Reduced Methyl Viologen Research Articles

Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum

Nitrate reductase from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harboring a bis(molybdopterin guanine dinucleotide) active site and a [4Fe-4S] cluster. Previous electron paramagnetic resonance (EPR) studies in both catalytic and inhibiting conditions showed that the molybdenum center has high coordination flexibility when reacted with reducing agents, substrates or inhibitors. As-prepared DdNapA samples, as well as those reacted with substrates and inhibitors, were crystallized and the corresponding structures were solved at resolutions ranging from 1.99 to 2.45 A. The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on that basis, the sixth molybdenum ligand, originally proposed to be an OH/OH(2) ligand, was assigned as a sulfur atom after refinement and analysis of the B factors of all the structures. This unexpected result was confirmed by a single-wavelength anomalous diffraction experiment below the iron edge (lambda = 1.77 A) of the as-purified enzyme. Furthermore, for six of the seven datasets, the S-S distance between the sulfur ligand and the Sgamma atom of the molybdenum ligand Cys(A140) was substantially shorter than the van der Waals contact distance and varies between 2.2 and 2.85 A, indicating a partial disulfide bond. Preliminary EPR studies under catalytic conditions showed an EPR signal designated as a turnover signal (g values 1.999, 1.990, 1.982) showing hyperfine structure originating from a nucleus of unknown nature. Spectropotentiometric studies show that reduced methyl viologen, the electron donor used in the catalytic reaction, does not interact directly with the redox cofactors. The turnover signal can be obtained only in the presence of the reaction substrates. With use of the optimized conditions determined by spectropotentiometric titration, the turnover signal was developed with (15)N-labeled nitrate and in D(2)O-exchanged DdNapA samples. These studies indicate that this signal is not associated with a Mo(V)-nitrate adduct and that the hyperfine structure originates from two equivalent solvent-exchangeable protons. The new coordination sphere of molybdenum proposed on the basis of our studies led us to revise the currently accepted reaction mechanism for periplasmic nitrate reductases. Proposals for a new mechanism are discussed taking into account a molybdenum and ligand-based redox chemistry, rather than the currently accepted redox chemistry based solely on the molybdenum atom.

Bioinspired Energy Conversion Systems for Hydrogen Production and Storage

AbstractRecent developments in photocatalytic hydrogen production by using artificial photosynthesis systems is described, together with those in hydrogen storage through the fixation of CO2 with H2. Hydrogen can be stored in the form of formic acid, which can be converted back to H2 in the presence of an appropriate catalyst. Electron donor–acceptor dyads are utilized as efficient photocatalysts to reduce methyl viologen (MV2+) by NADH (β‐nicotinamide adenine dinucleotide, reduced form) analogues to produce the methyl violgen radical cation that acts as an electron mediator for the production of hydrogen. Porphyrin‐monolayer‐protected gold clusters that enhance the light harvesting efficiency can also be used for the photocatalytic reduction of methyl viologen by NADH analogues. The use of a simple electron donor–acceptor dyad, the 9‐mesityl‐10‐methylacridinium ion (Acr+–Mes), enables the construction of a highly efficient photocatalytic hydrogen‐evolution system without an electron mediator such as MV2+, with poly(N‐vinyl‐2‐pyrrolidone)‐protected platinum nanoclusters (Pt–PVP) and NADH as a hydrogen‐evolution catalyst and an electron donor, respectively. Hydrogen thus produced can be stored in the form of formic acid (liquid) by fixation of CO2 with H2 in water by using ruthenium aqua complexes [RuII(η6‐C6Me6)(L)(OH2)]2+ [L = 2,2′‐bipyridine (bpy), 4,4′‐dimethoxy‐2,2′‐bipyridine (4,4′‐OMe‐bpy)] and iridium aqua complexes [IrIIICp*(L)(OH2)]2+ (Cp* = η5‐C5Me5, L = bpy, 4,4′‐OMe‐bpy) as catalysts at pH 3.0. Catalytic systems for the decomposition of HCOOH to H2 are also described. The combination of photocatalytic hydrogen generation with the catalytic fixation of CO2 with H2 and the decomposition of HCOOH back to H2 provides an excellent system for cutting CO2 emission.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Investigating the voltammetric reduction of methylviologen at gold and carbon based electrode materials. Evidence for a surface bound adsorption mechanism leading to electrode ‘protection’ using multi-walled carbon nanotubes

