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Bacterial genomes are a huge reservoir of genes encoding J‐domain protein co‐chaperones that recruit the molecular chaperone DnaK to assist protein substrates involved in survival, adaptation, or fitness. The atc operon of the aquatic mesophilic bacterium Shewanella oneidensis encodes the proteins AtcJ, AtcA, AtcB, and AtcC, and all of them, except AtcA, are required for growth at low temperatures. AtcJ is a short J‐domain protein that interacts with DnaK, but also with AtcC through its 21 amino acid C‐terminal domain. This interaction network is critical for cold growth. Here, we show that AtcJ represents a subfamily of short J‐domain proteins that (i) are found in several environmental, mostly aquatic, β‐ or ɣ‐proteobacteria and (ii) contain a conserved PX7W motif in their C‐terminal extension. Using a combination of NMR, biochemical and genetic approaches, we show that the hydrophobic nature of the tryptophan of the S. oneidensis AtcJ PX7W motif determines the strong AtcJ–AtcC interaction essential for cold growth. The AtcJ homologues are encoded by operons containing at least the S. oneidensis atcA, atcB, and atcC homologues. These findings suggest a conserved network of DnaK and Atc proteins necessary for low‐temperature growth and, given the variation in the atc operons, possibly for other biological functions. [ABSTRACT FROM AUTHOR]

CO dehydrogenases (CODHs) catalyse the reversible conversion between CO and CO2. Genomic analysis indicated that the metabolic functions of CODHs vary. The genome of Carboxydothermus hydrogenoformans encodes five CODHs (CODH-I-V), of which CODH-IV is found in a gene cluster near a peroxide-reducing enzyme. Our kinetic and crystallographic experiments reveal that CODH-IV differs from other CODHs in several characteristic properties: it has a very high affinity for CO, oxidizes CO at diffusion-limited rate over a wide range of temperatures, and is more tolerant to oxygen than CODH-II. Thus, our observations support the idea that CODH-IV is a CO scavenger in defence against oxidative stress and highlight that CODHs are more diverse in terms of reactivity than expected. [ABSTRACT FROM AUTHOR]

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Ni-containing CO dehydrogenases (CODHs) are very efficient metalloenzymes that catalyze the conversion between CO2 and CO. They are a source of inspiration for designing CO2-reduction catalysts and can also find direct use in biotechnology. They are deemed extremely sensitive to O2, but very little is known about this aspect of their reactivity. We investigated the reaction with O2 of Carboxydothermus hydrogenoformans (Ch) CODH II and the homologous, recently characterized CODH from Desulfovi-brio vulgaris (Dv) through protein film voltammetry and solution assays (in the oxidative direction). We found that O2 reacts very quickly with the active site of CODHs, generating species that reactivate upon reduction--this was unexpected. We observed that distinct CODHs exhibit different behaviors: Dv CODH reacts half as fast with O2 than Ch CODH, and only the former fully recovers the activity upon reduction. The results raise hope that fast CO/CO2 biological conversion may be feasible under aerobic conditions. [ABSTRACT FROM AUTHOR]

When enzymes are optimized for biotechnological purposes, the goal often is to increase stability or catalytic efficiency. However, many enzymes reversibly convert their substrate and product, and if one is interested in catalysis in only one direction, it may be necessary to prevent the reverse reaction. In other cases, reversibility may be advantageous because only an enzyme that can operate in both directions can turnover at a high rate even under conditions of low thermodynamic driving force. Therefore, understanding the basic mechanisms of reversibility in complex enzymes should help the rational engineering of these proteins. Here, we focus on NiFe hydrogenase, an enzyme that catalyzes H2 oxidation and production, and we elucidate the mechanism that governs the catalytic bias (the ratio of maximal rates in the two directions). Unexpectedly, we found that this bias is not mainly determined by redox properties of the active site, but rather by steps which occur on sites of the proteins that are remote from the active site. We evidence a novel strategy for tuning the catalytic bias of an oxidoreductase, which consists in modulating the rate of a step that is limiting only in one direction of the reaction, without modifying the properties of the active site. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Electrons are transferred over long distances along chains of FeS clusters in hydrogenases, mitochondrial complexes, and many other respiratory enzymes. It is usually presumed that electron transfer is fast in these systems, despite the fact that there has been no direct measurement of rates of FeS-to-FeS electron transfer in any respiratory enzyme. In this context, we propose and apply to NiFe hydrogenase an original strategy that consists of quantitatively interpreting the variations of steady-state activity that result from changing the nature of the FeS clusters which connect the active site to the redox partner, and/or the nature of the redox partner. Rates of intra- and intermolecular electron transfer are deduced from such large data sets. The mutation-induced variations of electron transfer rates cannot be explained by changes in intercenter distances and reduction potentials. This establishes that FeS-to-FeS rate constants are extremely sensitive to the nature and coordination of the centers. [ABSTRACT FROM AUTHOR]

