Jean Le Gall

Structure of a Dioxygen Reduction Enzyme from Desulfovibrio Gigas

Desulfovibrio gigas is a strict anaerobe that contains a well-characterized metabolic pathway that enables it to survive transient contacts with oxygen. The terminal enzyme in this pathway, rubredoxin:oxygen oxidoreductase (ROO) reduces oxygen to water in a direct and safe way. The 2.5 Å resolution crystal structure of ROO shows that each monomer of this homodimeric enzyme consists of a novel combination of two domains, a flavodoxin-like domain and a Zn-β-lactamase-like domain that contains a di-iron center for dioxygen reduction. This is the first structure of a member of a superfamily of enzymes widespread in strict and facultative anaerobes, indicating its broad physiological significance.

A candidate gene for hemochromatosis: frequency of the C282Y and H63D mutations

Abstract The gene whose alteration causes hereditary hemochromatosis (HFE according to the international nomenclature) was, more than 20 years ago, shown to map to 6p21.3. It has since escaped all efforts to identify it by positional cloning strategies. Quite recently, a gene named HLA-H was reported as being responsible for the disease. Two missense mutations, Cys282Tyr (C282Y) and His63Asp (H63D), were observed, but no proof was produced that the gene described is the hemochromatosis gene. To validate this gene as the actual site of the alteration causing hemochromatosis, we decided to look for the two mutations in 132 unrelated patients from Brittany. Our results indicate that more than 92% of these patients are homozygous for the C282Y mutation, and that all 264 chromosomes but 5 carry either mutation. These findings confirm the direct implication of HLA-H in hemochromatosis.

Dependance of sulfite reduction on a crystallized ferredoxin from Desulfovibrio gigas.

Abstract The isolation from Desulfovibrio desulfuricans of a ferredoxin has been shortly mentionned by Tagawa et al . (1962) , and Akagi (1965) has presented some evidence suggesting that reduction of sulfite by Clostridium nigrificans requires ferredoxin. It has also been shown that a particulate fraction from D. gigas was able to reduce sulfite if some soluble proteins were added to the preparation. This soluble protein could be separated into two protein fractions by treatment with DEAE cellulose. The acidic protein fraction was thought to contain ferredoxin ( Peck, 1966 ). Such data were strongly in favour of a ferredoxin requirement for the reduction of sulfite by D. gigas . In the present paper, the purification of a ferredoxin from D. gigas and its role in sulfite reduction are reported.

Structure-function relationship in hemoproteins: The role of cytochrome c3 in the reduction of colloidal sulfur by sulfate-reducing bacteria

Cytochromes c3 of different strains of sulfatereducing bacteria have been purified and tested for their capacity to reduce colloidal sulfur to hydrogen sulfide. The results are in good agreement with the activities reported for the whole cells. Cytochrome c3 is the sulfur reductase of some strains of sulfate-reducing bacteria such as Desulfovibrio desulfuricans Norway 4 and sulfate-reducing bacterium strain 9974 from which the sulfur reductase activity can be purified with the cytochrome c3. In contrast, Desulfovibrio vulgaris Hildenborough cytochrome c3 is inhibited by the product of the reaction namely hydrogen sulfide. Chloramphenicol has no effect on the sulfur reductase activity of D. desulfuricans Norway 4 when resting cells grown on lactate-sulfate medium are put in the presence of colloidal sulfur. This shows that the sulfur reductase activity is constitutive and corresponds to the fact that colloidal sulfur grown cells do not contain more cytochrome c3 (or another sulfur reductase) than lactate-sulfate-grown cells.

The Physiology of Sulphate-Reducing Bacteria

Publisher Summary The chapter discusses the physiology of sulphate-reducing bacteria. The chapter presents an account of their ecology and economic activities as relevant to the subject as their multiplication can have considerable ecological and economic consequences, and since these are due to their special physiology, in particular, the production of hydrogen sulphide. The sulphate-reducing bacteria form a physiologically distinctive group of anaerobic bacteria, their oxidative metabolism being based, not on fermentation, but on the reduction of sulphate or certain other inorganic sulphur compounds. Their physiology has broad analogies with that of the nitrate-reducing bacteria (denitrifying bacteria), but they are all exacting anaerobes and no examples of facultative aerobes are known. Some representatives of the group are capable of growth by non-respiratory processes involving dismutation of substrates, such as pyruvate, fumarate, or choline. Even when reducing sulphate, these organisms are completely unable to oxidize their carbon compounds, such as fatty acids; usually acetic acid plus carbon dioxide are the normal end products of carbon metabolism. The present chapter brings up to date all those publications on this group of bacteria, which had come to the authors attention by early 1972. Reviews of related subjects that have been published during this period, and which makes reference to these bacteria reviewing the general metabolism of sulphur bacteria. The chapter also discusses the chemical and biochemical activities of these bacteria.

