Homologous expression of a subcomplex of Pyrococcus furiosus hydrogenase that interacts with pyruvate ferredoxin oxidoreductase

Hydrogen gas is an attractive alternative fuel as it is carbon neutral and has higher energy content per unit mass than fossil fuels. The biological enzyme responsible for utilizing molecular hydrogen is hydrogenase, a heteromeric metalloenzyme requiring a complex maturation process to assemble its O(2)-sensitive dinuclear-catalytic site containing nickel and iron atoms. To facilitate their utility in applied processes, it is essential that tools are available to engineer hydrogenases to tailor catalytic activity and electron carrier specificity, and decrease oxygen sensitivity using standard molecular biology techniques. As a model system we are using hydrogen-producing Pyrococcus furiosus, which grows optimally at 100°C. We have taken advantage of a recently developed genetic system that allows markerless chromosomal integrations via homologous recombination. We have combined a new gene marker system with a highly-expressed constitutive promoter to enable high-level homologous expression of an engineered form of the cytoplasmic NADP-dependent hydrogenase (SHI) of P. furiosus. In a step towards obtaining 'minimal' hydrogenases, we have successfully produced the heterodimeric form of SHI that contains only two of the four subunits found in the native heterotetrameric enzyme. The heterodimeric form is highly active (150 units mg(-1) in H(2) production using the artificial electron donor methyl viologen) and thermostable (t(1/2) ～0.5 hour at 90°C). Moreover, the heterodimer does not use NADPH and instead can directly utilize reductant supplied by pyruvate ferredoxin oxidoreductase from P. furiosus. The SHI heterodimer and POR therefore represent a two-enzyme system that oxidizes pyruvate and produces H(2) in vitro without the need for an intermediate electron carrier.     

Construction and physiological studies of hydrogenase depleted mutants of Desulfovibrio fructosovorans

Desulfovibrio fructosovorans possesses two periplasmic hydrogenases (a nickel-iron and an iron hydrogenase) and a cytoplasmic NADP-dependent hydrogenase. The hydAB genes encoding the periplasmic iron hydrogenase were replaced, in the wild-type strain as well as in single mutants depleted of one of the other two hydrogenases, by the acc1 gene encoding resistance to gentamycin. Molecular characterization and remaining activity measurements of the resulting single and double mutants were performed. All mutated strains exhibited similar growth when H(2) was the electron donor but they grew differently on fructose, lactate or pyruvate as electron donors. Our results indicate that the loss of one enzyme might be compensated by another even though hydrogenases have different localization in the cells.     

Characterization of hydrogenase II from the hyperthermophilic archaeon Pyrococcus furiosus and assessment of its role in sulfur reduction

The fermentative hyperthermophile Pyrococcus furiosus contains an NADPH-utilizing, heterotetrameric (alphabetagammadelta), cytoplasmic hydrogenase (hydrogenase I) that catalyzes both H(2) production and the reduction of elemental sulfur to H(2)S. Herein is described the purification of a second enzyme of this type, hydrogenase II, from the same organism. Hydrogenase II has an M(r) of 320,000 +/- 20,000 and contains four different subunits with M(r)s of 52,000 (alpha), 39,000 (beta), 30,000 (gamma), and 24,000 (delta). The heterotetramer contained Ni (0.9 +/- 0.1 atom/mol), Fe (21 +/- 1.6 atoms/mol), and flavin adenine dinucleotide (FAD) (0.83 +/- 0.1 mol/mol). NADPH and NADH were equally efficient as electron donors for H(2) production with K(m) values near 70 microM and k(cat)/K(m) values near 350 min(-1) mM(-1). In contrast to hydrogenase I, hydrogenase II catalyzed the H(2)-dependent reduction of NAD (K(m), 128 microM; k(cat)/K(m), 770 min(-1) mM(-1)). Ferredoxin from P. furiosus was not an efficient electron carrier for either enzyme. Both H(2) and NADPH served as electron donors for the reduction of elemental sulfur (S(0)) and polysulfide by hydrogenase I and hydrogenase II, and both enzymes preferentially reduce polysulfide to sulfide rather than protons to H(2) using NADPH as the electron donor. At least two [4Fe-4S] and one [2Fe-2S] cluster were detected in hydrogenase II by electron paramagnetic resonance spectroscopy, but amino acid sequence analyses indicated a total of five [4Fe-4S] clusters (two in the beta subunit and three in the delta subunit) and one [2Fe-2S] cluster (in the gamma subunit), as well as two putative nucleotide-binding sites in the gamma subunit which are thought to bind FAD and NAD(P)(H). The amino acid sequences of the four subunits of hydrogenase II showed between 55 and 63% similarity to those of hydrogenase I. The two enzymes are present in the cytoplasm at approximately the same concentration. Hydrogenase II may become physiologically relevant at low S(0) concentrations since it has a higher affinity than hydrogenase I for both S(0) and polysulfide.     

