Rhodobactersphaeroides methionine sulfoxide reductase P reduces R- and S-diastereomers of methionine sulfoxide from a broad-spectrum of protein substrates.

Methionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as R- and S-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown to be absolutely stereospecific for the reduction of S-diastereomer and R-diastereomer, respectively. Recently, a new enzymatic system, MsrQ/MsrP which is conserved in all gram-negative bacteria, was identified as a key actor for the reduction of oxidized periplasmic proteins. The haem-binding membrane protein MsrQ transmits reducing power from the electron transport chains to the molybdoenzyme MsrP, which acts as a protein-MetO reductase. The MsrQ/MsrP function was well established genetically, but the identity and biochemical properties of MsrP substrates remain unknown. In this work, using the purified MsrP enzyme from the photosynthetic bacteria Rhodobacter sphaeroides as a model, we show that it can reduce a broad spectrum of protein substrates. The most efficiently reduced MetO is found in clusters, in amino acid sequences devoid of threonine and proline on the C-terminal side. Moreover, R. sphaeroides MsrP lacks stereospecificity as it can reduce both R- and S-diastereomers of MetO, similarly to its Escherichia coli homolog, and preferentially acts on unfolded oxidized proteins. Overall, these results provide important insights into the function of a bacterial envelop protecting system, which should help understand how bacteria cope in harmful environments.

Kinetics of substrate inhibition of periplasmic nitrate reductase.

Periplasmic nitrate reductase catalyzes the reduction of nitrate into nitrite using a mononuclear molybdenum cofactor that has nearly the same structure in all enzymes of the DMSO reductase family. In previous electrochemical investigations, we found that the enzyme exists in several inactive states, some of which may have been previously isolated and mistaken for catalytic intermediates. In particular, the enzyme slowly and reversibly inactivates when exposed to high concentrations of nitrate. Here, we study the kinetics of substrate inhibition and its dependence on electrode potential and substrate concentration to learn about the properties of the active and inactive forms of the enzyme. We conclude that the substrate-inhibited enzyme never significantly accumulates in the EPR-active Mo(+V) state. This conclusion is relevant to spectroscopic investigations where attempts are made to trap a Mo(+V) catalytic intermediate using high concentrations of nitrate.

Reductive activation in periplasmic nitrate reductase involves chemical modifications of the Mo-cofactor beyond the first coordination sphere of the metal ion.

In Rhodobacter sphaeroides periplasmic nitrate reductase NapAB, the major Mo(V) form (the "high g" species) in air-purified samples is inactive and requires reduction to irreversibly convert into a catalytically competent form (Fourmond et al., J. Phys. Chem., 2008). In the present work, we study the kinetics of the activation process by combining EPR spectroscopy and direct electrochemistry. Upon reduction, the Mo (V) "high g" resting EPR signal slowly decays while the other redox centers of the protein are rapidly reduced, which we interpret as a slow and gated (or coupled) intramolecular electron transfer between the [4Fe-4S] center and the Mo cofactor in the inactive enzyme. Besides, we detect spin-spin interactions between the Mo(V) ion and the [4Fe-4S](1+) cluster which are modified upon activation of the enzyme, while the EPR signatures associated to the Mo cofactor remain almost unchanged. This shows that the activation process, which modifies the exchange coupling pathway between the Mo and the [4Fe-4S](1+) centers, occurs further away than in the first coordination sphere of the Mo ion. Relying on structural data and studies on Mo-pyranopterin and models, we propose a molecular mechanism of activation which involves the pyranopterin moiety of the molybdenum cofactor that is proximal to the [4Fe-4S] cluster. The mechanism implies both the cyclization of the pyran ring and the reduction of the oxidized pterin to give the competent tricyclic tetrahydropyranopterin form.

Comparative genomic analysis provides insights into the evolution and niche adaptation of marine Magnetospira sp. QH-2 strain.

