In vitro activation of [FeFe] hydrogenase: new insights into hydrogenase maturation

The in vitro activation of the [FeFe] hydrogenase is accomplished by combining Escherichia coli cell extracts containing the heterologously expressed inactive HydA with extracts in which hydrogenase-specific maturation proteins HydE, HydF, and HydG are expressed in concert. Interestingly, the process of HydA activation occurs rapidly and in the absence of potential substrates, which suggests that the hydrogenase accessory proteins synthesize an H-cluster precursor that can be quickly transferred to the hydrogenase enzyme to affect activation. HydA activity is observed to be dependent on the protein fraction containing all three accessory proteins expressed in concert and cannot be accomplished with addition of heat-treated extract or extract filtrate, suggesting that the activation of the hydrogenase structural protein is mediated by interaction with the accessory assembly protein(s). These results represent the first important step in understanding the process of H-cluster assembly and provide significant insights into hydrogenase maturation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Orientation-selected ENDOR of the active center in Chromatium vinosum [NiFe] hydrogenase in the oxidized "ready" state

 Electron nuclear double resonance (ENDOR) was applied to study the active site of the oxidized "ready" state, Ni_r, in the [NiFe] hydrogenase of Chromatium vinosum. The magnetic field dependence of the EPR was used to select specific subsets of molecules contributing to the ENDOR response by stepping through the EPR envelope. Three hyperfine couplings could be clearly followed over the complete field range. Two protons, H1 and H2, display a very similar large isotropic coupling of 12.5 and 12.6 MHz, respectively. Their dipolar coupling is small (2.1 and 1.4 MHz, respectively). A third proton, H3, exhibits a small isotropic coupling of 0.5 MHz and a larger anisotropic contribution of 3.5 MHz. Based on a comparison with structural data obtained from X-ray crystallography of single crystals of hydrogenases from Desulfovibrio gigas and D. vulgaris and the known g-tensor orientation of Ni_r, an assignment of the ¹H hyperfine couplings could be achieved. H1 and H2 were assigned to the β-CH₂ protons of the bridging cysteine Cys533 and H3 could belong to a β-CH₂ proton of Cys68 or to a protonated cysteine (-SH) of Cys68 or Cys530. Received: 26 November 1998 / Accepted: 1 April 1999     

Effect of redox potential on activity of hydrogenase 1 and hydrogenase 2 in Escherichia coli

This report elucidates the distinctions of redox properties between two uptake hydrogenases in Escherichia coli. Hydrogen uptake in the presence of mediators with different redox potential was studied in cell-free extracts of E. coli mutants HDK103 and HDK203 synthesizing hydrogenase 2 or hydrogenase 1, respectively. Both hydrogenases mediated H(2) uptake in the presence of high-potential acceptors (ferricyanide and phenazine methosulfate). H(2) uptake in the presence of low-potential acceptors (methyl and benzyl viologen) was mediated mainly by hydrogenase 2. To explore the dependence of hydrogen consumption on redox potential of media in cell-free extracts, a chamber with hydrogen and redox ( E(h)) electrodes was used. The mutants HDK103 and HDK203 exhibited significant distinctions in their redox behavior. During the redox titration, maximal hydrogenase 2 activity was observed at the E(h) below -80 mV. Hydrogenase 1 had maximum activity in the E(h) range from +30 mV to +110 mV. Unlike hydrogenase 2, the activated hydrogenase 1 retained activity after a fast shift of redox potential up to +500 mV by ferricyanide titration and was more tolerant to O(2). Thus, two hydrogenases in E. coli are complementary in their redox properties, hydrogenase 1 functioning at higher redox potentials and/or at higher O(2) concentrations than hydrogenase 2.     

Unconsidered factors affecting hydrogenase activity measurement

The effects of sample geometry and enzyme concentration on the H2-evolving activity of hydrogenase from Thiocapsa roseopersicina was measured. The specific activity increased linearly with increasing interface area between the liquid and the gas phase. Enzyme concentration was varied over four orders of magnitude and within this range the apparent specific activity depended on hydrogenase concentration. The experimental findings have been interpreted by a mathematical model involving competing H2 consumption reactions. The observed phenomena interfere with the widely used hydrogenase assay so that most of the previously published specific activity values are underestimated and should be corrected. The systematic error due to these hitherto unspecified parameters can easily exceed 10 000%; therefore, a thorough standardization of the assay procedure is necessary in order to make the data from various laboratories comparable.     

