Influence of the protein structure surrounding the active site on the catalytic activity of [NiFeSe] hydrogenases

A combined experimental and theoretical study of the catalytic activity of a [NiFeSe] hydrogenase has been performed by H/D exchange mass spectrometry and molecular dynamics simulations. Hydrogenases are enzymes that catalyze the heterolytic cleavage or production of H₂. The [NiFeSe] hydrogenases belong to a subgroup of the [NiFe] enzymes in which a selenocysteine is a ligand of the nickel atom in the active site instead of cysteine. The aim of this research is to determine how much the specific catalytic properties of this hydrogenase are influenced by the replacement of a sulfur by selenium in the coordination of the bimetallic active site versus the changes in the protein structure surrounding the active site. The pH dependence of the D₂/H⁺ exchange activity and the high isotope effect observed in the Michaelis constant for the dihydrogen substrate and in the single exchange/double exchange ratio suggest that a “cage effect” due to the protein structure surrounding the active site is modulating the enzymatic catalysis. This “cage effect” is supported by molecular dynamics simulations of the diffusion of H₂ and D₂ from the outside to the inside of the protein, which show different accumulation of these substrates in a cavity next to the active site.     

Application of immobilized hydrogenase for the detritiation of water

Detritiation of contaminated water is an essential part of nuclear power production. Most promising methods used for this process are based on catalyzed hydrogen isotope exchange reactions. It is proposed herein to replace the platinum catalysts which are currently used in industry with immobilized hydrogenase. Whole bacterial cells of Alcaligenes eutrophus immobilized in calcium alginate or κ-carrageenan gels were found to be efficient catalysts of the reaction of hydrogen–tritium (H–T) exchange in both a batch tank reactor and in a column. The dependence of the reaction rate on the amount of immobilized cells in the system, and on the concentration of the cells in the matrix, indicate that enzymatic H–T exchange is not controlled by diffusion. Immobilized A. eutrophus cells are enzymatically active over a wide range of pH, with a broad maximum from pH 6.0 to 8.0, and are quite resistant to inhibitors of hydrogenases such as O₂ and CO. Upon increasing the temperature from 4 to 37°C, the rate of hydrogenase-catalyzed H–T exchange increases by a factor of 5. From the standpoint of catalytic efficiency, 1 g of PtO₂ is approximately equivalent to 10 g of cells (wet weight). In contrast of platinum-based catalysts, bacterial hydrogenases (1) are potentially inexpensive; (2) can be readily available in bulk quantities; (3) are maximally active in liquid water.     

Quantitative analysis of enzymatic fractionation of multiple substrate mixtures

The enzymatic conversion of mixtures of multiple substrates was studied quantitatively, based on established methodology used for the enzymatic kinetic resolution of racemic mixtures, involving the use of competitive factors: ratios of specificity constants (k_cat/K_M) of substrate pairs. The competitive factors of the substrates were defined in relation to a reference substrate. These competitive factors were used to predict the composition of the reaction mixture as a function of the degree of conversion of the reaction. The methodology was evaluated using three different lipases to hydrolyze a model mixture of four fatty acid methyl esters and for the esterification of a mixture of the same fatty acids in free form with ethanol. In most cases, the competitive factors determined from the initial phase of the reactions predicted the product composition during the rest of the reaction very well. The slowest reacting fatty acid was erucic acid (both in free form and as methyl ester), which was thus enriched in the remaining substrate fraction, while the other fatty acids: lauric acid, palmitic acid and oleic acid were converted faster. Simulations of the compositions of reaction mixtures with different values of the competitive factors were carried out to provide an overview of what could be achieved using enzymatic enrichment. Possible applications include reactions involving homologous substrates and mixtures of multiple isomers. The analysis presented provides guidelines that can be useful in the screening and development of enzymes for enzymatic enrichment applications. Biotechnol. Bioeng. 2013; 110: 78–86. © 2012 Wiley Periodicals, Inc.     