Magnetotactic bacteria (MTB) are capable of synthesizing intracellular organelles, the magnetosomes, that are membrane-bounded magnetite or greigite crystals arranged in chains. Although MTB are widely spread in various ecosystems, few axenic cultures are available, and only freshwater Magnetospirillum spp. have been genetically analysed. Here, we present the complete genome sequence of a marine magnetotactic spirillum, Magnetospira sp. QH-2. The high number of repeats and transposable elements account for the differences in QH-2 genome structure compared with other relatives. Gene cluster synteny and gene correlation analyses indicate that the insertion of the magnetosome island in the QH-2 genome occurred after divergence between freshwater and marine magnetospirilla. The presence of a sodium-quinone reductase, sodium transporters and other functional genes are evidence of the adaptive evolution of Magnetospira sp. QH-2 to the marine ecosystem. Genes well conserved among freshwater magnetospirilla for nitrogen fixation and assimilatory nitrate respiration are absent from the QH-2 genome. Unlike freshwater Magnetospirillum spp., marine Magnetospira sp. QH-2 neither has TonB and TonB-dependent receptors nor does it grow on trace amounts of iron. Taken together, our results show a distinct, adaptive evolution of Magnetospira sp. QH-2 to marine sediments in comparison with its closely related freshwater counterparts.

The oxygen sensor MgFnr controls magnetite biomineralization by regulation of denitrification in Magnetospirillum gryphiswaldense.

BACKGROUND: Magnetotactic bacteria are capable of synthesizing magnetosomes only under oxygen-limited conditions. However, the mechanism of the aerobic repression on magnetite biomineralization has remained unknown. In Escherichia coli and other bacteria, Fnr (fumarate and nitrate reduction regulator) proteins are known to be involved in controlling the switch between microaerobic and aerobic metabolism. Here, we report on an Fnr-like protein (MgFnr) and its role in growth metabolism and magnetite biomineralization in the alphaproteobacterium Magnetospirillum gryphiswaldense. RESULTS: Deletion of Mgfnr not only resulted in decreased N2 production due to reduced N2O reductase activity, but also impaired magnetite biomineralization under microaerobic conditions in the presence of nitrate. Overexpression of MgFnr in the WT also caused the synthesis of smaller magnetite particles under anaerobic and microaerobic conditions in the presence of nitrate. These data suggest that proper expression of MgFnr is required for WT-like magnetosome synthesis, which is regulated by oxygen. Analyses of transcriptional gusA reporter fusions revealed that besides showing similar properties to Fnr proteins reported in other bacteria, MgFnr is involved in the repression of the expression of denitrification genes nor and nosZ under aerobic conditions, possibly owing to several unique amino acid residues specific to MTB-Fnr. CONCLUSIONS: We have identified and thoroughly characterized the first regulatory protein mediating denitrification growth and magnetite biomineralization in response to different oxygen conditions in a magnetotactic bacterium. Our findings reveal that the global oxygen regulator MgFnr is a genuine O2 sensor. It is involved in controlling expression of denitrification genes and thereby plays an indirect role in maintaining proper redox conditions required for magnetite biomineralization.

Detrimental effect of the 6 His C-terminal tag on YedY enzymatic activity and influence of the TAT signal sequence on YedY synthesis.

