Nickel is essential for active hydrogenase in free-living Frankia isolated from Casuarina

Five free-living Frankia strains isolated from Casuarina were investigated for occurrence of hydrogenase activity. Nitrogenase activity (acetylene reduction) and hydrogen evolution were also evaluated. Acetylene reduction was recorded in all Frankia strains. None of the Frankia strains had any hydrogenase activity when grown on nickel-depleted medium and they released hydrogen in atmospheric air. After addition of nickel to the medium, the Frankia strains were shown to possess an active hydrogenase, which resulted in hydrogen uptake but no hydrogen evolution. The hydrogenase activity in Frankia strain KB5 increased from zero to 3.86 μ mol H₂ (mg protein)⁻¹ h⁻¹ after addition of up to 1.0 μ M Ni. It is likely that the hydrogenase activity could be enhanced even more as a response on further addition of Ni. It is indicated in this study that absence of hydrogenase activity in free-living Frankia isolated from Casuarina spp. is due to nickel deficiency. Frankia living in symbiosis with Casuarina spp. show hydrogenase activity. Therefore, the results also indicate that the hydrogenase to some extent is regulated by the host plant and/or that the host plant supplies the symbiotic microorganism with nickel. Moreover, the result shows that this Frankia is somewhat different from Frankia isolated from Alnus incana and Comptonia peregrina ., i.e., Frankia isolated from A. incana and C. peregrina showed a small hydrogen uptake activity even without addition of nickel.     

Hydrogenase in Frankia KB5: expression of and relation to nitrogenase

The localization and expression of the hydrogenase in free-living Frankia KB5 was investigated immunologically and by monitoring activity, focusing on its relationships with nitrogenase and H2. Immunological studies revealed that the large subunit of the hydrogenase in Frankia KB5 was modified post-translationally, and transferred into the membrane after processing. The large subunit was constitutively expressed and no correlation was found between hydrogenase activity and synthesis. Although H2 was not needed for induction of hydrogenase synthesis, exogenously added H2 triggered hydrogen uptake in medium containing nitrogen, i.e., in the hyphae. A correlation between nitrogenase activity and hydrogen uptake was found in cultures grown in media without nitrogen, but interestingly the two enzymes showed no co-regulation.     

The acetylene reduction assay inactivates root nodule uptake hydrogenase in some actinorhizal plants

Actinorhizal nodules do not usually evolve H₂ due to the action of an uptake hydrogenase. We have found that nodules of several Frankia symbioses evolved large amounts of H₂ gas when returned to air following exposure to 10 kPa C₂HT₂ during an acetylene reduction assay. Increased H₂ evolution in air persisted for several days when intact root systems of Alnus incana (L.) Moench (inoculated with Frankia UGL 011101) were treated with 10 kPa C.H₂ for 1 h. Full recovery of uptake hydrogenase activity required 4 to 8 days. Studies with crude homogenates of nodules of the same plants showed that hydrogenase (measured amperometrically with phenazine metho-sulfate as electron acceptor) was directly affected, since activity in treated nodules was only 10% of that in untreated nodules. A survey of actinorhizal symbioses revealed variation in the effect of an acetylene reduction assay on hydrogen metabolism. Nodules of three species, including Alnus rubra Bong, inoculated with Frankia HFPArD. showed complete inactivation of hydrogenase. H₂ evolution in air was 25% of the C₂H₂ reduction rate and H, evolution in Ar/O₂ was equal to the QH₂ reduction rate. Two symbioses, Ceanothus americanus L. (soil inoculant) and Batista glomerata Baill. (soil inoculant) showed no change following an acetylene reduction assay. A third group of symbioses showed an intermediate response.     