C-11 H-abstraction from linoleic acid, the rate-limiting step in lipoxygenase catalysis

[11,11-²H₂]-, [9,10,12,13-²H₄]-, [9,10,11,11,12,13-²H₆]- and unlabelled linoleic acids were incubated with pure lipoxygenase-1 from soya beans. The apparent rate constants of the overall reactions and the apparent Michaelis constants in air-equilibrated solutions at 25°C and pH 9.0 were obtained from Lineweaver-Burk plots. The apparent K_m-values were hardly affected by the type of substrate used. Substrates bearing ²H instead of ¹H at C-11 gave rise to considerable isotope effects, k_H/k_2H values being 8.7 and 9.3 for dideutero- and hexadeutero linoleate, respectively. From the observed isotope effects it was concluded, that H-abstraction from C-11 is the rate-determining step in the overall reaction. All substrates used gave identical product distributions. No measurable exchange of deuterium with solvent hydrogen occurred during oxygenation.     

Structural features of [NiFeSe] and [NiFe] hydrogenases determining their different properties: a computational approach

Hydrogenases are metalloenzymes that catalyze the reversible reaction \textH₂ \leftrightarrows 2\textH ⁺ + 2\texte ^- {\text{H}}_{2} \leftrightarrows 2{\text{H}}^{ + } + 2{\text{e}}^{ - } , being potentially useful in H₂ production or oxidation. [NiFeSe] hydrogenases are a particularly interesting subgroup of the [NiFe] class that exhibit tolerance to O₂ inhibition and produce more H₂ than standard [NiFe] hydrogenases. However, the molecular determinants responsible for these properties remain unknown. Hydrophobic pathways for H₂ diffusion have been identified in [NiFe] hydrogenases, as have proton transfer pathways, but they have never been studied in [NiFeSe] hydrogenases. Our aim was, for the first time, to characterize the H₂ and proton pathways in a [NiFeSe] hydrogenase and compare them with those in a standard [NiFe] hydrogenase. We performed molecular dynamics simulations of H₂ diffusion in the [NiFeSe] hydrogenase from Desulfomicrobium baculatum and extended previous simulations of the [NiFe] hydrogenase from Desulfovibrio gigas (Teixeira et al. in Biophys J 91:2035–2045, 2006). The comparison showed that H₂ density near the active site is much higher in [NiFeSe] hydrogenase, which appears to have an alternative route for the access of H₂ to the active site. We have also determined a possible proton transfer pathway in the [NiFeSe] hydrogenase from D. baculatum using continuum electrostatics and Monte Carlo simulation and compared it with the proton pathway we found in the [NiFe] hydrogenase from D. gigas (Teixeira et al. in Proteins 70:1010–1022, 2008). The residues constituting both proton transfer pathways are considerably different, although in the same region of the protein. These results support the hypothesis that some of the special properties of [NiFeSe] hydrogenases could be related to differences in the H₂ and proton pathways.     

A proton-deuterium exchange study of three types ofDesulfovibrio hydrogenases

Summary Hydrogenases are among the main enzymes involved in bacterial anaerobic corrosion of metals. The study of their mode of action is important for a full comprehension of this phenomenon. The three types ofDesulfovibrio hydrogenases [(Fe), (NiFe), (NiFeSe)] present different patterns in the pH dependence of their activity. The periplasmic enzyme fromDesulfovibrio salexigens and the cytoplasmic enzyme fromDesulfovibrio baculatus both have pH optima at 7.5 for H₂ uptake and 4.0 for H₂ evolution and H⁺–D₂ exchange reaction (measured by membrane-inlet mass-spectrometry). The H₂ to HD ratio at pH above 5.0 is higher than 1.0. The periplasmic hydrogenase fromD. gigas presents the same pH optimum (8.0) for the H⁺–D₂ exchange as for H₂ consumption. In contrast, the enzyme fromD. vulgaris has the highest activity in H₂ production and in the exchange at pH 5.0. Both hydrogenases have a H₂-to-HD ratio below 1.0.     

