Sulfate reduction is one of the earliest types of energy metabolism used by ancestral organisms to sustain life. Despite extensive studies, many questions remain about the way respiratory sulfate reduction is associated with energy conservation. A crucial enzyme in this process is the dissimilatory sulfite reductase (dSiR), which contains a unique siroheme-[4Fe4S] coupled cofactor. Here, we report the structure of desulfoviridin from Desulfovibrio vulgaris, in which the dSiR DsrAB (sulfite reductase) subunits are bound to the DsrC protein. with its conserved C-terminal cysteine reaching the distal side of the siroheme. We propose a novel mechanism for the process of sulfite reduction involving DsrAB, DsrC, and the DsrMKJOP membrane complex (a membrane complex with putative disulfide/thiol reductase activity), in which two of the six electrons for reduction of sulfite derive from the membrane quinone pool. These results show that DsrC is involved in sulfite reduction, which changes the mechanism of sulfate respiration. This has important implications for models used to date ancient sulfur metabolism based on sulfur isotope fractionations.The dissimilatory reduction of sulfur compounds is one of the earliest energy metabolisms detected on earth, at ϳ3.5 billion years ago (1, 2). At the end of the Archean (ϳ2.7 billion years ago), the advent of oxygenic photosynthesis led to a gradual increase in the levels of atmospheric oxygen, which in turn caused an increasing flux of sulfate to the oceans from weathering of sulfide minerals on land (3). As a consequence of this process, reduction of sulfate became a dominant biological process in the oceans, resulting in sulfidic anoxic conditions from about 2.5 to 0.6 billion years ago (3, 4). During this extended period, sulfate-reducing prokaryotes were main players in marine habitats where most evolutionary processes were taking place. Today, these organisms are still major contributors to the biological carbon and sulfur cycles, and their activities have important environmental and economic consequences.A key enzyme in sulfur-based energy metabolism is the dissimilatory sulfite reductase (dSiR), 3 which is present in organisms that reduce sulfate, sulfite, and other sulfur compounds. This enzyme is also found in some phototrophic and chemotrophic sulfur oxidizers, where it is proposed to operate in the reverse direction (reverse sulfite reductase, rSiR). The dSiR is minimally composed of two subunits, DsrA and DsrB, in an ϳ200-kDa ␣ 2 ␤ 2 arrangement. The dsrA and dsrB genes are paralogous and most likely arose from a very early gene duplication event that preceded the separation of the archaea and bacteria domains (5-8), in agreement with a very early onset of biological sulfite reduction. The dSiR belongs to a family of proteins that also include the assimilatory sulfite (aSiR) and nitrite (aNiR) reductases, the monomeric low molecular mass aSiRs, and other dSiRs like asrC and Fsr (9 -11). This family has in common a characteristic cofactor assembly that includes an iro...

Oxidation of membrane‐bound quinol molecules is a central step in the respiratory electron transport chains used by biological cells to generate ATP by oxidative phosphorylation. A novel family of cytochrome c quinol dehydrogenases that play an important role in bacterial respiratory chains was recognised in recent years. Here, we describe the first structure of a cytochrome from this family, NrfH from Desulfovibrio vulgaris, which forms a stable complex with its electron partner, the cytochrome c nitrite reductase NrfA. One NrfH molecule interacts with one NrfA dimer in an asymmetrical manner, forming a large membrane‐bound complex with an overall α4β2 quaternary arrangement. The menaquinol‐interacting NrfH haem is pentacoordinated, bound by a methionine from the CXXCHXM sequence, with an aspartate residue occupying the distal position. The NrfH haem that transfers electrons to NrfA has a lysine residue from the closest NrfA molecule as distal ligand. A likely menaquinol binding site, containing several conserved and essential residues, is identified.

Dissimilatory sulfite reductases (dSiRs) are crucial enzymes in bacterial sulfur-based energy metabolism, which are likely to have been present in some of the earliest life forms on Earth. Several classes of dSiRs have been proposed on the basis of different biochemical and spectroscopic properties, but it is not clear whether this corresponds to actual physiological or structural differences. Here, we describe the first structure of a dSiR from the desulforubidin class isolated from Desulfomicrobium norvegicum . The desulforubidin (Drub) structure is assembled as α 2 β 2 γ 2 , in which two DsrC proteins are bound to the core [DsrA] 2 [DsrB] 2 unit, as reported for the desulfoviridin (Dvir) structure from Desulfovibrio vulgaris . Unlike Dvir, four sirohemes and eight [4Fe–4S] clusters are present in Drub. However, the structure indicates that only two of the Drub coupled siroheme-[4Fe–4S] cofactors are catalytically active. Mass spectrometry studies of purified Drub and Dvir show that both proteins present different oligomeric complex forms that bind two, one, or no DsrC proteins, providing an explanation for conflicting spectroscopic and biochemical results in the literature, and further indicating that DsrC is not a subunit of dSiR, but rather a protein with which it interacts.

Summary Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of l-glutamate to α-ketoglutarate using NAD+ and/or NADP+ as a cofactor. Subunits of homo-hexameric bacterial enzymes comprise a substrate-binding Domain I followed by a nucleotide binding Domain II. The reaction occurs in a catalytic cleft between the two domains. Although conserved residues in the nucleotide-binding domains of various dehydrogenases have been linked to cofactor preferences, the structural basis for specificity in the glutamate dehydrogenase (GDH) family remains poorly understood. Here, the refined crystal structure of Escherichia coli GDH in the absence of reactants is described at 2.5Å resolution. Modelling of NADP+ in Domain II reveals the potential contribution of positively charged residues from a neighbouring α-helical hairpin to phosphate recognition. In addition, a serine residue that follows the P7 aspartate is presumed to form a hydrogen bond to the 2’-phosphate. Mutagenesis and kinetic analysis confirms the importance of these residues in NADP+ recognition. Surprisingly, one of the positively charged residues is conserved in all sequences of NAD+ dependent enzymes, but the conformations adopted by the corresponding regions in proteins whose structure has been solved preclude their contribution toward the co-ordination of the 2’-ribose phosphate of NADP+. These studies clarify the sequence/structure relationships in bacterial glutamate dehydrogenases, revealing that identical residues may specify different coenzyme preferences, depending on the structural context. Primary sequence alone is therefore not a reliable guide for predicting coenzyme specificity. We also consider how it is possible for a single sequence to accommodate both coenzymes in the dual specificity GDHs of animals.

The cytochrome c nitrite reductase (cNiR) isolated from Desulfovibrio vulgaris Hildenborough is a membrane-bound complex formed of NrfA and NrfH subunits. The catalytic subunit NrfA is a soluble pentahaem cytochrome c that forms a physiological dimer of about 120 kDa. The electron-donor subunit NrfH is a membrane-anchored tetrahaem cytochrome c of about 18 kDa molecular weight and belongs to the NapC/NirT family of quinol dehydrogenases, for which no structures are known. Crystals of the native cNiR membrane complex, solubilized with dodecylmaltoside detergent (DDM), were obtained using PEG 4K as precipitant. Anomalous diffraction data were measured at the Swiss Light Source to 2.3 Å resolution. Crystals belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 79.5, b = 256.7, c = 578.2 Å . Molecularreplacement and MAD methods were combined to solve the structure. The data presented reveal that D. vulgaris cNiR contains one NrfH subunit per NrfA dimer.

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