In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a molecular channel whose structure may determine the enzyme's selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallography and molecular dynamics, but kinetic data are scarce. Using protein film voltammetry, we determined and then compared the rates of inhibition by CO and O2 in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O2 toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quantitatively define the relations between diffusion, the Michaelis constant for H2 and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O2 because access to the active site is slow. [ABSTRACT FROM AUTHOR]

Hydrogenases catalyze the conversion between 2H+ + 2e- and H21. Most of these enzymes are inhibited by O2, which represents a major drawback for their use in biotechnological applications.12 Improving hydrogenase O2 tolerance is therefore a major contemporary challenge to allow the implementation of a sustainable hydrogen economy. We succeeded in improving O2 tolerance, which we define here as the ability of the enzyme to resist for several minutes to O2 exposure, by substituting with methionines small hydrophobic residues strongly conserved in the gas channel. Remarkably, the mutated enzymes remained active in the presence of an O2 concentration close to that found in aerobic solutions in equilibrium with air, while the wild type enzyme is inhibited in a few seconds. Crystallographic and spectroscopic studies showed that the structure and the chemistry at the active site are not affected by the mutations. Kinetic studies demonstrated that the inactivation is slower and reactivation faster in these mutants. We propose that in addition to restricting O2 diffusion to the active site of the enzyme, methionine may also interact with bound peroxide and provide an assisted escape route for H2O2 toward the gas channel. These results show for the first time that it is possible to improve O2-tolerance of [NiFe] hydrogenases, making possible the development of biohydrogen production systems. [ABSTRACT FROM AUTHOR]

Abstract: In NiFe hydrogenases, electrons are transferred from the active site to the redox partner via a chain of three Iron–Sulfur clusters. Intriguingly, the surface-exposed [4Fe4S] cluster has three cysteines and one histidine as a ligand, which is quite unusual. When this Histidine (H184 in Desulfovibrio fructosovorans) was changed into a glycine, the distal cubane was still assembled but the oxidative activity of the mutants was 3% of that of the WT. As glycine is not a cubane ligand, a water molecule is likely to stabilize the fourth iron atom, making this coordination position labile. It was then possible to exchange the water ligand with an exogenous ligand. Depending on the molecule tested, the enzyme exhibited various activity levels, making possible a modulation of the enzyme activity. [Copyright &y& Elsevier]

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 = µ1 M). 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. [ABSTRACT FROM AUTHOR]

In NiFe hydrogenases, electrons are transferred from the active site to the redox partner via a chain of three Iron-Sulfur clusters, and the surface-exposed [4Fe4S] cluster has an unusual His(Cys)3 ligation. When this Histidine (H184 in Desulfovibrio fructosovorans) is changed into a cysteine or a glycine, a distal cubane is still assembled but the oxidative activity of the mutants is only 1.5 and 3% of that of the WT, respectively. We compared the activities of the WT and engineered enzymes for H2 oxidation, H+ reduction and H/D exchange, under various conditions: (i) either with the enzyme directly adsorbed onto an electrode or using soluble redox partners, and (ii) in the presence of exogenous ligands whose binding to the exposed Fe of H184G was expected to modulate the properties of the distal cluster. Protein film voltammetry proved particularly useful to unravel the effects of the mutations on inter and intramolecular electron transfer (ET). We demonstrate that changing the coordination of the distal cluster has no effect on cluster assembly, protein stability, active-site chemistry and proton transfer; however, it slows down the first-order rates of ET to and from the cluster. All-sulfur coordination is actually detrimental to ET, and intramolecular (uphill) ET is rate determining in the glycine variant. This demonstrates that although [4Fe4S] clusters are robust chemical constructs, the direct protein ligands play an essential role in imparting their ability to transfer electrons. [ABSTRACT FROM AUTHOR]

We have used protein film voltammetry to study the NiFe hydrogenase from Desulfovibrio fructosovorans. We show how measurements of transient activity following the addition in the electrochemical cell of H2, CO, or O2 allow simple and virtually instantaneous determinations of the Michaelis constant, inhibition constant, or rate of inactivation, respectively, thus opening new opportunities to study the active site of NiFe hydrogenases. The binding and release of CO occur within a fraction of a second, and we determine and discuss how its affinity for the active site changes as the driving force for the H+/H2 reaction is continuously varied. Inactivation by O2 is a slow, bimolecular process (with pH-independent rate constant ≈ 3 × 104 s-1 M-1 at 40°C, under one atm of H2) that leads to a mixture of fully oxidized states, and unlike the case of CO inhibition, the active site is not fully protected by H2. This experimental approach could be used to study the reaction of other multicentered metalloenzymes with their gaseous substrates or inhibitors. [ABSTRACT FROM AUTHOR]

