Bacteria Foster Iron Cycling. Jacquelyn Savani University of California, Santa Barbara

• Petrobactin, a Photoreactive Siderophore K. Barbeau, G. Zhang, D. Live, A. Butler American Chemical Society 7 Aug. 2001