Characterization of the oxygen tolerance of a hydrogenase linked to a carbon monoxide oxidation pathway in Rubrivivax gelatinosus

A hydrogenase linked to the carbon monoxide oxidation pathway in Rubrivivax gelatinosus displays tolerance to O2. When either whole-cell or membrane-free partially purified hydrogenase was stirred in full air (21% O2, 79% N2), its H2 evolution activity exhibited a half-life of 20 or 6 h, respectively, as determined by an anaerobic assay using reduced methyl viologen. When the partially purified hydrogenase was stirred in an atmosphere containing either 3.3 or 13% O2 for 15 min and evaluated by a hydrogen-deuterium (H-D) exchange assay, nearly 80 or 60% of its isotopic exchange rate was retained, respectively. When this enzyme suspension was subsequently returned to an anaerobic atmosphere, more than 90% of the H-D exchange activity was recovered, reflecting the reversibility of this hydrogenase toward O2 inactivation. Like most hydrogenases, the CO-linked hydrogenase was extremely sensitive to CO, with 50% inhibition occurring at 3.9 microM dissolved CO. Hydrogen production from the CO-linked hydrogenase was detected when ferredoxins of a prokaryotic source were the immediate electron mediator, provided they were photoreduced by spinach thylakoid membranes containing active water-splitting activity. Based on its appreciable tolerance to O2, potential applications of this hydrogenase are discussed.     

Continuous monitoring, by mass spectrometry, of H2 production and recycling in Rhodopseudomonas capsulata.

Hydrogen evolution and consumption by cell and chromatophore suspensions of the photosynthetic bacterium Rhodopseudomonas capsulata was measured with a sensitive and specific mass spectrometric technique which directly monitors dissolved gases. H2 production by nitrogenase was inhibited by acetylene and restored by carbon monoxide. An H2 evolution activity coupled with HD formation and D2 uptake (H-D exchange) was unaffected by C2H2 and CO. Cultures lacking nitrogenase activity also exhibited H-D exchange activity, which was catalyzed by a membrane-bound hydrogenase present in the chromatophores of R. capsulata. A net hydrogen uptake, mediated by hydrogenase, was observed when electron acceptors such as CO2, O2, or ferricyanide were present in the medium.     

Intracellular Location and O2 Sensitivity of Uptake Hydrogenase in Azospirillum spp

Uptake hydrogenase activity of Azospirillum brasilense in vitro (cell-free extract) was very much more sensitive to O₂ than was that of A. amazonense, and the O₂ pressure optima for uptake hydrogenase activities were 0.01 and 0.4 to 3 kPa for A. brasilense and A. amazonense, respectively. The addition of superoxide dismutase did not increase uptake hydrogenase activity of A. brasilense either in vivo or in vitro. The O₂ uptake rates of A. brasilense and A. amazonense were nearly the same. Inhibition of A. brasilense O₂-dependent uptake hydrogenase activity by O₂ was highly reversible under the conditions tested. O₂ also markedly inhibited in vitro methylene blue-dependent uptake hydrogenase activity of A. brasilense, and this inhibition was highly reversible. It is concluded that the difference in O₂ tolerance of the uptake hydrogenases is not due to a difference in respiratory protection in the two species and may be due to inherent differences in the two enzymes. For the three species, A. brasilense, A. amazonense, and A. lipoferum, almost all the recovered methylene blue-dependent uptake hydrogenase activity was associated with the membrane fraction.     

Variation in nitrogenase and hydrogenase activity of alaska pea root nodules

Hydrogenase activity of root nodules in the symbiotic association between Pisum sativum L. and Rhizobium leguminosarum was determined by incubating unexcised nodules with tritiated H₂ and measuring tissue HTO. Hydrogenase activity saturated at 0.50 millimolar H₂ and was not inhibited by the presence of 0.10 atmosphere C₂H₂, which prevented H₂ evolution from nitrogenase. Total H₂ production from nitogenase was estimated as net H₂ evolution in air plus H₂ exchange in 0.10 atmosphere C₂H₂. Although such an estimate of nitrogenase function may not be quantitatively exact, due to uncertain relationships between H₂ exchange and H₂ uptake activity of hydrogenase, differences observed in H₂ exchange under various conditions represent an indication of changes in hydrogenase activity. Hydrogenase activity was lower in associations grown under higher photosynthetic photon flux densities and decreased relative to total H₂ production by nitrogenase. Total H₂ production and hydrogenase activity were maximum 28 days after planting. Thereafter, hydrogenase activity and H₂ production declined, but the potential proportion of nitrogenase-produced H₂ recovered by the uptake hydrogenase system increased. Of five R. leguminosarum strains tested two possessed hydrogenase activity. Strains which had the potential to reassimilate H₂ had significantly higher rates of N₂ reduction than those which did not exhibit hydrogenase activity.     