Cyclic voltammetry was recorded in an aqueous solution of 1,1′-dimethyl-4,4′ bipyridinium dichloride (“methylviologen” or “paraquat”) at a variety of electrode materials namely gold, boron doped diamond (BDD), edge (eppg) and basal plane (bppg) pyrolytic graphite electrode. Previous mechanisms reported in the literature to describe the observed voltammetry are critically assessed and an alternative mechanistic interpretation is presented involving evidence for surface adsorption of intermediate species in the redox behaviour of methylviologen (MV) rather than any disproportionation or comproportionation as was previously claimed for the specific case of gold electrodes. Various electrodes were then modified with multi-walled carbon nanotubes (MWCNTs) where the formation of solid MV0 on the electrode surface was prevented due to preferential adsorption of MV0 on the MWCNTs. Thus coating an electrode with MWCNTs can protect the electrode surface from “fouling”, providing scope for diffusion dominated voltammetry at electrodes where surface inhibition has hitherto dominated.

Evidence for a Radical Mechanism of the Dechlorination of Chlorinated Propenes Mediated by the Tetrachloroethene Reductive Dehalogenase of Sulfurospirillum multivorans

The reductive dehalogenation of chlorinated propenes was studied with the tetrachloroethene reductive dehalogenase purified from Sulfurospirillum multivorans to obtain indications for a radical mechanism of this reaction. When reduced methyl viologen (MV), which is a radical cation, was applied as electron donor for the reduction of different chloropropenes, a significant part of MV could not be rereduced with Ti(III) citrate, indicating that a part of the MV was consumed in a side reaction. Mass spectrometric analysis of assays with MV as electron donor revealed the formation of side products, the masses of which might account for the formation of adducts from a chloropropenyl radical and reduced methyl viologen. With Ti(III) citrate as sole electron donor, 2,3-dichloropropene was reduced and as a side product, 2,5-dichloro-1,5-hexadiene was formed demonstrating that the reductive dechlorination of 2,3-dichloropropene proceeds via a radical reaction mechanism. The results support different dehalogenation mechanisms forthe reductive dechlorination of chloropropenes and halogenated ethenes.

Photoinduced Release of Nitroxyl and Nitric Oxide from Diazeniumdiolates

Aqueous photochemistry of diazen-1-ium-1,2,2-triolate (Angeli's anion) and (Z)-1[N-(3-aminopropyl)-N-(3-aminopropyl)amino]diazen-1-ium-1,2-diolate (DPTA NONOate) has been investigated by laser kinetic spectroscopy. In neutral aqueous solutions, 266 nm photolysis of these diazeniumdiolates generates a unique spectrum of primary products including the ground-state triplet (3NO-) and singlet (1HNO) nitroxyl species and nitric oxide (NO*). Formation of these spectrophotometrically invisible products is revealed and quantitatively assayed by analyzing a complex set of their cross-reactions leading to the formation of colored intermediates, the N2O2*- radical and N3O3- anion. The experimental design employed takes advantage of the extremely slow spin-forbidden protic equilibration between 3NO- and 1HNO and the vast difference in their reactivity toward NO*. To account for the kinetic data, a novel combination reaction, 3NO-+1HNO, is introduced, and its rate constant of 6.6x10(9) M-1 s-1 is measured by competition with the reduction of methyl viologen by 3NO-. The latter reaction occurring with 2.1x10(9) M-1 s-1 rate constant and leading to the stable, colored methyl viologen radical cation is useful for detection of 3NO-. The distributions of the primary photolysis products (Angeli's anion: 22% 3NO-, 58% 1HNO, and 20% NO*; DPTA NONOate: 3% 3NO-, 12% 1HNO, and 85% NO*) show that neither diazeniumdiolate is a highly selective photochemical generator of nitroxyl species or nitric oxide, although the selectivity of DPTA NONOate for NO* generation is clearly greater.