Occurrence and localization of two distinct hydrogenases in the heterocystous cyanobacterium Anabaena sp. strain 7120

Two distinct types of hydrogenase occur in Anabaena 7120 and are distinguishable in whole filaments by the application of selective assay methods. A reversible hydrogenase occurs both in heterocysts and vegetative cells and can be selectively assayed by measuring H2 evolution from reduced methyl viologen. Activities in aerobically grown filaments were low but could be increased by 2 to 3 orders of magnitude by growing cells microaerobically. The presence of the reversible hydrogenase was independent of the N2-fixing properties of the organism, and activity did not respond to added H2 in the culture. Illumination was necessary during derepression of the reversible hydrogenase, and addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea increased the amount of enzyme that was synthesized. An uptake hydrogenase occurred only in heterocysts of aerobically grown filaments, but a small amount of activity also was present in the vegetative cells of filaments grown microaerobically with 20% H2. It was assayed selectively by measuring an oxyhydrogen reaction at atmospheric levels of O2. Additional uptake hydrogenase could be elicited by including H2 or by removing O2 from the sparging gas of a culture.     

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.     

Regulation of hydrogenase in Rhizobium japonicum.

Factors that regulate the expression of an H2 uptake system in free-living cultures of Rhizobium japonicum have been investigated. Rapid rates of H2 uptake by R. japonicum were obtained by incubation of cell suspensions in a Mg-phosphate buffer under a gas phase of 86.7% N2, 8.3% H2, 4.2% CO2, and 0.8% O2. Cultures incubated under conditions comparable with those above, with the exception that Ar replaced H2, showed no hydrogenase activity. When H2 was removed after initiation of hydrogenase derepression, further increase in hydrogenase activity ceased. Nitrogenase activity was not essential for expression of hydrogenase activity. All usable carbon substrates tested repressed hydrogenase formation, but none of them inhibited hydrogenase activity. No effect on hydrogenase formation was observed from the addition of KNO3 or NH4Cl at 10 mM. Oxygen repressed hydrogenase formation, but did not inhibit activity of the enzyme in whole cells. The addition of rifampin or chloramphenicol to derepressed cultures resulted in inhibition of enzyme formation similar to that observed by O2 repression. The removal of CO2 during derepression caused a decrease in the rate of hydrogenase formation. No direct effect of CO2 on hydrogenase activity was observed.     

Effect of O2 on vesicle formation, acetylene reduction, and O2-uptake kinetics in Frankia sp. HFPCcI3 isolated from Casuarina cunninghamiana

The effect of the partial pressure of oxygen (PO2) on the formation of vesicles, which are thought to be the site of N2 fixation in Frankia, was studied in HFPCcI3, an effective isolate from Casuarina cunninghamiana. Unlike other actinorhizal root nodules, vesicles are not produced by the endophyte in Casuarina nodules. However, in culture under aerobic conditions, large, phase-bright vesicles are formed in HFPCcI3 within 20 h following removal of NH+4 from the culture medium and reach peak numbers within 72 to 96 h. In vivo acetylene reduction activity parallels vesicle formation. Optimum rates of acetylene reduction in short-term assays occurred at 20% O2 (0.2 atm (1 atm = 101.325 kPa] in the gas phase. O2 uptake (respiration) determined polarographically showed diffusion-limited kinetics and remained unsaturated by O2 until 300 microM O2. In contrast, respiration in NH+4-grown cells was saturated by O2 between 8 and 10 microM O2. These results indicate the presence of a diffusion barrier associated with the vesicles. Vesicle development was repressed in cells incubated in N-free media sparged with gas mixtures with PO2 between 0.001 and 0.003 atm. Nitrogenase was induced under these conditions, but acetylene reduction was extremely O2 sensitive. The kinetics of O2 uptake as a function of dissolved O2 concentration in avesicular cells were similar to those in NH+4-grown cells indicating the lack of a diffusion barrier. These results demonstrate that vesicle formation and the development of the O2 protection mechanisms of nitrogenase are regulated by ambient PO2 in HFPCcI3.     

Uptake hydrogenase activity and ATP formation in Rhizobium leguminosarum bacteroids.