The effect of sulfur compounds on H<Subscript>2</Subscript> evolution/consumption reactions,mediated by various hydrogenases,in the purple sulfur bacterium, <Emphasis Type="Italic">Thiocapsa roseopersicina</Emphasis>

The influence of reduced sulfur compounds (including stored S⁰) on H₂ evolution/consumption reactions in the purple sulfur bacterium, Thiocapsa roseopersicina BBS, was studied using mutants containing only one of the three known [NiFe] hydrogenase enzymes: Hox, Hup or Hyn. The observed effects depended on the kind of hydrogenase involved. The mutant harbouring Hox hydrogenase was able to use S₂O₃²⁻, SO₃²⁻, S²⁻ and S⁰ as electron donors for light-dependent H₂ production. Dark H₂ evolution from organic substrates via Hox hydrogenase was inhibited by S⁰. Under light conditions, endogenous H₂ uptake by Hox or Hup hydrogenases was suppressed by S compounds. СО₂-dependent H₂ uptake by Hox hydrogenase in the light required the additional presence of S compounds, unlike the Hup-mediated process. Dark H₂ consumption via Hyn hydrogenase was connected to utilization of S⁰ as an electron acceptor and resulted in the accumulation of H₂S. In wild type BBS, with high levels of stored S⁰, dark H₂ production from organic substrates was significantly lower, but H₂S accumulation significantly higher, than in the mutant GB1121(Hox⁺). There is a possibility that H₂ produced via Hox hydrogenase is consumed by Hyn hydrogenase to reduce S⁰.     

Kinetic parameters for hydrogen evolution by the NAD-linked hydrogenase of Alcaligenes eutrophus

The hydrogen-evolving reaction of the purified soluble NAD-linked hydrogenase of Alcaligenes eutrophus was used to determine kinetic parameters of the enzyme. The H₂-evolving activity with methyl viologen as electron mediator was 20-fold as compared to that with NADH. In the assay with dithionite-reduced methyl viologen (K _m 0.7 mM) the hydrogenase was most active at a redox potential of –560 mV and exhibited a pH optimum of 7.0. The K _m for protons, the second substrate for H₂ evolution, was 6.2 nM. With electrochemically reduced methyl viologen the pH optimum was shifted to pH 6.0. Double-reciprocal plots of reaction rates versus proton concentrations intercepted at the ordinate for different methyl viologen concentrations. At different pH values such an intercept was also observed with the dye as the varied substrate. The kinetic data are diagnostic for an ordered bisubstrate mechanism where both substrates are bound before the product H₂ is released. Hydrogenase coupled to thylakoid membranes resulted in a constant H₂ evolution rate over 6 h. The system appeared to be limited by the capacity of the thylakoid membranes.     

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.     

Ascorbate accumulation during sulphur deprivation and its effects on photosystem II activity and H2 production of the green alga Chlamydomonas reinhardtii

In nature, H₂ production in Chlamydomonas reinhardtii serves as a safety valve during the induction of photosynthesis in anoxia, and it prevents the over‐reduction of the photosynthetic electron transport chain. Sulphur deprivation of C. reinhardtii also triggers a complex metabolic response resulting in the induction of various stress‐related genes, down‐regulation of photosynthesis, the establishment of anaerobiosis and expression of active hydrogenase. Photosystem II (PSII) plays dual role in H₂ production because it supplies electrons but the evolved O₂ inhibits the hydrogenase. Here, we show that upon sulphur deprivation, the ascorbate content in C. reinhardtii increases about 50‐fold, reaching the mM range; at this concentration, ascorbate inactivates the Mn‐cluster of PSII, and afterwards, it can donate electrons to tyrozin Z⁺ at a slow rate. This stage is followed by donor‐side‐induced photoinhibition, leading to the loss of charge separation activity in PSII and reaction centre degradation. The time point at which maximum ascorbate concentration is reached in the cell is critical for the establishment of anaerobiosis and initiation of H₂ production. We also show that ascorbate influenced H₂ evolution via altering the photosynthetic electron transport rather than hydrogenase activity and starch degradation.     