Photosensitized Reduction of Methyl Viologen Using Eosin-Y in Presence of a Sacrificial Electron Donor in Water-Alcohol Mixture

Photoreduction of methyl viologen (MV2+) by eosin-Y (EY2−) in the presence of triethanolamine (TEOA) has been investigated in water–methanol mixture by means of steady-state photolysis and laser-flash photolysis in the visible/near-infrared regions. The complete conversion to the persistent methyl viologen radical cation (MV·+) was observed in the presence of lower concentrations of EY2− and excess TEOA. By laser-flash photolysis measurements, electron transfer was confirmed to occur from the triplet state of EY2− [3(EY2−)*] to MV2+ in the rate constants of ca 2.0 × 1010M−1 s−1. The rates and efficiencies of production of MV·+ were found to be dependent on solvent compositions and concentrations of MV2+ ionic salt and TEOA. The back electron transfer reaction from MV·+ to EY·− was retarded in the presence of TEOA, which supports that EY2− is reproduced by accepting an electron from TEOA. In the presence of excess TEOA, the indirect formation of MV·+ from EY·3− which was produced by accepting an electron from TEOA, was confirmed. The contributions of both the oxidative and reductive routes of 3(EY2−)* for the MV·+ formation have been confirmed.

Purification and characterization of putative alkaline [Ni–Fe] hydrogenase from unicellular marine green alga, Tetraselmis kochinensis NCIM 1605

Hydrogenase enzyme from the unicellular marine green alga Tetraselmis kochinensis NCIM 1605 was purified 467 fold to homogeneity. The molecular weight was estimated to be approximately 89kDa by SDS-PAGE. This enzyme consists of two subunits with molecular masses of approximately 70 and approximately 19kDa. The hydrogenase was found to contain 10g atoms of Fe and 1g of atom of Ni per mole of protein. The specific activity of hydrogen evolution was 50micromol H(2)/mg/h of enzyme using reduced methyl viologen as an electron donor. This hydrogenase enzyme has pI value approximately 9.6 representing its alkaline nature. The absorption spectrum of the hydrogenase enzyme showed an absorption peak at 425nm indicating that the enzyme had iron-sulfur clusters. The total of 16 cysteine residues were found per mole of enzyme under the denaturing condition and 20 cysteine residues in reduced denatured enzyme indicating that it has two disulfide bridges.

Insights into the Mechanism of N2O Reduction by Reductively Activated N2O Reductase from Kinetics and Spectroscopic Studies of pH Effects

Nitrous oxide reductase (N2OR) from Achromobacter cycloclastes (Ac) can be reductively activated with reduced methyl viologen over a broad range of pH. Activated Ac N2OR displays a complex activity profile as a function of the pH at which catalytic turnover is measured. Spectroscopic and steady-state kinetics data suggest that [H+] has multiple effects on both the activation and the catalytic reactions. These experimental results are in good agreement with previous theoretical studies, which suggested that the transition state is stabilized by H-bonding interactions between the active site and an N2O-derived intermediate bound to the catalytic CuZ cluster.

Electronic Interplay on Illuminated Aqueous Carbon Nanohorn−Porphyrin Ensembles

Tetracationic water-soluble porphyrin (H(2)P(4+)) has been immobilized by pi-pi stacking interactions onto the skeleton of carbon nanohorns (CNH), without disrupting their pi-electronic network. The stable aqueous solution of the CNH-H(2)P(4+) nanoensemble was examined by both electron microscopy and spectroscopic techniques. The efficient fluorescence quenching of the H(2)P(4+) moiety in the CNH-H(2)P(4+) nanoensemble was probed by steady-state as well as time-resolved fluorescence emission spectroscopy, suggesting charge separation from the photoexcited H(2)P(4+) to CNH. In the presence of methyl viologen dication (MV(2+)) and a hole trap, accumulation of the reduced species of methyl viologen was observed by the photoillumination of CNH-H(2)P(4+), suggesting that the electron migration from the initially formed charge-separated state takes place. Transient absorption spectroscopy gave further insights on the transient species such as the charge-separated state (CNH(*-))-(H(2)P(4+))(*+), which was consumed in the presence of MV(2+) and hole shifter, leaving the reduced methyl viologen.

Electrochemical analysis of the reduction of ferritin using oxidized methyl viologen

The electrochemical analysis was carried out by the electrochemical reduction of horse spleen ferritin (HoSF) using oxidized methyl viologen. The reduction and oxidation peaks of the methyl viologen and released Fe appeared on the cyclic voltammogram. The Fe was released on reduction of the HoSF by the methyl viologen. The effect of oxidized methyl viologen concentration and the ferritin solution pH on reduction of the HoSF were determined. The reduction of the ferritin procedure occurred by applying a potential that reduced the oxidized methyl viologen, and the HoSF was reduced by the reduced methyl viologen.