BACKGROUND: YedY, a molybdoenzyme belonging to the sulfite oxidase family, is found in most Gram-negative bacteria. It contains a twin-arginine signal sequence that is cleaved after its translocation into the periplasm. Despite a weak reductase activity with substrates such as dimethyl sulfoxide or trimethylamine N-oxide, its natural substrate and its role in the cell remain unknown. Although sequence conservation of the YedY family displays a strictly conserved hydrophobic C-terminal residue, all known studies on Escherichia coli YedY have been performed with an enzyme containing a 6 histidine-tag at the C-terminus which could hamper enzyme activity. RESULTS: In this study, we demonstrate that the tag fused to the C-terminus of Rhodobacter sphaeroides YedY is detrimental to the enzyme's reductase activity and results in an eight-fold decrease in catalytic efficiency. Nonetheless this C-terminal tag does not influence the properties of the molybdenum active site, as assayed by EPR spectroscopy. When a cleavable His-tag was fused to the N-terminus of the mature enzyme in the absence of the signal sequence, YedY was expressed and folded with its cofactor. However, when the signal sequence was added upstream of the N-ter tag, the amount of enzyme produced was approximately ten-fold higher. CONCLUSION: Our study thus underscores the risk of using a C-terminus tagged enzyme while studying YedY, and presents an alternative strategy to express signal sequence-containing enzymes with an N-terminal tag. It brings new insights into molybdoenzyme maturation in R. sphaeroides showing that for some enzymes, maturation can occur in the absence of the signal sequence but that its presence is required for high expression of active enzyme.

Coproporphyrin excretion and low thiol levels caused by point mutation in the Rhodobacter sphaeroides S-adenosylmethionine synthetase gene.

A spontaneous mutant of Rhodobacter sphaeroides f. sp. denitrificans IL-106 was found to excrete a large amount of a red compound identified as coproporphyrin III, an intermediate in bacteriochlorophyll and heme synthesis. The mutant, named PORF, is able to grow under phototrophic conditions but has low levels of intracellular cysteine and glutathione and overexpresses the cysteine synthase CysK. The expression of molybdoenzymes such as dimethyl sulfoxide (DMSO) and nitrate reductases is also affected under certain growth conditions. Excretion of coproporphyrin and overexpression of CysK are not directly related but were both found to be consequences of a diminished synthesis of the key metabolite S-adenosylmethionine (SAM). The wild-type phenotype is restored when the gene metK encoding SAM synthetase is supplied in trans. The metK gene in the mutant strain has a mutation leading to a single amino acid change (H145Y) in the encoded protein. This point mutation is responsible for a 70% decrease in intracellular SAM content which probably affects the activities of numerous SAM-dependent enzymes such as coproporphyrinogen oxidase (HemN); uroporphyrinogen III methyltransferase (CobA), which is involved in siroheme synthesis; and molybdenum cofactor biosynthesis protein A (MoaA). We propose a model showing that the attenuation of the activities of SAM-dependent enzymes in the mutant could be responsible for the coproporphyrin excretion, the low cysteine and glutathione contents, and the decrease in DMSO and nitrate reductase activities.

Dependence of catalytic activity on driving force in solution assays and protein film voltammetry: insights from the comparison of nitrate reductase mutants.

Rhodobacter sphaeroides periplasmic nitrate reductase (Rs NapAB) is one of the enzymes whose assays give odd results: in spectrophotometric assays with methyl viologen as the electron donor, the activity increases as the reaction progresses, whereas the driving force provided by the soluble redox partner decreases; in protein film voltammetry (PFV), whereby the enzyme directly exchanges electrons with an electrode, the activity of NapAB decreases at large overpotential, whereas a monotonic increase is expected [Elliott, S. J., et al. (2002) Biochim. Biophys. Acta 1555, 54-59]. The relations between these phenomena and the catalytic mechanism are still debated. By studying NapAB mutants, we found that the peculiar dependences of electrochemical and solution activities on driving force are greatly affected by substituting certain amino acids that are located in the vicinity of the active site (M153, Q384, R392); this led us to establish and discuss the relation between the experimental parameters of the electrochemical and spectrophotometric assays: we show that the rate of reduction of the enzyme (which depends on the electrode potential or on the concentration of reduced MV) modulates the activity of the enzyme, but the "solution potential" does not. Our results also support the view that the complex profiles of activity versus potential are fingerprints of the active site chemistry, rather than direct consequences of changes in the redox states of relays that are remote from the active site.

Reduction of Protein Bound Methionine Sulfoxide by a Periplasmic Dimethyl Sulfoxide Reductase.

