The first crystal structure of a native di-iron center in an iron-storage protein (bacterio)ferritin is reported. The protein, isolated from the anaerobic bacterium Desulfovibrio desulfuricans, has the unique property of having Fe-coproporphyrin III as its heme cofactor. The three-dimensional structure of this bacterioferritin was determined in three distinct catalytic/redox states by X-ray crystallography (at 1.95, 2.05 and 2.35 A resolution), corresponding to different intermediates of the di-iron ferroxidase site. Conformational changes associated with these intermediates support the idea of a route for iron entry into the protein shell through a pore that passes through the di-iron center. Molecular surface and electrostatic potential calculations also suggest the presence of another ion channel, distant from the channels at the three- and four-fold axes proposed as points of entry for the iron atoms.

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Flavodiiron proteins have emerged in the last two decades as a newly discovered family of oxygen and/or nitric oxide reductases widespread in the three life domains, and present in both aerobic and anaerobic organisms. Herein we present the main features of these fascinating enzymes, with a particular emphasis on the metal sites, as more appropriate for this special issue in memory of the exceptional bioinorganic scientist R. J. P. Williams who pioneered the notion of (metal) element availability-driven evolution. We also compare the flavodiiron proteins with the other oxygen and nitric oxide reductases known until now, highlighting how throughout evolution Nature arrived at different solutions for similar functions, in some cases adding extra features, such as energy conservation. These enzymes are an example of the (bioinorganic) unpredictable diversity of the living world.

The crystal structure of a DNA-binding protein from starved cells (Dps) (DR2263) from Deinococcus radiodurans was determined in two states: a native form, to 1.1-A resolution, and one soaked in an iron solution, to 1.6-A resolution. In comparison with other Dps proteins, DR2263 has an extended N-terminal extension, in both structures presented here, a novel metal binding site was identified in this N-terminal extension and was assigned to bound zinc. The zinc is tetrahedrally coordinated and the ligands, that belong to the N-terminal extension, are two histidines, one glutamate and one aspartate residue, which are unique to this protein within the Dps family. In the iron-soaked crystal structure, a total of three iron sites per monomer were found: one site corresponds to the ferroxidase centre with structural similarities to those found in other Dps family members; the two other sites are located on the two different threefold axes corresponding to small pores in the Dps sphere, which may possibly form the entrance and exit channels for iron storage.

Hybrid cluster proteins (HCP) contain two types of Fe/S clusters, namely a [4Fe-4S] 2؉/1؉ or [2Fe-2S] 2؉/1؉ cluster and a novel type of hybrid cluster, [4Fe-2S-2O], in the as-isolated state. Although first isolated from anaerobic sulfate-reducing bacteria, the analysis of the genomic sequences reveals that genes encoding putative hybrid cluster proteins are present in a wide range of organisms, aerobic, anaerobic, or facultative, from the Bacteria, Archaea, and Eukarya domains. Despite a detailed spectroscopic and structural characterization, the precise physiological function of these proteins remained unknown. The present work shows that the transcription of the Escherichia coli hcp gene is induced by hydrogen peroxide, and this induction is regulated by the redox-sensitive transcriptional activator, OxyR. The E. coli hcp mutant strain exhibits higher sensitivity to hydrogen peroxide, a behavior that reverts to the wild type phenotype once a plasmid carrying the hcp gene is reintroduced. Furthermore, the purified HCPs from E. coli and Desulfovibrio desulfuricans ATCC 27774 show an alternative enzymatic activity, which under physiological conditions exhibited K m values for hydrogen peroxide (ϳ0.3 mM) within the range of other peroxidases. Altogether, the results reveal that HCP is involved in oxidative stress protection.Hybrid cluster proteins (HCP), 2 first isolated from the sulfate-reducing bacteria Desulfovibrio vulgaris (1) and Desulfovibrio desulfuricans (2), contain an unusual iron-sulfur cluster, which in the as-isolated (partially oxidized) state is a mixed oxygen-iron-sulfur cluster, [4Fe-2S-2O], the so-called hybrid cluster, and a cubane [4Fe-4S] 2ϩ/1ϩ cluster (3) or a dinuclear [2Fe-2S] 2ϩ/1ϩ cluster (4). HCPs are widespread among the three life domains, as genes encoding for orthologs of HCP are observed in a wide range of distinct organisms such as enterobacteria, clostridia, Bacteroides, Cyanobacteria, Bacillus sps., methanogens, and protozoa, e.g. Entamoeba (5).In Escherichia coli HCP was detected by immunoblotting in cells grown anaerobically with nitrate or nitrite (4). In accordance, the transcription of hcp from E. coli (6, 7), Salmonella enterica serovar typhimurium (8), Shewanella oneidensis (9), and D. vulgaris (10) was found to be elevated in response to nitrogen oxides such as nitrate, nitrite, or S-nitrosoglutathione. Interestingly, it was observed in the microarray profile of Erwinia chrysanthemi that the transcription level of hcp is highly increased upon plant infection (11). The expression profile of the colonizer of the human urinary tract E. coli strain 83972 in patients carrying urinary infections also showed upregulation of hcp (12).Moreno-Vivian and collaborators (13) reported the increase of anaerobic tolerance to hydroxylamine of E. coli cells overproducing Rhodobacter capsulatus HCP. However, the optimal conditions of the oxygen-sensitive hydroxylamine reductase activity of R. capsulatus HCP (13), as well of the E. coli HCP (14), could only be found at pH 9. The anaerobical...