Development of a viologen-based microtiter plate assay for the analysis of oxyanion reductase activity: Application to the membrane-bound selenate reductase from Enterobacter cloacae SLD1a-1

The membrane-bound selenate reductase of Enterobacter cloacae SLD1a-1 is purified in low yield and has relatively low activity in the pure form compared to that of other oxyanion reductases, such as the membrane-bound and periplasmic nitrate reductases. A microtiter plate assay based on the original quartz cuvette viologen assay of Jones and Garland (R.W. Jones, P.B. Garland, Biochem. J 164 (1977) 199–211) was developed specifically for analysis of such low-abundant, labile oxyanion reductases. The plate assay detects the enzyme-dependent reoxidation of reduced methyl viologen spectrophotometrically at 600 nm. The assay is quick, uses a minimal sample volume (<5 μl), can simultaneously test a range of alternative substrates, and permits activity measurements on multiple samples. We demonstrate the accuracy and versatility of the microtiter plate assay by application to the kinetic analysis, inhibition, and pH optimization of the membrane-bound selenate reductase from E. cloacae SLD1a-1. Results show that the membrane–bound selenate reductase has optimum activity at pH ∼ 8 and its active site is able to accommodate larger inhibitory complexes resulting in mixed-type inhibition, in the presence of selenate and potassium thiocyanate.

Expression and characterization of the assimilatory NADH-nitrite reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1

A nas gene region from Rhodobacter capsulatus E1F1 containing the putative nasB gene for nitrite reductase was previously cloned. The recombinant His(6)-NasB protein overproduced in E. coli showed nitrite reductase activity in vitro with both reduced methyl viologen and NADH as electron donors. The apparent K ( m ) values for nitrite and NADH were 0.5 mM and 20 microM, respectively, at the pH and temperature optima (pH 9 and 30 degrees C). The optical spectrum showed features that indicate the presence of FAD, iron-sulfur cluster and siroheme as prosthetic groups, and nitrite reductase activity was inhibited by sulfide and iron reagents. These results indicate that the phototrophic bacterium R. capsulatus E1F1 possesses an assimilatory NADH-nitrite reductase similar to that described in non-phototrophic organisms.

Temperature dependence of nitrate reductase in the psychrophilic unicellular alga Koliella antarctica and the mesophilic alga Chlorella sorokiniana

Temperature responses of nitrate reductase (NR) were studied in the psychrophilic unicellular alga, Koliella antarctica, and in the mesophilic species, Chlorella sorokiniana. Enzymes from both species were purified to near homogeneity by Blue Sepharose (Pharmacia, Uppsala, Sweden) affinity chromatography and high-resolution anion-exchange chromatography (MonoQ; Pharmacia; Uppsala, Sweden). Both enzymes have a subunit molecular mass of 100 kDa, and K. antarctica NR has a native molecular mass of 367 kDa. NR from K. antarctica used both NADPH and NADH, whereas NR from C. sorokiniana used NADH only. Both NRs used reduced methyl viologen (MVH) or benzyl viologen (BVH). In crude extracts, maximal NADH and MVH-dependent activities of cryophilic NR were found at 15 and 35 degrees C, respectively, and retained 77 and 62% of maximal activity, respectively, at 10 degrees C. Maximal NADH and MVH-dependent activities of mesophilic NR, however, were found at 25 and 45 degrees C, respectively, with only 33 and 23% of maximal activities being retained at 10 degrees C. In presence of 2 microM flavin adenine dinucleotide (FAD), activities of cryophilic NADH:NR and mesophilic NADH:NR were stable up to 25 and 35 degrees C, respectively. Arrhenius plots constructed with cryophilic and mesophilic MVH:NR rate constants, in both presence or absence of FAD, showed break points at 15 and 25 degrees C, respectively. Essentially, similar results were obtained for purified enzymes and for activities measured in crude extracts. Factors by which the rate increases by raising temperature 10 degrees C (Q10) and apparent activation energy (E(a)) values for NADH and MVH activities measured in enzyme preparations without added FAD differed slightly from those measured with FAD. Overall thermal features of the NADH and MVH activities of the cryophilic NR, including optimal temperatures, heat inactivation (with/without added FAD) and break-point temperature in Arrhenius plots, are all shifted by about 10 degrees C towards lower temperatures than those of the mesophilic enzyme. Transfer of electrons from NADH to nitrate occurs via all three redox centres within NR molecule, whereas transfer from MVH requires Mo-pterin prosthetic group only; therefore, our results strongly suggest that structural modification(s) for cold adaptation affect thermodynamic properties of each of the functional domains within NR holoenzyme in equal measure.