In proteins, methionine (Met) can be oxidized into Met sulfoxide (MetO). The ubiquitous methionine sulfoxide reductases (Msr) A and B are thiol-oxidoreductases reducing MetO. Reversible Met oxidation has a wide range of consequences, from protection against oxidative stress to fine-tuned regulation of protein functions. Bacteria distinguish themselves by the production of molybdenum-containing enzymes reducing MetO, such as the periplasmic MsrP which protects proteins during acute oxidative stress. The versatile dimethyl sulfoxide (DMSO) reductases were shown to reduce the free amino acid MetO, but their ability to reduce MetO within proteins was never evaluated. Here, using model oxidized proteins and peptides, enzymatic and mass spectrometry approaches, we showed that the Rhodobacter sphaeroides periplasmic DorA-type DMSO reductase reduces protein bound MetO as efficiently as the free amino acid L-MetO and with catalytic values in the range of those described for the canonical Msrs. The identification of this fourth type of enzyme able to reduce MetO in proteins, conserved across proteobacteria and actinobacteria, suggests that organisms employ enzymatic systems yet undiscovered to regulate protein oxidation states.

Tuning the redox properties of a [4Fe-4S] center to modulate the activity of Mo-bisPGD periplasmic nitrate reductase.

Molybdoenzymes are ubiquitous in living organisms and catalyze, for most of them, oxidation-reduction reactions using a large range of substrates. Periplasmic nitrate reductase (NapAB) from Rhodobacter sphaeroides catalyzes the 2-electron reduction of nitrate into nitrite. Its active site is a Mo bis-(pyranopterin guanine dinucleotide), or Mo-bisPGD, found in most prokaryotic molybdoenzymes. A [4Fe-4S] cluster and two c-type hemes form an intramolecular electron transfer chain that deliver electrons to the active site. Lysine 56 is a highly conserved amino acid which connects, through hydrogen-bonds, the [4Fe-4S] center to one of the pyranopterin ligands of the Mo-cofactor. This residue was proposed to be involved in the intramolecular electron transfer, either defining an electron transfer pathway between the two redox cofactors, and/or modulating their redox properties. In this work, we investigated the role of this lysine by combining site-directed mutagenesis, activity assays, redox titrations, EPR and HYSCORE spectroscopies. Removal of a positively-charged residue at position 56 strongly decreased the redox potential of the [4Fe-4S] cluster at pH 8 by 230 mV to 400 mV in the K56H and K56M mutants, respectively, thus affecting the kinetics of electron transfer from the hemes to the [4Fe-4S] center up to 5 orders of magnitude. This effect was partly reversed at acidic pH in the K56H mutant likely due to protonation of the imidazole ring of the histidine. Overall, our study demonstrates the critical role of a charged residue from the second coordination sphere in tuning the reduction potential of the [4Fe-4S] cluster in RsNapAB and related molybdoenzymes.

Major Mo(V) EPR signature of Rhodobacter sphaeroides periplasmic nitrate reductase arising from a dead-end species that activates upon reduction. Relation to other molybdoenzymes from the DMSO reductase family.