Comparison of the membrane-bound and detergent-solubilised hydrogenase from paracoccus denitrificans. Isolation of the hydrogenase.

The hydrogenase from Paracoccus denitrificans is an integral membrane protein and has been solubilised by Triton X-100. The membrane-bound and detergent-solubilised forms of the enzyme have been compared. Both forms of the enzyme show a pH optimum for reduction of benzyl viologen at pH 8.5--9.0 and are both inhibited by concentrations of NaCl greater than 30 mM. An Arrhenius plot of the activity of hydrogenase in the membrane shows no 'break'. The form of the Arrhenius plot and the activation energy are not significantly changed on solubilisation of the enzyme. The Km and V values for benzyl viologen, methyl viologen and H2 are unaltered when the enzyme is extracted from the membrane. Therefore, solubilisation of hydrogenase from the membrane by Triton X-400 is unlikely to disrupt the native conformation of the enzyme. The detergent-solubilised hydrogenase has subsequently been purified using ammonium sulphate precipitation, sucrose density gradient centrifugation and chromatography on hydroxyapatite. The overall yield of activity is 23%, with a final purification of over 100-fold.     

Application of hydrogenase in biotechnological conversions

Evidence will be presented in this review article that the application of hydrogenase has large biotechnological possibilities. Our investigations show: Fast reaction of hydrogenase at an electrode surface to reduce H+; Photochemical production of H2 by hydrogenase by photosensitized Ru-complexes dissolved in reversed micellar membranes and vectorial H+ transport through the membrane to the water phase; The production of fine chemicals in reversed micelles by a system containing specific enzymes, hydrogenase and H2. The rules to obtain maximal conversion rates with this system will be presented.     

The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase.

The roles of the product of the hoxZ gene immediately downstream of the hydrogenase gene (hoxKG) in Azotobacter vinelandii were investigated by constructing and characterizing a mutant with the center of the hoxZ gene deleted. The strain lacking the functional hoxZ gene product exhibited a low rate of H2 oxidation with O2 as the electron acceptor relative to that of the wild-type strain. Nevertheless, when the enzyme was exogenously activated and methylene blue was used as the electron acceptor from hydrogenase, rates of H2 oxidation comparable to those in the wild-type strain were observed. These results suggest that the gene product of hoxZ plays a role in activating and maintaining hydrogenase in a reduced active state. The product of hoxZ could also be the linkage necessary for transfer of electrons from H2 to the electron transport chain.     

Immobilization of hydrogenase on glass beads

Hydrogenase from $\text{Clostridium pasteurianum}$ was immobilized on glass beads by four different methods. The sensitivity of the native and bound enzyme to oxygen was examined. Hydrogenase bound to succinyl glass proved to be the most stable to oxygen. All bound enzymes were active with ferredoxin as a substrate and evolved hydrogen in a chloroplast-ferredoxin-hydrogenase system driven by light.     

Metallocenter assembly of the hydrogenase enzymes

The biosynthesis of the [NiFe]- and [FeFe]-hydrogenase enzymes requires the activities of multiple proteins that assemble the intricate metallocenters on the enzyme precursor proteins in an energy-dependent process. These accessory proteins include enzymes that synthesize the non-protein iron ligands as well as metallochaperones for the delivery of nickel to the [NiFe]-hydrogenase. Over the past few years many of these proteins have been examined in vitro. The biochemical properties, in the context of the earlier genetic studies, provide a basis for assigning function to the individual accessory proteins and mapping out the sequential steps of the metallocenter assembly pathways. This framework will serve as a foundation for detailed mechanistic analysis of these complex biomolecular factories.     