A new function of the Desulfovibrio vulgaris Hildenborough [Fe] hydrogenase in the protection against oxidative stress

Sulfate-reducing bacteria, like Desulfovibrio vulgaris Hildenborough, have developed a set of reactions allowing them to survive in oxic environments and even to reduce molecular oxygen to water. D. vulgaris contains a cytoplasmic superoxide reductase (SOR) and a periplasmic superoxide dismutase (SOD) involved in the elimination of superoxide anions. To assign the function of SOD, the periplasmic [Fe] hydrogenase activity was followed in both wild-type and sod deletant strains. This activity was lower in the strain lacking the SOD than in the wild-type when the cells were exposed to oxygen for a short time. The periplasmic SOD is thus involved in the protection of sensitive iron-sulfur-containing enzyme against superoxide-induced damages. Surprisingly, production of the periplasmic [Fe] hydrogenase was higher in the cells exposed to oxygen than in those kept in anaerobic conditions. A similar increase in the amount of [Fe] hydrogenase was observed when an increase in the redox potential was induced by addition of chromate. Viability of the strain lacking the gene encoding [Fe] hydrogenase after exposure to oxygen for 1 h was lower than that of the wild-type. These data reveal for the first time that production of the periplasmic [Fe] hydrogenase is up-regulated in response to an oxidative stress. A new function of the periplasmic [Fe] hydrogenase in the protective mechanisms of D. vulgaris Hildenborough toward an oxidative stress is proposed.     

Development of a cell-free system reveals an oxygen-labile step in the maturation of [NiFe]-hydrogenase 2 of Escherichia coli

By combining extracts from strains lacking genes encoding either the maturation enzymes or the large subunits of hydrogenases 1, 2 and 3 we could reconstitute in vitro under strictly anaerobic conditions 10-15% of the hydrogenase activity present in wild type Escherichia coli extracts. Purified, unprocessed Strep-tagged variants of the hydrogenase 2 large subunit, HybC, isolated from either a ΔhybD (encoding the hydrogenase 2-specific protease) mutant or a strain deficient in HypF could also be matured to active, processed enzyme using this system. These studies reveal that minimally one step early on the hydrogenase maturation pathway is oxygen-labile.     

Characterization of hydrogenase from the hyperthermophilic archaebacterium, Pyrococcus furiosus

The archaebacterium, Pyrococcus furiosus, grows optimally at 100 degrees C by a fermentative type metabolism in which H2 and CO2 are the only detectable products. The organism also reduces elemental sulfur (S0) to H2S. Cells grown in the absence of S0 contain a single hydrogenase, located in the cytoplasm, which has been purified 350-fold to apparent homogeneity. The yield of H2 evolution activity from reduced methyl viologen at 80 degrees C was 40%. The hydrogenase has a Mr value of 185,000 +/- 15,000 and is composed of three subunits of Mr 46,000 (alpha), 27,000 (beta), and 24,000 (gamma). The enzyme contains 31 +/- 3 g atoms of iron, 24 +/- 4 g atoms of acid-labile sulfide, and 0.98 +/- 0.05 g atoms of nickel/185,000 g of protein. The H2-reduced hydrogenase exhibits an electron paramagnetic resonance (EPR) signal at 70 K typical of a single [2Fe-2S] cluster, while below 15 K, EPR absorption is observed from extremely fast relaxing iron-sulfur clusters. The oxidized enzyme is EPR silent. The hydrogenase is reversibly inhibited by O2 and is remarkably thermostable. Most of its H2 evolution activity is retained after a 1-h incubation at 100 degrees C. Reduced ferredoxin from P. furiosus also acts as an electron donor to the enzyme, and a 350-fold increase in the rate of H2 evolution is observed between 45 and 90 degrees C. The hydrogenase also catalyzes H2 oxidation with methyl viologen or methylene blue as the electron acceptor. The temperature optimum for both H2 oxidation and H2 evolution is greater than 95 degrees C. Arrhenius plots show two transition points at approximately 60 and approximately 80 degrees C independent of the mode of assay. That occurring at 80 degrees C is associated with a dramatic increase in H2 production activity. The enzyme preferentially catalyzes H2 production at all temperatures examined and appears to represent a new type of "evolution" hydrogenase.     