Desulfoferrodoxin (Dfx), a small iron protein containing two mononuclear iron centres (designated centre I and II), was shown to complement superoxide dismutase (SOD) deficient mutants of Escherichia coli[Pianzzola, M.J., Soubes M. & Touati, D. (1996) J. Bacteriol.178, 6736–6742]. Furthermore, neelaredoxin, a protein from Desulfovibrio gigas containing an iron site similar to centre II of Dfx, was recently shown to have a significant SOD activity [Silva, G., Oliveira, S., Gomes, C.M., Pacheco, I., Liu, M.Y., Xavier, A.V., Teixeira, M., Le Gall, J. & Rodrigues‐Pousada, C. (1999) Eur. J. Biochem. 259, 235–243]. Thus, the SOD activity of Dfx isolated from the sulphate‐reducing bacterium Desulfovibrio desulfuricans ATCC 27774 was studied. The protein exhibits a SOD activity of 70 U·mg−1, which increases approximately 2.5‐fold upon incubation with cyanide. Cyanide binds specifically to Dfx centre II, yielding a low‐spin iron species with g‐values at 2.27 (g⊥) and 1.96 (g∥). Upon reaction of fully oxidized Dfx with the superoxide generating system xanthine/xanthine oxidase, Dfx centres I and II become partially reduced, suggesting that Dfx operates by a redox cycling mechanism, similar to those proposed for other SODs.  Evidence for another SOD in D. desulfuricans is also presented – this enzyme is inhibited by cyanide, and N‐terminal sequence data strongly indicates that it is an analogue to Cu,Zn‐SODs isolated from other sources. This is the first indication that a Cu‐containing protein may be present in a sulphate‐reducing bacterium.

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The class II chelatases associated with heme, siroheme, and cobalamin biosynthesis are structurally related enzymes that insert a specific metal ion (Fe 2þ or Co 2þ ) into the center of a modified tetrapyrrole (protoporphyrin or sirohydrochlorin). The structures of two related class II enzymes, CbiX S from Archaeoglobus fulgidus and CbiK from Salmonella enterica, that are responsible for the insertion of cobalt along the cobalamin biosynthesis pathway are presented in complex with their metallated product. A further structure of a CbiK from Desulfovibrio vulgaris Hildenborough reveals how cobalt is bound at the active site. The crystal structures show that the binding of sirohydrochlorin is distinctly different to porphyrin binding in the protoporphyrin ferrochelatases and provide a molecular overview of the mechanism of chelation. The structures also give insights into the evolution of chelatase form and function. Finally, the structure of a periplasmic form of Desulfovibrio vulgaris Hildenborough CbiK reveals a novel tetrameric arrangement of its subunits that are stabilized by the presence of a heme b cofactor. Whereas retaining colbaltochelatase activity, this protein has acquired a central cavity with the potential to chaperone or transport metals across the periplasmic space, thereby evolving a new use for an ancient protein subunit.enzyme mechanism | tetrapyrrole biosynthesis