The role of uptake hydrogenase was studied in Rhizobium leguminosarum bacteroids from the nodules of Pisum sativum L. cv. Homesteader. Uptake hydrogenase activity, measured by the 3H2 uptake method, was dependent on O-consumption and was similar to H2 uptake measured by gas chromatography. Km for O2 of 0.0007 atm (0.0709 kPa) and a Km for H2 of 0.0074 atm (0.7498, kPa) were determined. H2 increased the rate of endogenous respiration by isolates with uptake hydrogenase (Hup+) but had no effect on an isolate lacking uptake hydrogenase (Hup-). A survey of 14 Hup+ isolates indicated a wide range of H2 uptake activities. Four of the isolates tested had activities similar to or higher than those found in two Hup+ Rhizobium japonicum strains. H2 uptake was strongly coupled to ATP formation in only 5 of the 14 isolates. H2 increased the optimal O2 level of C2H2 reduction by 0.01 atm and permitted enhanced C2H2 reduction at O2 levels above the optimum in both a coupled and an uncoupled isolate. At suboptimal O2 concentrations a small enhancement of C2H2 reduction by H2 was seen in two out of three isolates in which H2 oxidation was coupled to ATP formation. Thus, the main function of uptake hydrogenase in R. leguminosarum appears to be in the protection of nitrogenase from O2 damage.     

Hydrogenase activities in cyanobacteria

In the unicellular Anacystis nidulans, the expression of both the H2-uptake (with phenazine methosulfate or methylene blue as the electron acceptor) and H2-evolution (with methyl viologen reduced by Na2S2O4) was dependent on Ni in the culture medium. In extracts from Anacystis and Anabaena 7119, H2-evolution and uptake activities were strongly inhibited by Cu2+, p-chloromercuribenzoate and HgCl2 suggesting that at least one functional SH-group is involved in catalysis by hydrogenase. Extracts from the N2-fixing Anabaena 7119 contained two different hydrogenase fractions which could be separated by chromatography on DE-52 cellulose using a linear NaCl concentration gradient. The fraction eluting with 0.13 M NaCl from the column catalyzed only the uptake of H2 with methylene blue as the electron acceptor but virtually not the evolution of H2 ("uptake" hydrogenase fraction). The fraction eluting at a NaCl strength of 0.195 M catalyzed both H2-uptake with methylene blue and H2-evolution with reduced methyl viologen ("reversible" hydrogenase fraction). Growth under anaerobic conditions drastically enhanced the activity levels of the "reversible" but not of the "uptake" hydrogenase fraction. The "uptake" hydrogenase but not the "reversible" protein was activated by reduced thioredoxin. It is suggested that thioredoxin activates the H2-uptake by the membrane-bound "uptake" hydrogenase also in intact cells. The occurrence of the number of hydrogenases in cyanobacteria will be reevaluated.     

Comparative characterization of two distinct hydrogenases from Anabaena sp. strain 7120

Two distinct hydrogenases, hereafter referred to as "uptake" and "reversible" hydrogenase, were extracted from Anabaena sp. strain 7120 and partially purified. The properties of the two enzymes were compared in cell-free extracts. Uptake hydrogenase was largely particulate, and although membrane bound, it could catalyze an oxyhydrogen reaction. Particulate and solubilized uptake hydrogenase could catalyze H2 uptake with a variety of artificial electron acceptors which had midpoint potentials above 0 mV. Reversible hydrogenase was soluble, could donate electrons rapidly to electron acceptors of both positive and negative midpoint potential, and could evolve H2 rapidly when provided with reduced methyl viologen. Uptake hydrogenase was irreversibly inactivated by O2, whereas reversible hydrogenase was reversibly inactivated and could be reactivated by exposure to dithionite or H2. Reversible hydrogenase was stable to heating at 70 degrees C, but uptake hydrogenase was inactivated with a half-life of 12 min at this temperature. Uptake hydrogenase was eluted from Sephadex G-200 in a single peak of molecular weight 56,000, whereas reversible hydrogenase was eluted in two peaks with molecular weights of 165,000 and 113,000. CO was competitive with H2 for each enzyme; the Ki's for CO were 0.0095 atm for reversible hydrogenase and 0.039 atm for uptake hydrogenase. The pH optima for H2 evolution and H2 uptake by reversible hydrogenase were 6 and 9, respectively. Uptake hydrogenase existed in two forms with pH optima of 6 and 8.5. Both enzymes had very low Km's for H2, and neither was inhibited by C2H2.     

Regulation of two nickel-requiring (inducible and constitutive) hydrogenases and their coupling to nitrogenase in Methylosinus trichosporium OB3b.