By analysing the results of experiments carried out with two FeFe hydrogenases and several “channel mutants” of a NiFe hydrogenase, we demonstrate that whether or not hydrogen evolution is significantly inhibited by H2 is not a consequence of active site chemistry, but rather relates to H2 transport within the enzyme. [ABSTRACT FROM AUTHOR]

We studied the mechanism of aerobic inactivation of Desulfovibrio fructosovorans nickel-iron (NiFe) hydrogenase by quantitatively examining the results of electrochemistry, EPR and FTIR experiments. They suggest that, contrary to the commonly accepted mechanism, the attacking O2 is not incorporated as an active site ligand but, rather, acts as an electron acceptor. Our findings offer new ways toward the understanding of O2 inactivation and O2 tolerance in NiFe hydrogenases. [ABSTRACT FROM AUTHOR]

Abstract: Hydrogenase electrodes for hydrogen oxidation are elaborated by an innovative immobilization strategy of [NiFe] hydrogenases from Desulfovibrio fructosovorans on highly porous single-walled (SWCNT) and multi-walled (MWCNT) carbon nanotube electrodes. The bioelectrode fabrication involved the adsorption of hydrogenase and amphiphilic pyrrole monomer functionalized by a methylviologen moiety on the nanotube deposits. The electropolymerization of the adsorbed monomer then leads to the enzyme entrapment in polypyrrole film surrounding the nanotubes. In addition, the redox polypyrrole achieves an efficient electrical wiring of hydrogenase on SWCNT and MWCNT electrodes via a mediated electron transfer. The latter configuration showed improved performances in catalytic responses (up to 0.30±0.01mAcm−2) at stationary electrodes due to the more appropriate wettability of MWCNTs. This led to a better coating of the nanostructured surface and thus, to an enhanced mediated electron transfer between the enzyme and the nanotubes. [Copyright &y& Elsevier]

CO dehydrogenases (CODHs) catalyse the reversible conversion between CO and CO2. Genomic analysis indicated that the metabolic functions of CODHs vary. The genome of Carboxydothermus hydrogenoformans encodes five CODHs (CODH-I-V), of which CODH-IV is found in a gene cluster near a peroxide-reducing enzyme. Our kinetic and crystallographic experiments reveal that CODH-IV differs from other CODHs in several characteristic properties: it has a very high affinity for CO, oxidizes CO at diffusion-limited rate over a wide range of temperatures, and is more tolerant to oxygen than CODH-II. Thus, our observations support the idea that CODH-IV is a CO scavenger in defence against oxidative stress and highlight that CODHs are more diverse in terms of reactivity than expected. [ABSTRACT FROM AUTHOR]

CO‐Dehydrogenasen (CODHs) katalysieren die reversible Umsetzung zwischen CO und CO2. Genomische Analysen deuten darauf hin, dass CODHs unterschiedliche metabolische Funktionen haben. Das Genom von Carboxydothermus hydrogenoformans kodiert fünf CODHs (CODH‐I–V), von denen CODH‐IV in einem Gencluster gemeinsam mit einem Peroxid‐reduzierenden Enzym vorliegt. Unsere kinetischen und kristallographischen Analysen zeigen, dass sich CODH‐IV von anderen CODHs durch charakteristische Merkmale unterscheidet: CODH‐IV hat eine hohe Affinität für CO, oxidiert CO mit diffusionslimitierter Geschwindigkeit über einen weiten Temperaturbereich und ist toleranter gegen O2 als CODH‐II. Daher stützen unsere Beobachtungen die Idee, dass CODH‐IV als CO‐Fänger bei der Abwehr von oxidativem Stress agiert und unterstreichen, dass CODHs in Bezug auf ihre Reaktivität diverser als erwartet sind. CO‐Dehydrogenasen (CODHs) katalysieren die reversible Umwandlung zwischen CO und CO2. CODH‐IV unterscheidet sich dabei von anderen CODHs, indem es als CO‐Scavenger zur Abwehr von oxidativem Stress fungiert. Dies unterstreicht, dass CODHs in Bezug auf ihre Reaktivität diverser sind als erwartet. [ABSTRACT FROM AUTHOR]