Control of Carbon and Electron Flow in Clostridium acetobutylicum Fermentations: Utilization of Carbon Monoxide to Inhibit Hydrogen Production and to Enhance Butanol Yields

Extracts prepared from non-solvent-producing cells of Clostridium acetobutylicum contained methyl viologen-linked hydrogenase activity (20 U/mg of protein at 37°C) but did not display carbon monoxide dehydrogenase activity. CO addition readily inhibited the hydrogenase activity of cell extracts or of viable metabolizing cells. Increasing the partial pressure of CO (2 to 10%) in unshaken anaerobic culture tube headspaces significantly inhibited (90% inhibition at 10% CO) both growth and hydrogen production by C. acetobutylicum. Growth was not sensitive to low partial pressures of CO (i.e., up to 15%) in pH-controlled fermentors (pH 4.5) that were continuously gassed and mixed. CO addition dramatically altered the glucose fermentation balance of C. acetobutylicum by diverting carbon and electrons away from H₂, CO₂, acetate, and butyrate production and towards production of ethanol and butanol. The butanol concentration was increased from 65 to 106 mM and the butanol productivity (i.e., the ratio of butanol produced/total acids and solvents produced) was increased by 31% when glucose fermentations maintained at pH 4.5 were continuously gassed with 85% N₂-15% CO versus N₂ alone. The results are discussed in terms of metabolic regulation of C. acetobutylicum saccharide fermentations to achieve maximal butanol or solvent yield.     

Purification and Characterization of Membrane-Associated Hydrogenase from the Deep-Sea Epsilonproteobacterium Hydrogenimonas thermophila

Membrane-associated hydrogenase was purified from the chemolithoautotrophic epsilonproteobacterium Hydrogenimonas thermophila at 152-fold purity. The hydrogenase was found to be localized in the periplasmic space, and was easily solubilized with 0.1% Triton X-100 treatment. Hydrogen oxidation activity was 1,365 μmol H₂/min/mg of protein at 80 °C at pH 9.0, with phenazine methosulphate as the electron acceptor. Hydrogen production activity was 900 μmol H₂/min/mg of protein at 80 °C and pH 6.0, with reduced methyl viologen as the electron donor. The hydrogenase from this organism showed higher oxygen tolerance than those from other microorganisms showing hydrogen oxidation activity. The structural genes of this hydrogenase, which contains N-terminal amino acid sequences from both small and large subunits of purified hydrogenase, were successfully elucidated. The hydrogenase from H. thermophila was found to be phylogenetically related with H₂ uptake hydrogenases from pathogenic Epsilonproteobacteria.     

Effect of anaerobiosis on photosynthetic reactions and nitrogen metabolism of algae with and without hydrogenase