Requirement of nickel metabolism proteins HypA and HypB for full activity of both hydrogenase and urease in Helicobacter pylori

The nickel-containing enzymes hydrogenase and urease require accessory proteins in order to incorporate properly the nickel atom(s) into the active sites. The Helicobacter pylori genome contains the full complement of both urease and hydrogenase accessory proteins. Two of these, the hydrogenase accessory proteins HypA (encoded by hypA) and HypB (encoded by hypB), are required for the full activity of both the hydrogenase and the urease enzymes in H. pylori. Under normal growth conditions, hydrogenase activity is abolished in strains in which either hypA (HypA:kan) or hypB (HypB:kan) have been interrupted by a kanamycin resistance cassette. Urease activity in these strains is 40 (HypA:kan)- and 200 (HypB:kan)-fold lower than for the wild-type (wt) strain 43504. Nickel supplementation in the growth media restored urease activity to almost wt levels. Hydrogenase activity was restored to a lesser extent, as has been observed for hyp mutants in other (H(2)-oxidizing) bacteria. Expression levels of UreB (the urease large subunit) were not affected by inactivation of either hypA or hypB, as determined by immunoblotting. Urease activity was not affected by lesions in the genes for either the hydrogenase accessory proteins HypD or HypF or the hydrogenase large subunit structural gene, indicating that the urease deficiency was not caused by lack of hydrogenase activity. When crude extracts of wt, HypA:kan and HypB:kan were separated by anion exchange chromatography, the urease-containing fractions of the mutant strains contained about four (HypA:kan)- and five (HypB:kan)-fold less nickel than did the urease from wt, indicating that the lack of urease activity in these strains results from a nickel deficiency in the urease enzyme.     

Effects of Deletion of Genes Encoding Fe-Only Hydrogenase of Desulfovibrio vulgaris Hildenborough on Hydrogen and Lactate Metabolism

The physiological properties of a hyd mutant of Desulfovibrio vulgaris Hildenborough, lacking periplasmic Fe-only hydrogenase, have been compared with those of the wild-type strain. Fe-only hydrogenase is the main hydrogenase of D. vulgaris Hildenborough, which also has periplasmic NiFe- and NiFeSe-hydrogenases. The hyd mutant grew less well than the wild-type strain in media with sulfate as the electron acceptor and H(2) as the sole electron donor, especially at a high sulfate concentration. Although the hyd mutation had little effect on growth with lactate as the electron donor for sulfate reduction when H(2) was also present, growth in lactate- and sulfate-containing media lacking H(2) was less efficient. The hyd mutant produced, transiently, significant amounts of H(2) under these conditions, which were eventually all used for sulfate reduction. The results do not confirm the essential role proposed elsewhere for Fe-only hydrogenase as a hydrogen-producing enzyme in lactate metabolism (W. A. M. van den Berg, W. M. A. M. van Dongen, and C. Veeger, J. Bacteriol. 173:3688-3694, 1991). This role is more likely played by a membrane-bound, cytoplasmic Ech-hydrogenase homolog, which is indicated by the D. vulgaris genome sequence. The physiological role of periplasmic Fe-only hydrogenase is hydrogen uptake, both when hydrogen is and when lactate is the electron donor for sulfate reduction.     

The HypB protein from Bradyrhizobium japonicum can store nickel and is required for the nickel-dependent transcriptional regulation of hydrogenase

The HypB protein from Bradyrhizobium japonicum is a metal-binding GTPase required for hydrogenase expression. In-frame mutagenesis of hypB resulted in strains that were partially or completely deficient in hydrogenase expression, depending on the degree of disruption of the gene. Complete deletion of the gene yielded a strain (JHΔEg) which lacked hydrogenase activity under all conditions tested, including the situation as bacteroids from soybean nodules. Mutant strain JHΔ23H lacking only the N-terminal histidine-rich region (38 amino acids deleted, 23 of which are His residues) expressed partial hydrogenase activity. The activity of strain JHΔ23H was low in comparison to the wild type in 10–50 nM nickel levels, but could be cured to nearly wild-type levels by including 50 μM nickel during the derepression incubation. Studies on strains harbouring the hup promoter–lacZ fusion plasmid showed that the complete deletion of hypB nearly abolished hup promoter activity, whereas the histidine deletion mutant had 60% of the wild-type promoter activity in 50 μM NiCl₂. Further evidence that HypB is required for hup promoter-binding activity was obtained from gel-shift assays. HypB could not be detected by immunoblotting when the cells were cultured heterotrophically, but when there was a switch to microaerobic conditions (1% partial pressure O₂, 10% partial pressure H₂) HypB was detected, and its expression preceded hydrogenase synthesis by 3–6 h. ⁶³Ni accumulation by whole cells showed that both of the mutant strains accumulate less nickel than the wild-type strain at all time points tested during the derepression incubation. Wild-type cultures that received nickel during the HypB expression-specific period and were then washed and derepressed for hydrogenase without nickel had activities comparable to those cells that were derepressed for hydrogenase with nickel for the entire time period. In contrast to the wild type, strain JHΔ23H cultures supplied with nickel only during the HypB expression period achieved hydrogenase activities that were 30% of those cultures supplied with nickel for the entire hydrogenase derepression period. These results indicate that the loss of the metal-binding area of HypB causes a decrease in the ability of the cells to sequester and store nickel for later use in one or more hydrogenase expression steps.     