Kinetic investigations of the mechanism of dihydrogen driven catalytic reduction of methylene blue, safranine O, methyl viologen and ferricyanide using platinum carbonyl cluster anions (Chini-clusters) as catalyst

[Bu 4N] 2[Pt 12(CO) 24] ( 1) catalyses the selective reduction of electron acceptors ( S), methylene blue (MB +), safranine O (Saf +), methyl viologen (MV 2+) and ferricyanide by dihydrogen. Macroscopic rate investigations for the cationic substrates in DMF, and for ferricyanide in DMSO have been carried out. In all cases, k obs is given by k 1 [ S] + k 2, indicating that there are two catalytic cycles. In one of them, the formation of a complex between S and [Pt 12(CO) 24] 2− in the rate determining step (rate constant k 1) is followed by electron transfer and/or other fast steps. In the other catalytic cycle, the rate determining step (rate constant k 2) involves formation of the solvated cluster anion [Pt 12(CO) 24] 2−. The solvated cluster then undergoes fast reduction by dihydrogen and other reactions. The relative contributions of these two cycles depend on the substrate, and for MB +, Saf +, MV 2+ and [Fe(CN) 6] 3− the contribution of the second cycle is about 99%, 55%, 77% and 97%, respectively. Both k 1 and k 2 of ferricyanide are about three orders of magnitude smaller than those of the cationic electron acceptors. The rates of reduction of MB + and Saf + have also been studied in the presence of added water. Rates increase as the presence of water provides an additional pathway for the reduction of [Pt 12(CO) 24] 2− to [Pt 9(CO) 18] 2−.

The effect of the concentration of platinum nanocolloid stabilizer on the rate of catalytic reactions in aqueous solution

The reduction of methyl viologen with hydrogen in the presence of NaOH and neptunium (VI) ions with formic acid in aqueous solutions catalyzed by platinum nanocolloids is studied. It is established that the rate of reaction with the participation of methyl viologen exponentially decreases with an increase in the concentration of a colloid stabilizer (sodium polyacrylate). The rate of neptunium reduction catalyzed by platinum nanoparticles of various sizes also decreases with an increase in the polyacrylate concentration to 4 × 10−3 M. A further increase in the content of stabilizer leads to some acceleration of the reaction. The mechanism of the effect of the stabilizer concentration on the catalytic activity of platinum nanocolloids is discussed.

Characterization of membrane-bound nitrate reductase from denitrifying bacteriaOchrobactrum anthropi SY509

In this study, we have purified and characterized the membrane bound nitrate reductase obtained from the denitrifying bacteria,Ochrobactrum anthropi SY509, which was isolated from soil samples.O. anthropi SY509 can grow in minimal medium using nitrate as a nitrogen source. We achieved an overall purification rate of 15-fold from the protein extracted from the membrane fraction, with a recovery of approximately 12% of activity. The enzyme exhibited its highest level of activity at pH 5.5, and the activity was increased up to 70°C. Periplasmic and cytochromic proteins, including nitrite and nitrous oxide reductase, were excluded during centrifugation and were verified using enzyme essay. Reduced methyl viologen was determined to be the most efficient electron donor among a variety of anionic and cationic dyestuffs, which could be also used as an electron donor with dimethyl dithionite. The effects of purification and storage conditions on the stability of enzyme were also investigated. The activity of the membrane-bound nitrate reductase was stably maintained for over 2 weeks in solution. To maintain the stability of enzyme, the cell was disrupted using sonication at low temperatures, and enzyme was extracted by hot water without any surfactant. The purified enzyme was stored in solution with no salt to prevent any significant losses in activity levels.