Compartmentalisation of [FeFe]‐hydrogenase maturation in Chlamydomonas reinhardtii

Molecular hydrogen (H₂) can be produced in green microalgae by [FeFe]‐hydrogenases as a direct product of photosynthesis. The Chlamydomonas reinhardtii hydrogenase HYDA1 contains a catalytic site comprising a classic [4Fe4S] cluster linked to a unique 2Fe sub‐cluster. From in vitro studies it appears that the [4Fe4S] cluster is incorporated first by the housekeeping FeS cluster assembly machinery, followed by the 2Fe sub‐cluster, whose biosynthesis requires the specific maturases HYDEF and HYDG. To investigate the maturation process in vivo, we expressed HYDA1 from the C. reinhardtii chloroplast and nuclear genomes (with and without a chloroplast transit peptide) in a hydrogenase‐deficient mutant strain, and examined the cellular enzymatic hydrogenase activity, as well as in vivo H₂ production. The transformants expressing HYDA1 from the chloroplast genome displayed levels of H₂ production comparable to the wild type, as did the transformants expressing full‐length HYDA1 from the nuclear genome. In contrast, cells equipped with cytoplasm‐targeted HYDA1 produced inactive enzyme, which could only be activated in vitro after reconstitution of the [4Fe4S] cluster. This indicates that the HYDA1 FeS cluster can only be built by the chloroplastic FeS cluster assembly machinery. Further, the expression of a bacterial hydrogenase gene, CPI, from the C. reinhardtii chloroplast genome resulted in H₂‐producing strains, demonstrating that a hydrogenase with a very different structure can fulfil the role of HYDA1 in vivo and that overexpression of foreign hydrogenases in C. reinhardtii is possible. All chloroplast transformants were stable and no toxic effects were seen from HYDA1 or CPI expression.     

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.     

Solid/gas biocatalysis: an appropriate tool to study the influence of organic components on kinetics of lipase-catalyzed alcoholysis

The influence of the addition of an extra component in a gaseous reaction medium, on the kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by immobilized lipase B from Candida antarctica was studied in a continuous solid/gas reactor. In this reactor, the solid phase is composed of a packed enzymatic sample, which is percolated by gaseous nitrogen, simultaneously carrying gaseous substrates and additional components to the enzyme while removing reaction products. The system permits to set thermodynamic activity of all gaseous components (substrates or not) independently at the desired values. This allows in particular to study the influence of an extra added component at a constant thermodynamic activity value, contrary to classical solid/liquid system, which involves large variations of thermodynamic activity of added solvent, when performing full kinetic studies. Alcohol inhibition constant (K_I) and methyl propionate and propanol dissociation constants (K_MP and K_P) have been determined in the solid/gas reactor in the presence of 2-methyl-2-butanol, and compared with values previously obtained in the absence of added component and in the presence of water. Complementary experiments were carried out in the presence of an apolar compound (hexane) and led to the conclusion that the effect of added organic component on lipase-catalyzed alcoholysis is related to their competitive inhibitory character towards first substrate methyl propionate. The comparison of data obtained in liquid or with gaseous 2-methyl-2-butanol shows that lower K_MP and K_I are found in gaseous medium, which would correspond on the one hand to a lower acylation rate k₂, and on the other hand to a higher binding rate k₁ between substrate and free enzyme in gaseous medium.     

A proteomic view of the facultatively chemolithoautotrophic lifestyle of Ralstonia eutropha H16

Ralstonia eutropha H16 is an H₂‐oxidizing, facultative chemolithoautotroph. Using 2‐DE in conjunction with peptide mass spectrometry we have cataloged the soluble proteins of this bacterium during growth on different substrates: (i) H₂ and CO₂, (ii) succinate and (iii) glycerol. The first and second conditions represent purely lithoautotrophic and purely organoheterotrophic nutrition, respectively. The third growth regime permits formation of the H₂‐oxidizing and CO₂‐fixing systems concomitant to utilization of an organic substrate, thus enabling mixotrophic growth. The latter type of nutrition is probably the relevant one with respect to the situation faced by the organism in its natural habitats, i.e. soil and mud. Aside from the hydrogenase and Calvin‐cycle enzymes, the protein inventories of the H₂‐CO₂‐ and succinate‐grown cells did not reveal major qualitative differences. The protein complement of the glycerol‐grown cells resembled that of the lithoautotrophic cells. Phosphoenolpyruvate (PEP) carboxykinase was present under all three growth conditions, whereas PEP carboxylase was not detectable, supporting earlier findings that PEP carboxykinase is alone responsible for the anaplerotic production of oxaloacetate from PEP. The elevated levels of oxidative stress proteins in the glycerol‐grown cells point to a significant challenge by ROS under these conditions. The results reported here are in agreement with earlier physiological and enzymological studies indicating that R. eutropha H16 has a heterotrophic core metabolism onto which the functions of lithoautotrophy have been grafted.     