Summary The effect of anaerobic (N₂+CO₂) pre-incubation in the dark on photosynthetic reactions (O₂ evolution, measured manometrically and with the oxygraph; fluorescence; and photoproduction of H₂, measured with the mass spectrometer) was studied in algae with hydrogenase (strains of Chlorella fusca, C. kessleri, C. vulgaris f. tertia, and Ankistrodesmus braunii) and in algae without hydrogenase (strains of Chlorella vulgaris, C. saccharophila, and C. minutissima).The inhibition by anaerobic incubation of photosynthetic O₂ evolution is much stronger in algae without hydrogenase than it is in algae with hydrogenase. The effect of anaerobiosis is most pronounced at rather low light intensity (about 1000 lux), in acid medium (pH 4), and after prolonged anaerobic incubation in the dark (about 20 h). These results indicate that the presence of hydrogenase might be ecologically advantageous for algae under certain conditions.Chlorophyll fluorescence showed the fastest response to anaerobic incubation, and the most pronounced difference between algae with and without hydrogenase. After only 30 min under N₂+CO₂, fluorescence in algae with hydrogenase starts with a peak and decreases within 10 to 20 sec to a rather low steady-state level which is only slightly higher than that found under aerobic conditions. In algae without hydrogenase, fluorescence is rather low during the first 1 to 2 sec and then rises to a higher steady-state level which is much higher than that of the aerobic controls. This indicates an inhibition due to anaerobiosis of photosystem II in algae without hydrogenase.Algae with hydrogenase can react in different ways during the first minutes of illumination. In some cases there is an immediate photoproduction of H₂, which is followed after a few minutes by photosynthetic O₂ evolution; in other algae there is a simultaneous production of H₂ and O₂ from the very beginning; in a few experiments there was no photoproduction of H₂ at all, and in this case there was no photosynthetic O₂ evolution either. Thus, photoproduction of H₂ seems to be the process which normally enables algae with hydrogenase to oxidise and thereby activate their photosynthetic electron transport system after anaerobic incubation.A mass spectrometric search for nitrogen fixation (using N₂ and acetylene) in eucaryotic green algae gave negative results, even with species containing hydrogenase and under anaerobic conditions.     

Low spin quantitation of NiFeC EPR signal from carbon monoxide dehydrogenase is not due to damage incurred during protein purification

Evidence is presented that the O₂-sensitive, nickel- and iron-containing enzyme carbon monoxide dehydrogenase from Clostridium thermoaceticum was purified without significantly inactivating either its CO oxidation or CO/acetyl-CoA exchange activities. All CO oxidation activity from the crude extract was recovered in the purified enzyme (and side fractions). The exchange activity could not be quantified similarly, because the crude extract and early purification step fractions exhibited little or no exchange activity. Later purification fractions exhibited much more exchange activity, suggesting that an inhibitor was present in the impure fractions. The NiFeC EPR signal intensity was used as an indicator of the enzyme's capacity to catalyze exchange. This signal was extremely sensitive to oxygen; exposure to as little as 0.5 equiv/mol enzyme dimer resulted in substantial loss of intensity. The NiFeC intensities at each step in the purification were virtually invariant, indicating that the enzyme had not been exposed to oxygen and had not been inactivated towards catalyzing exchange. The ability to purify carbon monoxide dehydrogenase (CODH) without inactivating nearly any of the molecules suggests that it is quite stable under anaerobic conditions. The purified enzyme, which could not have lost functional metal ions during purification, contained 1.9 Ni and 11.3 Fe, similar to previous reports. The NiFeC EPR signal intensity from each purification fraction (0.2 spins/mol enzyme dimer) was as low as from previous preparations, indicating that its low spin quantitation is not the result of damage incurred during purification. If the low intensity arises from heterogeneity as proposed earlier, the heterogeneity must originate prior to purification.     

HupUV proteins of Rhodobacter capsulatus can bind H2: evidence from the H-D exchange reaction.

The H-D exchange reaction has been measured with the D2-H2O system, for Rhodobacter capsulatus JP91, which lacks the hupSL-encoded hydrogenase, and R. capsulatus BSE16, which lacks the HupUV proteins. The hupUV gene products, expressed from plasmid pAC206, are shown to catalyze an H-D exchange reaction distinguishable from the H-D exchange due to the membrane-bound, hupSL-encoded hydrogenase. In the presence of O2, the uptake hydrogenase of BSE16 cells catalyzed a rapid uptake and oxidation of H2, D2, and HD present in the system, and its activity (H-D exchange, H2 evolution in presence of reduced methyl viologen [MV+]) depended on the external pH, while the H-D exchange due to HupUV remained insensitive to external pH and O2. These data suggest that the HupSL dimer is periplasmically oriented, while the HupUV proteins are in the cytoplasmic compartment.     