The membrane-bound hydrogenase of Alcaligenes eutrophus: II. Localization and immunological comparison with other hydrogenase systems

Immunological comparison of the soluble and the membrane-bound hydrogenase of Alcaligenes eutrophus revealed no common antigenic determinants shared by the native proteins, however, a small amount of cross-reacting material was detected after freezing and thawing. Immune precipitation assays supported previous observations indicating the membrane-bound hydrogenase to be localized in the outer surface of the cytoplasmic membrane.The membrane-bound hydrogenases of A. eutrophus and Pseudomonas pseudoflava showed close immunological relationship, and material cross-reacting to both antisera was found in membrane extracts of all hydrogen-oxidizing strains of Pseudomonas, Alcaligenes and Aquaspirillum. Material cross-reacting to the membrane-bound hydrogenase of Xanthobacter autotrophicus GZ 29 was found only in a few hydrogen-oxidizing bacteria. Material cross-reacting to the soluble hydrogenase of A. eutrophus was detected in strains of A. eutrophus and A. ruhlandii only.Comparison of the membrane-bound hydrogenase of A. eutrophus, P. pseudoflava and X. autotrophicus with hydrogenases of other physiological bacterial groups revealed serological relationship to the membrane-bound hydrogenases of the hydrogen bacteria and of Chromatium vinosum only. The results are discussed in terms of physiological, taxonomical, and evolutionary aspects.     

Nitrate reductase activity in nodules of pea inoculated with hydrogenase positive and hydrogenase negative strains of Rhizobium leguminosarum

The nitrate reductase (NR, EC 1.6.6.1) activity in root nodules formed by hydrogenase positive (Hup⁺) and hydrogenase negative (Hup⁻) Rhizobium leguminosarum strains was examined in symbioses with the pea cultivar Alaska ( Pisum sativum L.), Rates of activity were determined by the in vivo assay in nodules from plants that were only N₂-dependent or grown in the presence of 2 m M KNO₃. The rates varied widely among strains, regardless of the Hup phenotype of the R. leguminosarum strain used for inoculation, but the overall results indicated that nodules formed by Hup⁻ strains accumulated more nitrite in the incubation medium than did those with Hup⁻ phenotypes. Total plant dry weight and reduced nitrogen content of pea plants grown in the presence of 2 m M KNO₃ and inoculated with single Hup⁺ and Hup⁻ R. leguminosarum strains were statistically different among some strains. These observations suggest that the possible advantages derived from the presence of the Hup system on whole plant growth may be counteracted by the higher rates of NR activity in the Hup⁻ strains in the R. leguminosarum -pea symbiosis.     

Methyl viologen hydrogenase II, a new member of the hydrogenase family from Methanobacterium thermoautotrophicum delta H.

Two methyl viologen hydrogenase (MVH) enzymes from Methanobacterium thermoautotrophicum delta H have been separated (resolution, Rs at 1.0) on a Mono Q column after chromatography on DEAE-Sephacel and Superose 6 Prep Grade. The newly discovered MVH (MVH II) was eluted at 0.5 M NaCl with a linear gradient of 0.45 to 0.65 M NaCl (100 ml). The previously described MVH (MVH I) eluted in a NaCl gradient at 0.56 M. The specific activities of MVH I and MVH II were 184.8 and 61.3 U/mg of protein, respectively, when enzyme activity was compared at pH 7.5, the optimal pH for MVH II. Gel electrophoresis in nondenaturing systems indicated that MVH I and MVH II had a similar molecular mass of 145 kDa. Denatured MVH II showed four protein bands (alpha, 50 kDa; beta, 44 kDa; gamma, 36 kDa; delta, 15 kDa), similar to MVH I. The N-terminal amino acid sequences of the alpha, gamma, and delta subunits of MVH II were identical with the sequences of the equivalent subunits of MVH I. However, the N-terminal amino acid sequence of the beta subunit of MVH II was totally different from the sequence of the beta subunit of MVH I. Both MVH I and MVH II had the same optimal temperature of 60 degrees C for maximum activity. The pH optima of MVH I and MVH II were 9.0 and 7.5, respectively. Most of the divalent metal ions tested significantly inhibited MVH I activity, but MVH II activity was only partially inhibited by some divalent cations. Both hydrogenases were shown to be stable for over 8 days at --20 degrees C under anaerobic conditions. When exposed to air, 90% of MVH I activity was lost within 2 min; however, MVH II lost only 50% of its activity in 3 h.