The primary and three-dimensional structures of a nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774 reveal a new member of the Hmc family

BACKGROUND Haem-containing proteins are directly involved in electron transfer as well as in enzymatic functions. The nine-haem cytochrome c (9Hcc), previously described as having 12 haem groups, was isolated from cells of Desulfovibrio desulfuricans ATCC 27774, grown under both nitrate- and sulphate-respiring conditions. RESULTS Models for the primary and three-dimensional structures of this cytochrome, containing 292 amino acid residues and nine haem groups, were derived using the multiple wavelength anomalous dispersion phasing method and refined using 1.8 A diffraction data to an R value of 17.0%. The nine haem groups are arranged into two tetrahaem clusters, with Fe-Fe distances and local protein fold similar to tetrahaem cytochromes c3, while the extra haem is located asymmetrically between the two clusters. CONCLUSIONS This is the first known three-dimensional structure in which multiple copies of a tetrahaem cytochrome c3-like fold are present in the same polypeptide chain. Sequence homology was found between this cytochrome and the C-terminal region (residues 229-514) of the high molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough (DvH Hmc). A new haem arrangement in domains III and IV of DvH Hmc is proposed. Kinetic experiments showed that 9Hcc can be reduced by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774, but that this reduction is faster in the presence of tetrahaem cytochrome c3. As Hmc has never been found in D. desulfuricans ATCC 27774, we propose that 9Hcc replaces it in this organism and is therefore probably involved in electron transfer across the membrane.

Regulation of the reduction of sulfite and thiosulfate by ferredoxin, flavodoxin and cytochrome cc′3 in extracts of the sulfate reducer Desulfovibrio gigas

Abstract Cytochrome cc′3, flavodoxin and ferredoxin are able to stimulate the reduction of thiosulfate by molecular H2 in an extract of the sulfate reducer Desulfovibrio gigas. Only flavodoxin and ferredoxin will stimulate the reduction of sulfite by the same extract, whereas cytochrome cc′3 is specific for thiosulfate reduction. Sulfite accumulates during the early stage of thiosulfate reduction and is reduced only when thiosulfate has disappeared. The results are discussed in terms of the regulation, by these different electron carriers, of the electron flow from H2 to the various sulfur compounds acting as terminal electron acceptors in sulfate-reducing bacteria.

Cytochrome c-551.5 (c7) from Desulfuromonas acetoxidans.

Cytochrome c-551.5 of the anaerobic sulfur-reducing bacterium Desulfuromonas acetoxidans has been purified to homogeneity and characterized. It elicits absorption bands at 551.5, 522.5 and 418 nm in the reduced form; the absorptivity ratio Aalpha(red)/A280nm(ox) equals 3.8 for the pure preparation. The molecular weight was estimated to be 9800 by gel filtration. Determination of the amion acid composition and analysis of the N-terminal amino acid sequence showed the cytochrome to be identical with the threehaem cytochrome c-551.5 (c7) isolated from the syntrophic mixed culture Chloropseudomonas ethylica strain 2K. The occurrence of multihaem cytochromes c in bacteria is discussed.

Could a Diiron-Containing Four-Helix-Bundle Protein Have Been a Primitive Oxygen Reductase?