The exchange activities of [Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) from methanogenic archaea in comparison with the exchange activities of [FeFe] and [NiFe] hydrogenases

[Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) catalyzes the reversible reduction of methenyltetrahydromethanopterin (methenyl-H₄MPT⁺) with H₂ to methylene-H₄MPT, a reaction involved in methanogenesis from H₂ and CO₂ in many methanogenic archaea. The enzyme harbors an iron-containing cofactor, in which a low-spin iron is complexed by a pyridone, two CO and a cysteine sulfur. [Fe] hydrogenase is thus similar to [NiFe] and [FeFe] hydrogenases, in which a low-spin iron carbonyl complex, albeit in a dinuclear metal center, is also involved in H₂ activation. Like the [NiFe] and [FeFe] hydrogenases, [Fe] hydrogenase catalyzes an active exchange of H₂ with protons of water; however, this activity is dependent on the presence of the hydride-accepting methenyl-H₄MPT⁺. In its absence the exchange activity is only 0.01% of that in its presence. The residual activity has been attributed to the presence of traces of methenyl-H₄MPT⁺ in the enzyme preparations, but it could also reflect a weak binding of H₂ to the iron in the absence of methenyl-H₄MPT⁺. To test this we reinvestigated the exchange activity with [Fe] hydrogenase reconstituted from apoprotein heterologously produced in Escherichia coli and highly purified iron-containing cofactor and found that in the absence of added methenyl-H₄MPT⁺ the exchange activity was below the detection limit of the tritium method employed (0.1 nmol min⁻¹ mg⁻¹). The finding reiterates that for H₂ activation by [Fe] hydrogenase the presence of the hydride-accepting methenyl-H₄MPT⁺ is essentially required. This differentiates [Fe] hydrogenase from [FeFe] and [NiFe] hydrogenases, which actively catalyze H₂/H₂O exchange in the absence of exogenous electron acceptors.     

A new mechanistic model for an O2-protected electron-bifurcating hydrogenase,Hnd from Desulfovibrio fructosovorans

The genome of the sulfate-reducing and anaerobic bacterium Desulfovibrio fructosovorans encodes different hydrogenases. Among them is Hnd, a tetrameric cytoplasmic [FeFe] hydrogenase that has previously been described as an NADP-specific enzyme (Malki et al., 1995). In this study, we purified and characterized a recombinant Strep-tagged form of Hnd and demonstrated that it is an electron-bifurcating enzyme. Flavin-based electron-bifurcation is a mechanism that couples an exergonic redox reaction to an endergonic one allowing energy conservation in anaerobic microorganisms. One of the three ferredoxins of the bacterium, that was named FdxB, was also purified and characterized. It contains a low-potential (E_m?=??450?mV) [4Fe4S] cluster. We found that Hnd was not able to reduce NADP⁺, and that it catalyzes the simultaneous reduction of FdxB and NAD⁺. Moreover, Hnd is the first electron-bifurcating hydrogenase that retains activity when purified aerobically due to formation of an inactive state of its catalytic site protecting against O₂ damage (H_inact). Hnd is highly active with the artificial redox partner (methyl viologen) and can perform the electron-bifurcation reaction to oxidize H₂ with a specific activity of 10?μmol of NADH/min/mg of enzyme. Surprisingly, the ratio between NADH and reduced FdxB varies over the reaction with a decreasing amount of FdxB reduced per NADH produced, indicating a more complex mechanism than previously described. We proposed a new mechanistic model in which the ferredoxin is recycled at the hydrogenase catalytic subunit.     

Characterization of the hydrogen-deuterium exchange activities of the energy-transducing HupSL hydrogenase and H(2)-signaling HupUV hydrogenase in Rhodobacter capsulatus

Rhodobacter capsulatus synthesizes two homologous protein complexes capable of activating molecular H(2), a membrane-bound [NiFe] hydrogenase (HupSL) linked to the respiratory chain, and an H(2) sensor encoded by the hupUV genes. The activities of hydrogen-deuterium (H-D) exchange catalyzed by the hupSL-encoded and the hupUV-encoded enzymes in the presence of D(2) and H(2)O were studied comparatively. Whereas HupSL is in the membranes, HupUV activity was localized in the soluble cytoplasmic fraction. Since the hydrogenase gene cluster of R. capsulatus contains a gene homologous to hoxH, which encodes the large subunit of NAD-linked tetrameric soluble hydrogenases, the chromosomal hoxH gene was inactivated and hoxH mutants were used to demonstrate the H-D exchange activity of the cytoplasmic HupUV protein complex. The H-D exchange reaction catalyzed by HupSL hydrogenase was maximal at pH 4. 5 and inhibited by acetylene and oxygen, whereas the H-D exchange catalyzed by the HupUV protein complex was insensitive to acetylene and oxygen and did not vary significantly between pH 4 and pH 11. Based on these properties, the product of the accessory hypD gene was shown to be necessary for the synthesis of active HupUV enzyme. The kinetics of HD and H(2) formed in exchange with D(2) by HupUV point to a restricted access of protons and gasses to the active site. Measurement of concentration changes in D(2), HD, and H(2) by mass spectrometry showed that, besides the H-D exchange reaction, HupUV oxidized H(2) with benzyl viologen, produced H(2) with reduced methyl viologen, and demonstrated true hydrogenase activity. Therefore, not only with respect to its H(2) signaling function in the cell, but also to its catalytic properties, the HupUV enzyme represents a distinct class of hydrogenases.     