Enzymes of the DMSO reductase family use a mononuclear Mo-bis(molybdopterin) cofactor (MoCo) to catalyze a variety of oxo-transfer reactions. Much functional information on nitrate reductase, one of the most studied members of this family, has been gained from EPR spectroscopy, but this technique is not always conclusive because the signature of the MoCo is heterogeneous, and which signals correspond to active species is still unsure. We used site-directed mutagenesis, EPR and protein film voltammetry to demonstrate that the MoCo in R. sphaeroides periplasmic nitrate reductase (NapAB) is subject to an irreversible reductive activation process whose kinetics we precisely define. This activation quantitatively correlates with the disappearance of the so-called "Mo(V) high-g" EPR signal, but this reductive process is too slow to be part of the normal catalytic cycle. Therefore, in NapAB, this most intense and most commonly observed signature of the MoCo arises from a dead-end, inactive state that gives a catalytically competent species only after reduction. This activation proceeds, even without substrate, according to a reduction followed by an irreversible nonredox step, both of which are pH independent. An apparently similar process occurs in other nitrate reductases (both assimilatory and membrane bound), and this also recalls the redox cycling procedure, which activates periplasmic DMSO reductases and simplifies their spectroscopic signatures. Hence we propose that heterogeneity at the active site and reductive activation are common properties of enzymes from the DMSO reductase family. Regarding NapAB, the fact that we could detect no Mo EPR signal upon reoxidizing the fully reduced enzyme suggests that the catalytically active form of the Mo(V) is thermodynamically unstable, as is the case for other enzymes of the DMSO reductase family. Our original approach, which combines spectroscopy and protein film voltammetry, proves useful for discriminating the forms of the active site on the basis of their catalytic properties. This could be applied to other enzymes for which the question arises as to the catalytic relevance of certain long-lived, spectroscopically characterized species.

Crystal structures of an Extracytoplasmic Solute Receptor from a TRAP transporter in its open and closed forms reveal a helix-swapped dimer requiring a cation for alpha-keto acid binding.

BACKGROUND: The import of solutes into the bacterial cytoplasm involves several types of membrane transporters, which may be driven by ATP hydrolysis (ABC transporters) or by an ion or H+ electrochemical membrane potential, as in the tripartite ATP-independent periplasmic system (TRAP). In both the ABC and TRAP systems, a specific periplasmic protein from the ESR family (Extracytoplasmic Solute Receptors) is often involved for the recruitment of the solute and its presentation to the membrane complex. In Rhodobacter sphaeroides, TakP (previously named SmoM) is an ESR from a TRAP transporter and binds alpha-keto acids in vitro. RESULTS: We describe the high-resolution crystal structures of TakP in its unliganded form and as a complex with sodium-pyruvate. The results show a limited "Venus flytrap" conformational change induced by substrate binding. In the liganded structure, a cation (most probably a sodium ion) is present and plays a key role in the association of the pyruvate to the protein. The structure of the binding pocket gives a rationale for the relative affinities of various ligands that were tested from a fluorescence assay. The protein appears to be dimeric in solution and in the crystals, with a helix-swapping structure largely participating in the dimer formation. A 30 A-long water channel buried at the dimer interface connects the two ligand binding cavities of the dimer. CONCLUSION: The concerted recruitment by TakP of the substrate group with a cation could represent a first step in the coupled transport of both partners, providing the driving force for solute import. Furthermore, the unexpected dimeric structure of TakP suggests a molecular mechanism of solute uptake by the dimeric ESR via a channel that connects the binding sites of the two monomers.

Effects of slow substrate binding and release in redox enzymes: theory and application to periplasmic nitrate reductase.

For redox enzymes, the technique called protein film voltammetry makes it possible to determine the entire profile of activity against driving force by having the enzyme exchanging directly electrons with the rotating-disc electrode onto which it is adsorbed. Both the potential location of the catalytic response and its detailed shape report on the sequence of catalytic events, electron transfers and chemical steps, but the models that have been used so far to decipher this signal lack generality. For example, it was often proposed that substrate binding to multiple redox states of the active site may explain that turnover is greater in a certain window of electrode potential, but no fully analytical treatment has been given. Here, we derive (i) the general current equation for the case of reversible substrate binding to any redox states of a two-electron active site (as exemplified by flavins and Mo cofactors), (ii) the quantitative conditions for an extremum in activity to occur, and (iii) the expressions from which the substrate-concentration dependence of the catalytic potential can be interpreted to learn about the kinetics of substrate binding and how this affects the reduction potential of the active site. Not only does slow substrate binding and release make the catalytic wave shape highly complex, but we also show that it can have important consequences which will escape detection in traditional experiments: the position of the wave (this is the driving force that is required to elicit catalysis) departs from the reduction potential of the active site even at the lowest substrate concentration, and this deviation may be large if substrate binding is irreversible. This occurs in the reductive half-cycle of periplasmic nitrate reductase where irreversibility lowers the driving force required to reduce the active site under turnover conditions and favors intramolecular electron transfer from the proximal [4Fe4S]+ cluster to the active site Mo(V).