Photochemical and enzymatic synthesis of formic acid from CO 2 with chlorophyll and dehydrogenase system

Photochemical and enzymatic formic acid synthesis from CO 2 gas system with the combination of formate dehydrogenase (FDH) from Saccharomyces cerevisiae and methylviologen (MV 2+) reduction by the visible light photosensitisation of Mg chlorophyll- a (Mg Chl- a) is developed. Photochemical formic acid produced from CO 2 by adding FDH to the MV 2+ photoreduction system using photosensitisation of Mg Chl- a in the presence of NADPH as an electron donating reagent, and 56 μM of formic acid from CO 2 gas is produced after 4 h irradiation (1.0 M = 1.0 mol dm −3).

Chemical modification studies of tryptophan, arginine and lysine residues in maize chloroplast ferredoxin:sulfite oxidoreductase

The ferredoxin-dependent sulfite reductase from maize was treated, in separate experiments, with three different covalent modifiers of specific amino acid side chains. Treatment with the tryptophan-modifying reagent, N-bromosuccinimide (NBS), resulted in a loss of enzymatic activity with both the physiological donor for the enzyme, reduced ferredoxin, and with reduced methyl viologen, a non-physiological electron donor. Formation of the 1:1 ferredoxin/sulfite reductase complex prior to treating the enzyme with NBS completely protected the enzyme against the loss of both activities. Neither the secondary structure, nor the oxidation-reduction midpoint potential (Em) values of the siroheme and [4Fe-4S] cluster prosthetic groups of sulfite reductase, nor the binding affinity of the enzyme for ferredoxin were affected by NBS treatment. Treatment of sulfite reductase with the lysine-modifying reagent, N-acetylsuccinimide, inhibited the ferredoxin-linked activity of the enzyme without inhibiting the methyl viologen-linked activity. Complex formation with ferredoxin protects the enzyme against the inhibition of ferredoxin-linked activity produced by treatment with N-acetylsuccinimide. Treatment of sulfite reductase with N-acetylsuccinimide also decreased the binding affinity of the enzyme for ferredoxin. Treatment of sulfite reductase with the arginine-modifying reagent, phenylglyoxal, inhibited both the ferredoxin-linked and methyl viologen-linked activities of the enzyme but had a significantly greater effect on the ferredoxin-dependent activity than on the reduced methyl viologen-linked activity. The effects of these three inhibitory treatments are consistent with a possible role for a tryptophan residue the catalytic mechanism of sulfite reductase and for lysine and arginine residues at the ferredoxin-binding site of the enzyme.

Immobilization of Active Hydrogenases by Encapsulation in Polymeric Porous Gels

Hydrogenases encapsulated in porous polymeric silica gels retain significant levels of hydrogen production activity when compared to hydrogenases in solution using reduced methyl viologen as an electron donor. Encapsulated hydrogenases remain active after storage at room temperature for longer than four weeks and are less sensitive to proteolytic digestion. Nanoscopic confinement of active hydrogenases in solids paves the way for their potential use in hydrogen producing catalytic materials applications.

Enlarging the gas access channel to the active site renders the regulatory hydrogenase HupUV of Rhodobacter capsulatus O2 sensitive without affecting its transductory activity

In the photosynthetic bacterium Rhodobacter capsulatus, the synthesis of the energy-producing hydrogenase, HupSL, is regulated by the substrate H2, which is detected by a regulatory hydrogenase, HupUV. The HupUV protein exhibits typical features of [NiFe] hydrogenases but, interestingly, is resistant to inactivation by O2. Understanding the O2 resistance of HupUV will help in the design of hydrogenases with high potential for biotechnological applications. To test whether this property results from O2 inaccessibility to the active site, we introduced two mutations in order to enlarge the gas access channel in the HupUV protein. We showed that such mutations (Ile65-->Val and Phe113-->Leu in HupV) rendered HupUV sensitive to O2 inactivation. Also, in contrast with the wild-type protein, the mutated protein exhibited an increase in hydrogenase activity after reductive activation in the presence of reduced methyl viologen (up to 30% of the activity of the wild-type). The H2-sensing HupUV protein is the first component of the H2-transduction cascade, which, together with the two-component system HupT/HupR, regulates HupSL synthesis in response to H2 availability. In vitro, the purified mutant HupUV protein was able to interact with the histidine kinase HupT. In vivo, the mutant protein exhibited the same hydrogenase activity as the wild-type enzyme and was equally able to repress HupSL synthesis in the absence of H2.