Aerobically purified hydrogenase from Azotobacter vinelandii: activity, activation, and spectral properties

The hydrogenase from Azotobacter vinelandii is typically purified under anaerobic conditions. In this work, the hydrogenase was purified aerobically. The yields were low (about 2%) relative to those of the anaerobic purification (about 20%). The rate of enzyme activity depended upon the history of the enzyme. The enzyme preparations were active as isolated in H2 oxidation, and isotope exchange. The activity increased during the assay to a new maximal level (turnover activation). Treatment with reductants (e.g., H2, dithionite, dithiothreitol, indigo carmine) resulted in greater activation (reductant activation). Activation of the hydrogenase was accompanied by decrease in visible light absorption (300-600 nm) with maximal decreases at 450 and 345 nm which indicated the reduction of iron-sulfur clusters. The aerobically purified hydrogenase was susceptible to irreversible inactivation by cyanide. Pretreatment with acetylene did not influence activation of the hydrogenase. Once activated, the aerobically purified hydrogenase was indistinguishable from the anaerobically purified hydrogenase with respect to the catalytic properties tested.     

Interaction of the active site of the Ni–Fe–Se hydrogenase from Desulfovibrio vulgaris Hildenborough with carbon monoxide and oxygen inhibitors

The study of Ni–Fe–Se hydrogenases is interesting from the basic research point of view because their active site is a clear example of how nature regulates the catalytic function of an enzyme by the change of a single residue, in this case a cysteine, which is replaced by a selenocysteine. Most hydrogenases are inhibited by CO and O₂. In this work we studied these inhibition processes for the Ni–Fe–Se hydrogenase from Desulfovibrio vulgaris Hildenborough by combining catalytic activity measurements, followed by mass spectrometry or chronoamperometry, with Fourier transform IR spectroscopy experiments. The results show that the CO inhibitor binds to Ni in both conformations of the active site of this hydrogenase in a way similar to that in standard Ni–Fe hydrogenases, although in one of the CO-inhibited conformations the active site of the Ni–Fe–Se hydrogenase is more protected against the attack by O₂. The inhibition of the Ni–Fe–Se hydrogenase activity by O₂ could be explained by oxidation of the terminal cysteine ligand of the active-site Ni, instead of the direct attack of O₂ on the bridging site between Ni and Fe.     

Partial purification and characterization of the first hydrogenase isolated from a thermophilic sulfate-reducing bacterium.

A soluble [NiFe] hydrogenase has been partially purified from the obligate thermophilic sulfate-reducing bacterium Thermodesulfobacterium mobile. A 17% purification yield was obtained after four chromatographic steps and the hydrogenase presents a purity index (A398 nm/A277 nm) equal to 0.21. This protein appears to be 75% pure on SDS-gel electrophoresis showing two major bands of molecular mass around 55 and 15 kDa. This hydrogenase contains 0.6-0.7 nickel atom and 7-8 iron atoms per mole of enzyme and has a specific activity of 783 in the hydrogen uptake reaction, of 231 in the hydrogen production assay and of 84 in the deuterium-proton exchange reaction. The H2/HD ratio is lower than one in the D2-H+ exchange reaction. The enzyme is very sensitive to NO, relatively little inhibited by CO but unaffected by NO2-. The EPR spectrum of the native hydrogenase shows the presence of a [3Fe-4S] oxidized cluster and of a Ni(III) species.     