Conversion of D-xylose into xylitol by xylose reductase from Candida pelliculosa coupled with the oxidoreductase system of methanogen strain HU

Summary A preliminary test for the enzymatic conversion of D-xylose into xylitol by the intact cells of Candida pelliculosa (xylose reductase) coupled with the intact cells of a formate-utilizing methanogen strain HU (hydrogenase and F₄₂₀-NADP oxidoreductase) was conducted by using H₂ as an electron donor of NADP⁺. In the system, NADP(H) was well regenerated via the methanogen cells and about 90% conversion of xylose to xylitol (ca. 8 g/l) could be achieved at 35°C and pH 7.5 after 24 h incubation.     

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.     

The utilization of molecular hydrogen by the blue-green alga Anabaena cylindrica

The blue-green alga Anabaena cylindrica is found to consume molecular hydrogen in a hydrogenase dependent reaction. This hydrogen uptake proceeds in the dark and is strictly dependent on oxygen, thus representing a Knallgas reactions. Its rate is almost as high as that of the endogenous respiration in Anabaena. Studies with inhibitors reveal that hydrogen is utilized via the complete respiratory chain providing additional energy for the alga. CO plus C₂H₂ completely block the Knallgas reaction which explains the previously reported considerable increase in the total H₂ formation representing the difference between the nitrogenase-dependent H₂-evolution and the reutilization of the gas catalysed by the hydrogenase in intact Anabaena.H₂ is able to support the C₂H₂-reduction in the dark in a reaction again strictly dependent on oxygen. Moreover, H₂ is also consumed in experiments carried out under far red light and in the presence of dichlorophenyl-dimenthyl-urea (DCMU) where the energy for nitrogen fixation is no longer provided by respiration but by cyclic photophosphorylation. Under these conditions, H₂ is found to supply electrons for the formation of C₂H₄ from C₂H₂ in a reaction no longer dependent on the presence of oxygen. Moreover, in these experiments, the presence of H₂ stabilizes the C₂H₂-reduction activity against the deleterious effect of oxygen.Thus, this communication provides evidence for a triplicate function of the H₂-uptake catalysed by hydrogenase in intact Anabaena which is (a) to provide energy by the Knallgas reaction, (b) to supply reducing equivalents for nitrogenase, (c) to protect nitrogenase from damage by oxygen.Abbreviations DCMU N-(3,4-dichlorophenyl)N,N-dimethylurea - DNP 2-4-dinitrophenol - FCCP carbonylcyanid-p-trifluormethoxyphenyl-hydrazone(=p-CF₃-CCP) - Chl chlorophyll     

Properties of the hydrogenase system in Rhizobium japonicum bacteroids

The hydrogenase system which catalyzes the oxyhydrogen reaction in soybean nodules produced by strains of $\text{Rhizobium japonicum}$ is located in the bacteroids. The hydrogenase complex in intact bacteroids has an apparent K_m for H₂ of 2.8 μM and an apparent K_m for O₂ of 1.3 μM. The addition of hydrogen to bacteroids increases oxygen uptake but decreases respiratory CO₂ production, indicating a conservation of endogenous substrates. After correction for the effect of hydrogen on endogenous respiration a ratio of 1.9 ± 0.1 for H₂ to O₂ uptake was determined. Bacteroids from greenhouse or field-grown soybeans that evolved hydrogen showed no measurable oxyhydrogen reaction activity whereas consistent activity was demonstrated by bacteroids from soybean nodules that evolved little or no H₂.