Growth of magnetotactic sulfate-reducing bacteria in oxygen concentration gradient medium.

Although dissimilatory sulfate-reducing bacteria (SRB) are generally described as strictly anaerobic organisms with regard to growth, several reports have shown that some SRB, particularly Desulfovibrio species, are quite resistant to O2 . For example, SRB remain viable in many aerobic environments while some even reduce O2 to H2 O. However, reproducible aerobic growth of SRB has not been unequivocally documented. Desulfovibrio magneticus is a SRB that is also a magnetotactic bacterium (MTB). MTB biomineralize magnetosomes which are intracellular, membrane-bounded, magnetic iron mineral crystals. The ability of D. magneticus to grow aerobically in several different media under air where an O2 concentration gradient formed, or under O2 -free N2 gas was tested. Under air, cells grew as a microaerophilic band of cells at the oxic-anoxic interface in media lacking sulfate. These results show that D. magneticus is capable of aerobic growth with O2 as a terminal electron acceptor. This is the first report of consistent, reproducible aerobic growth of SRB. This finding is critical in determining important ecological roles SRB play in the environment. Interestingly, the crystal structure of the magnetite crystals of D. magneticus grown under microaerobic conditions showed significant differences compared with those produced anaerobically providing more evidence that environmental parameters influence magnetosome formation.

Heterologous expression of membrane proteins: choosing the appropriate host.

BACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein.

Reassessing the strategies for trapping catalytic intermediates during nitrate reductase turnover.

We examined the kinetics of nitrate reduction by periplasmic nitrate reductase (Nap) by using protein film voltammetry and solution assays. We demonstrate that, under turnover conditions, the enzyme exists as a mixture of active and inactive forms which interconvert on a time scale that is much slower than turnover. The dead-end species accumulates under mildly reducing conditions and at high nitrate concentration, resulting in substrate inhibition and in an uncommon hysteresis in the voltammetric signature. Solution assays with two electron donors having different reduction potentials fully support the electrochemical results. This illustrates the consequences of the high flexibility of the active site molybdenum coordination sphere and questions the conclusions from earlier studies in which attempts were made to trap catalytic intermediates of Nap in experiments carried out under turnover conditions at very high substrate concentration.

Access to the active site of periplasmic nitrate reductase: insights from site-directed mutagenesis and zinc inhibition studies.

The periplasmic nitrate reductase (NapAB), a member of the DMSO reductase superfamily, catalyzes the first step of the denitrification process in bacteria. In this heterodimer, a di-heme NapB subunit is associated to the catalytic NapA subunit that binds a [4Fe-4S] cluster and a bis(molybdopterin guanine dinucleotide) cofactor. Here, we report the kinetic characterization of purified mutated heterodimers from Rhodobacter sphaeroides. By combining site-directed mutagenesis, redox potentiometry, EPR spectroscopy, and enzymatic characterization, we investigate the catalytic role of two conserved residues (M153 and R392) located in the vicinity of the molybdenum active site. We demonstrate that M153 and R392 are involved in nitrate binding: the Vm measured on the M153A and R392A mutants are similar to that measured on the wild-type enzyme, whereas the Km for nitrate is increased 10-fold and 200-fold, respectively. The use of an alternative enzymatic assay led us to discover that NapAB is uncompetitively inhibited by Zn2+ ions (Ki' = 1 microM). We used this property to further probe the active site access in the mutant enzymes. It is proposed that R392 acts as a filter by preventing a direct reduction of the Mo atom by small reducing molecules and partially protecting the active site against zinc inhibition. In addition, we show that M153 is a key residue mediating this inhibition likely by coordinating Zn2+ ions via its sulfur atom. This residue is not conserved in the DMSO reductase superfamily while it is conserved in the periplasmic nitrate reductase family. Zinc inhibition is therefore likely to be specific and restricted to periplasmic nitrate reductases.