Determination of Hydrogenase in Free-living Cultures of Rhizobium japonicum and Energy Efficiency of Soybean Nodules

A sensitive tritium exchange assay was applied to the Rhizobium system for measuring the expression of uptake hydrogenase in free-living cultures of Rhizobium japonicum. Hydrogenase was detected about 45 hours after inoculation of cultures maintained under microaerophilic conditions (about 0.1% O₂). The tritium exchange assay was used to screen a variety of different strains of R. japonicum (including major production strains) with the findings that about 30% of the strains expressed hydrogenase activity with identical results being observed using an alternative assay based on uptake of H₂. The relative efficiency of intact soybean nodules inoculated with 10 different rhizobial strains gave results identical to those obtained using free-living cultures. The tritium exchange assay provides an easy, quick, and accurate assessment of H₂ uptake efficiency of intact nodules.     

CO-dependent H2 evolution by Rhodospirillum rubrum: Role of CODH:CooF complex

Upon exposure to CO during anaerobic growth, the purple phototrophic bacterium Rhodospirillum rubrum expresses a CO-oxidizing H₂ evolving enzymatic system. The CO-oxidizing enzyme, carbon monoxide dehydrogenase (CODH), has been purified and extensively characterized. However the electron transfer pathway from CODH to the CO-induced hydrogenase that evolves H₂ is not well understood. CooF is an Fe-S protein that is the proposed mediator of electron transfer between CODH and the CO-induced hydrogenase. Here we present the spectroscopic and biochemical properties of the CODH:CooF complex. The characteristic EPR signals observed for CODH are largely insensitive to CooF complexation. Metal analysis and EPR spectroscopy show that CooF contains 2 Fe₄S₄ clusters. The observation of 2 Fe₄S₄ clusters for CooF contradicts the prediction of 4 Fe₄S₄ clusters based on analysis of the amino acid sequence of CooF and structural studies of CooF homologs. Comparison of in vivo and in vitro CO-dependent H₂ evolution indicates that ∼ 90% of the activity is lost upon cell lysis. We propose that the loss of two labile Fe-S clusters from CooF during cell lysis may be responsible for the low in vitro CO-dependent H₂ evolution activity. During the course of these studies, a new assay for CODH:CooF was developed using membranes from an R. rubrum mutant that did not express CODH:CooF, but expressed high levels of the CO-induced hydrogenase. The assay revealed that the CO-induced hydrogenase requires the presence of CODH:CooF for optimal H₂ evolution activity.     

Physiological reactions of the reversible hydrogenase from anabaena 7120

The reversible hydrogenase from Anabaena 7120 appeared when O₂ was continuously removed from a growing culture. Activity increased further when cells were incubated under argon in the dark or in the light plus 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Hydrogenase existed in an inactive state during periods of O₂ evolution. It could be reductively activated by exposure to reduced methyl viologen or by dark, anaerobic incubation. Hydrogenase-containing cells evolved H₂ slowly during dark anaerobic incubations, and the rate of H₂ evolution was increased by illumination with low intensity light. Light enhancement of H₂ evolution was of short duration and was eliminated by the ferredoxin antagonist disalicylidene diaminopropane. Physiological acceptors that supported H₂ uptake included NO₃⁻, NO₂⁻, and HSO₃⁻, and light had a slight influence on the rate of H₂ uptake with these acceptors. Low levels of O₂ supported H₂ uptake, but higher concentrations of O₂ inactivated the hydrogenase. Hydrogen uptake with HCO₃⁻ as acceptor was the most rapid reaction measured, and it was strictly light-dependent. It occurred only at low light intensities, and higher light intensities restored normal O₂-evolving photosynthesis. It is suggested that hydrogenase is present to capture exogenous H₂ as a source of reducing equivalents during growth in anaerobic environments.     

Identification of a membrane-bound hydrogenase of Desulfovibrio vulgaris (Hildenborough)

Two hydrogenase activities from Desulfovibrio vulgaris (Hildenborough) could be distinguished immunologically and biochemically. The first activity, described as hydrogenase I, corresponded to the soluble enzyme located in the periplasmic space of D. vulgaris. Hydrogenase I had a high specific activity and was sensitive to inhibition by CO. The second activity, hydrogenase II, was located in the membrane fraction, had a lower specific activity and was not affected by CO. The enzymes exhibited different electrophoretic mobilities in polyacrylamide gels, and reacted differently when exposed to proteases. Antibodies raised against purified periplasmic hydrogenase of D. vulgaris reacted with hydrogenase I, but not with hydrogenase II.