Genetic disruption of both Chlamydomonas reinhardtii [FeFe]-hydrogenases: Insight into the role of HYDA2 in H₂ production

Chlamydomonas reinhardtii (Chlamydomonas throughout) encodes two [FeFe]-hydrogenases, designated HYDA1 and HYDA2. While HYDA1 is considered the dominant hydrogenase, the role of HYDA2 is unclear. To study the individual functions of each hydrogenase and provide a platform for future bioengineering, we isolated the Chlamydomonas hydA1-1, hydA2-1 single mutants and the hydA1-1 hydA2-1 double mutant. A reverse genetic screen was used to identify a mutant with an insertion in HYDA2, followed by mutagenesis of the hydA2-1 strain coupled with a H(2) chemosensor phenotypic screen to isolate the hydA1-1 hydA2-1 mutant. Genetic crosses of the hydA1-1 hydA2-1 mutant to wild-type cells allowed us to also isolate the single hydA1-1 mutant. Fermentative, photosynthetic, and in vitro hydrogenase activities were assayed in each of the mutant genotypes. Surprisingly, analyses of the hydA1-1 and hydA2-1 single mutants, as well as the HYDA1 and HYDA2 rescued hydA1-1 hydA2-1 mutant demonstrated that both hydrogenases are able to catalyze H(2) production from either fermentative or photosynthetic pathways. The physiology of both mutant and complemented strains indicate that the contribution of HYDA2 to H(2) photoproduction is approximately 25% that of HYDA1, which corresponds to similarly low levels of in vitro hydrogenase activity measured in the hydA1-1 mutant. Interestingly, enhanced in vitro and fermentative H(2) production activities were observed in the hydA1-1 hydA2-1 strain complemented with HYDA1, while maximal H(2)-photoproduction rates did not exceed those of wild-type cells.     

A novel and remarkably thermostable ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus.

The archaebacterium Pyrococcus furiosus is a strict anaerobe that grows optimally at 100 degrees C by a fermentative-type metabolism in which H2 and CO2 are the only detectable products. A ferredoxin, which functions as the electron donor to the hydrogenase of this organism was purified under anaerobic reducing conditions. It had a molecular weight of approximately 12,000 and contained 8 iron atoms and 8 cysteine residues/mol but lacked histidine or arginine residues. Reduction and oxidation of the ferredoxin each required 2 electrons/mol, which is consistent with the presence of two [4Fe-4S] clusters. The reduced protein gave rise to a broad rhombic electronic paramagnetic resonance spectrum, with gz = 2.10, gy = 1.86, gx = 1.80, and a midpoint potential of -345 mV (at pH 8). However, this spectrum represented a minor species, since it quantitated to only approximately 0.3 spins/mol. P. furiosus ferredoxin is therefore distinct from other ferredoxins in that the bulk of its iron is not present as iron-sulfur clusters with an S = 1/2 ground state. The apoferredoxin was reconstituted with iron and sulfide to give a protein that was indistinguishable from the native ferredoxin by its iron content and electron paramagnetic resonance properties, which showed that the novel iron-sulfur clusters were not artifacts of purification. The reduced ferredoxin also functioned as an electron donor for H2 evolution catalyzed by the hydrogenase of the mesophilic eubacterium Clostridium pasteurianum. P. furiosus ferredoxin was resistant to denaturation by sodium dodecyl sulfate (20%, wt/vol) and was remarkably thermostable. Its UV-visible absorption spectrum and electron carrier activity to P. furiosus hydrogenase were unaffected by a 12-h incubation of 95 degrees C.