Dioxygen, which became abundant in the earths atmosphere approximately 2 ± 3 billion years ago, is a molecule with a very high oxidising power. Since dioxygen has a poor reactivityÐdue to unfavourable thermodynamic and kinetic factorsÐliving organisms could only take advantage of the oxic atmosphere by developing appropriate enzymes that can efficiently utilise it, that is, developing safe high-energy-yielding processes. At the present time, a large variety of proteins that are capable of utilising either dioxygen or its reduced species is known, and their evolution, as well as that of the metabolic pathways they integrate, is a challenging issue. The present work raises the hypothesis that a four-helix-bundle diiron-containing protein may have been an early oxygen reductase operating in the first stages of the transition between the anoxic and the present oxic atmosphere. This protein would have the required features to perform dioxygen chemistry, namely the potential to harbour a binuclear transition metal site. Such a system allows the complete reduction of dioxygen to water and would play an important role in oxygen defence mechanisms in primordial anaerobes. This hypothesis is based on data here reported concerning the oxygen reductase activity of the diiron protein rubrerythrin, together with sequenceand structure-based data of protein phylogeny between this and other diiron proteins. Interestingly, analysis of the available data depicts an evolutionary relationship between such an early system and the alternative oxidases present in extant eukaryotes. Our knowledge about the diiron carboxylate protein family has expanded considerably in the last years, with an increase in both sequence and structure data. 4] This family comprises the R2 subunit of ribonucleotide reductase (RNR R2), the hydroxylase subunit of the soluble methane monooxygenase (MMOH), (bacterio)ferritin ((B)FR), rubrerythrin (RR), stearoyl-acyl carrier D desaturase (D desaturase) and hemerythrin (HR). In all cases, carboxylates and oxide or hydroxide ions bridge the diiron site, surrounded by a four-helix-bundle protein fold. These enzymes have quite a large functional diversity, but they have an oxygenactivating step in common that, with the exception of hemerythrin, drives subsequent redox reactions. Plant alternative oxidase (AOX) is a non-energy-transducing terminal oxygen reductase operating in mitochondria and chloroplasts, which is reduced by ubiquinol and catalyses the four-electron reduction of dioxygen to water. 6] Despite numerous efforts, a pure preparation of this protein could never be obtained, thus preventing the elucidation of the chemical properties of its catalytic site. On the basis of sequence comparisons of several alternative oxidases, it has been suggested that they contain a diiron site as a reactive centre in a four-helix-bundle conformation. This hypothesis was recently further analysed by Andersson and Nordlund who revised the initially proposed structural model. These authors, considering additional sequences from alternative oxidases, presented a new model for this protein which likens it even more to the diiron carboxylate proteins. The amino acid sequence identity between the diiron carboxylate proteins is in general too low to allow the use of a conventional phylogenetic analysis. However, structure-oriented local sequence alignments, comprising only the regions of the four helices aligned according to homologous residues in the metal-binding site, can successfully be used to infer general phylogenetic relationships between these proteins. This approach was followed and the study of more than 50 sequences from diiron carboxylate proteins was undertaken, combining sequence and structure data for the structurally conserved region of the four-helix bundle (Figure 1). These structure-

Linkage disequilibrium and extended haplotypes in the HLA-A to D6S105 region: implications for mapping the hemochromatosis gene (HFE).

The hemochromatosis gene (HFE) maps to 6p21.3, in close linkage with the HLA Class I genes. Linkage disequilibrium (LD) studies were designed to narrow down the most likely candidate region for HFE, as an alternative to traditional linkage analysis. However, both the HLA-A and D6S105 subregions, which are situated 2–3 cM and approximately 3 Mb apart, have been suggested to contain HFE. The present report extends our previous study based upon the analysis of a large number of HFE and normal chromosomes from 66families of Breton ancestry. In addition to the previously used RFLP markers spanning the 400-kb surrounding HLA-A, we examined three microsatellites: D6S510, HLA-F, and D6S105. Our combined data not only confirm a peak of LD at D6S105, but also reveal a complex pattern of LD over the i82 to D6S105 interval. Within our ethnically well-defined population of Brittany, the association of HFE with D6S105 is as great as that with HLA-A, while the internal markers display a lower LD. Fine haplotype analysis enabled us to identify two categories of haplotypes segregating with HFE. In contrast to the vast majority of normal haplotypes, 50% of HFE haplotypes are completely conserved over the HLA-A to D6S105 interval. These haplotypes could have been conserved through recombination suppression, selective forces and/or other evolutionary factors. This particular haplotypic configuration might account for the apparent inconsistencies between genetic linkage and LD data, and additionally greatly complicates positional cloning of HFE through disequilibrium mapping.