Two uptake hydrogenases were found in the obligate methanotroph Methylosinus trichosporium OB3b; one was constitutive, and a second was induced by H2. Both hydrogenases could be assayed by measuring methylene blue reduction anaerobically or by coupling their activity to nitrogenase acetylene reduction activity in vivo in an O2-dependent reaction. The H2 concentration for half-maximal activity of the inducible and constitutive hydrogenases in both assays was 0.01 and 0.5 bar (1 and 50 kPa), respectively, making it easy to distinguish these enzymes from one another both in vivo and in vitro. Hydrogen uptake was shown to be coupled to ATP synthesis in methane-starved cells. Methane, methanol, formate, succinate, and glucose all repressed the H2-mediated synthesis of the inducible hydrogenase. Furthermore, this enzyme was only expressed in N-starved cultures and was repressed by NH4+ and NO3-; synthesis of the constitutive hydrogenase was not affected by excess N in the growth medium. In nickel-free, EDTA-containing medium, the activities of these two enzymes were negligible; however, both enzyme activities appeared rapidly following the addition of nickel to the culture. Chloramphenicol, when added along with nickel, had no effect on the rapid appearance of either the constitutive or inducible activity, indicating that nickel is not required for synthesis of the hydrogenase apoproteins. These observations all suggest that these hydrogenases are nickel-containing enzymes. Finally, both hydrogenases were soluble and could be fractionated by 20% ammonium sulfate; the constitutive enzyme remained in the supernatant solution, while the inducible enzyme was precipitated under these conditions.     

Aerobic, inactive forms of Azotobacter vinelandii hydrogenase: activation kinetics and insensitivity to C2H2 inhibition

Azotobacter vinelandii hydrogenase (EC class 1.12), either purified or membrane-associated, was obtained aerobically in an inactive state. The kinetics of activation by treatment with a reductant (H2 or dithionite) were determined. Three distinct phases of the activation were observed. Aerobically prepared, inactive hydrogenase was insensitive to acetylene inhibition, but could be rendered acetylene-sensitive by reduction with dithionite. These findings indicate that acetylene inhibition of hydrogenase requires catalytically active enzyme.     

Nitrogen fixation and biomass production in symbioses between Alnus incana and Frankia strains with different hydrogen metabolism

Three different strains of Frankia , the pure cultures AvcI1 and CpI1 and a local strain (crushed nodule inoculum), were compared in symbiosis with one clone of Alnus incana (L.) Moench. Hydrogen metabolism, nitrogenase (EC 1.7.99.2) activity and relative efficiency of nitrogenase were studied as well as growth and nitrogen content of the plants. The local Frankia strain showed no measurable hydrogen uptake but high H₂-evolution. No H₂-evolution was detected in Frankia AvcI1 because of its hydrogenase activity. CpI1 also had hydrogenase, although only a very small H₂-evolution was detected at the end of the growth period. Hydrogenase activity was detected both in pure cultures and nodule homogenates of CpI1 and AvcI1. Growth, biomass production and nitrogen content were highest in alders inoculated with Frankia AvcI1 while the lowest values were found for alders living in symbiosis with the local Frankia strain. The presence of hydrogenase in Frankia seemed to be benefical for growth and biomass production in the alders. However, the strains also differed with respect to spore formation. The local strain, but not AvcI1 and CpI1, formed spores in the root nodules.     

Nif- Hup- mutants of Rhizobium japonicum.

Two H2 uptake-negative (Hup-) Rhizobium japonicum mutants were obtained that also lacked symbiotic N2 fixation (acetylene reduction) activity. One of the mutants formed green nodules and was deficient in heme. Hydrogen oxidation activity in this mutant could be restored by the addition of heme plus ATP to crude extracts. Bacteroid extracts from the other mutant strain lacked hydrogenase activity and activity for both of the nitrogenase component proteins. Hup+ revertants of the mutant strains regained both H2 uptake ability and nitrogenase activity.     