Genetic and biochemical evidence for the involvement of a molybdenum-dependent enzyme in one of the selenite reduction pathways of Rhodobacter sphaeroides f. sp. denitrificans IL106.

Selenite reduction in Rhodobacter sphaeroides f. sp. denitrificans was observed under photosynthetic conditions, following a 100-h lag period. This adaptation period was suppressed if the medium was inoculated with a culture previously grown in the presence of selenite, suggesting that selenite reduction involves an inducible enzymatic pathway. A transposon library was screened to isolate mutants affected in selenite reduction. Of the eight mutants isolated, two were affected in molybdenum cofactor synthesis. These moaA and mogA mutants showed an increased duration of the lag phase and a decreased rate of selenite reduction. When grown in the presence of tungstate, a well-known molybdenum-dependent enzyme (molybdoenzyme) inhibitor, the wild-type strain displayed the same phenotype. The addition of tungstate in the medium or the inactivation of the molybdocofactor synthesis induced a decrease of 40% in the rate of selenite reduction. These results suggest that several pathways are involved and that one of them involves a molybdoenzyme. Although addition of nitrate or dimethyl sulfoxide (DMSO) to the medium increased the selenite reduction activity of the culture, neither the periplasmic nitrate reductase NAP nor the DMSO reductase is the implicated molybdoenzyme, since the napA and dmsA mutants, with expression of nitrate reductase and DMSO reductase, respectively, eliminated, were not affected by selenite reduction. A role for the biotine sulfoxide reductase, another characterized molybdoenzyme, is unlikely, since its overexpression in a defective strain did not restore the selenite reduction activity.

In Rhodobacter sphaeroides respiratory nitrate reductase, the kinetics of substrate binding favors intramolecular electron transfer.

The respiratory nitrate reductase (NapAB) from Rb. sphaeroides is a periplasmic molybdenum-containing enzyme which belongs to the DMSO reductase family. We report a study of NapAB by protein film voltammetry (PFV), and we present the first quantitative interpretation of the complex redox-state dependence of activity that has also been observed with other related enzymes. The model we use to fit the data assumes that binding of substrate partly limits turnover and is faster and weaker when the Mo ion is in the V oxidation state than when it is fully reduced. We explain how the presence in the catalytic cycle of such slow chemical steps coupled to electron transfer to the active site decreases the driving force required to reduce the MoV ion and makes exergonic the last intramolecular electron-transfer step (between the proximal cubane and the Mo cofactor). Importantly, comparison is made with all Mo enzymes for which PFV data are available, and we emphasize general features of the energetics of the catalytic cycles in enzymes of the DMSO reductase family.

Structural and redox plasticity in the heterodimeric periplasmic nitrate reductase.

The structure of the respiratory nitrate reductase (NapAB) from Rhodobacter sphaeroides, the periplasmic heterodimeric enzyme responsible for the first step in the denitrification process, has been determined at a resolution of 3.2 A. The di-heme electron transfer small subunit NapB binds to the large subunit with heme II in close proximity to the [4Fe-4S] cluster of NapA. A total of 57 residues at the N- and C-terminal extremities of NapB adopt an extended conformation, embracing the NapA subunit and largely contributing to the total area of 5,900 A(2) buried in the complex. Complex formation was studied further by measuring the variation of the redox potentials of all the cofactors upon binding. The marked effects observed are interpreted in light of the three-dimensional structure and depict a plasticity that contributes to an efficient electron transfer in the complex from the heme I of NapB to the molybdenum catalytic site of NapA.