H2 consumption by Escherichia coli coupled via hydrogenase 1 or hydrogenase 2 to different terminal electron acceptors

Hydrogen uptake in the presence of various terminal electron acceptors was examined in Escherichia coli mutants synthesizing either hydrogenase 1 or hydrogenase 2. Both hydrogenases mediated nitrate-dependent H2 consumption but neither of them was coupled with nitrite. Unlike hydrogenase 2, hydrogenase 1 demonstrated poor activity with electron acceptors of low midpoint redox potential. Oxygen-linked H2 uptake via hydrogenase 1 was observed over a wide range of air concentrations. Hydrogenase 2 catalyzed this reaction only at low air concentrations. Thus, hydrogenase 1 works in cells at higher redox potential, being more tolerant to oxygen than hydrogenase 2.     

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.     

Occurrence and activity of hydrogenase in symbiotic Frankia from field-collected Alnus incana

Occurrence and activity of the hydrogen uptake enzyme were studied in root nodule homogenates made from plants of Alnus incana (L.) Moench collected from field sites in the northern part of Sweden. Nitrogenase (EC 1.7.99.2) activity (estimated by acetylene reduction) and hydrogen evolution were studied in excised nodules. All Frankia sources showed acetylene reduction activity, and possessed a hydrogen uptake system. Hydrogen uptake in nodule homogenates from the Frankia sources measured at 23.8 μM H₂ ranged from 0.04 to 5.0 μmol H₂ (g fresh weight nodule)⁻¹ h⁻¹. The H₂ uptake capacity of nodule homogenates from one of the Frankia sources was almost 8 times higher than the hydrogen evolution from nitrogenase, both expressed on a nodule fresh weight basis. Frankia sources from field sites 6 and 11 showed K_m for H₂ of 13.0 and 23.6 μM H₂, respectively. This indicates similarities in the hydrogen uptake enzymes in the two Frankia sources. It is concluded that hydrogen uptake is a common characteristic in Frankia.     

Measurement in vivo of hydrogenase-catalysed hydrogen evolution in the presence of nitrogenase enzyme in cyanobacteria.

A method was devised that allows measurement in vivo of hydrogenase-catalysed H2 evolution from the cyanobacterium Anabaena cylindrica, independent of nitrogenase activity, which is also present. Addition of low concentrations of reduced Methyl Viologen (1-10mM) to intact heterocystous filaments of the organism resulted in H2 evolution, but produced conditions giving total inhibition of nitrogenase (acetylene-reducing and H2-evolving) activity. That the H2 formed under these conditions was not contributed to by nitrogenase was also supported by the observation that its rate of formation was similar in the dark or with Ar replaced by N2 in the gas phase, and also in view of the pattern of H2 evolution at very low Methyl Viologen concentrations. Conclusive evidence that the H2 formed in the presence of Methyl Viologen was solely hydrogenase-mediated was its evolution even from nitrogenase-free (non-heterocystous) cultures; by contrast 'uptake' hydrogenase activity in such cultures was greatly decreased. The hydrogenase activity was inhibited by CO and little affected by acetylene. Finally the hydrogenase activity was shown to be relatively constant at different stages during the batch growth of the organism, as opposed to nitrogenase activity, which varied.     

Nickel affects expression of the nickel-containing hydrogenase of Alcaligenes latus.

The effects of nickel on the expression of hydrogenase in the hydrogen-oxidizing bacterium Alcaligenes latus were studied. In the absence of added nickel, both hydrogenase activity, measured as O2-dependent H2 uptake, and hydrogenase protein, measured in a Western immunoblot, were very low compared with the levels in cells induced for hydrogenase in the presence of nickel. Hydrogenase activity and protein levels were dependent on the added nickel concentration and were saturated at 30 nM added Ni2+. The amount of hydrogenase protein in a culture at a given nickel concentration was calculated from the H2 uptake activity of the culture at that Ni2+ concentration. Between 0 and 30 nM added Ni2+, the amount of hydrogenase protein (in nanomoles) was stoichiometric with the amount of added Ni2+. Thus, all of the added Ni2+ could be accounted for in hydrogenase. Between 0 and 50 nM added Ni2+, all the Ni present in the cultures was associated with the cells after 12 h; above 50 nM added Ni2+, some Ni remained in the medium. No other divalent metal cations tested were able to substitute for Ni2+ in the formation of active hydrogenase. We suggest two possible mechanisms for the regulation of hydrogenase activity and protein levels by nickel.