Evidence for three distinct hydrogenase activities in Rhodospirillum rubrum

Inducer, inhibitor, and mutant studies on three hydrogenase activities of Rhodospirillum rubrum indicate that they are mediated by three distinct hydrogenase enzymes. Uptake hydrogenase mediates H2 uptake to an unknown physiological acceptor or methylene blue and is maximally synthesized during autotrophic growth in light. Formate-linked hydrogenase is synthesized primarily during growth in darkness or when light becomes limiting, and links formate oxidation to H2 production. Carbon-monoxide-linked hydrogenase is induced whenever CO is present and couples CO oxidation to H2 evolution. The enzymes can be expressed singly or conjointly depending on growth conditions, and the inhibitor or inducer added. All three hydrogenases can use methyl viologen as the mediator for both the H2 evolution and H2 uptake reactions while displaying distinct pH optima, reversibility, and sensitivity to C2H2 gas. Yet, we present evidence that the CO-linked hydrogenase, unlike the uptake hydrogenase, does not link to methylene blue as the electron acceptor. These differences allow conditions to be established to quantitatively assay each hydrogenase independently of the others both in vivo and in vitro.     

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.     

Hydrogen oxidation activity in membranes from Rhizobium japonicum

Membranes capable of oxidizing H₂ with O₂ as terminal acceptor were obtained from free-living Rhizobium japonicum. Membranes contained highest H₂-uptake specific activities when isolated in the presence of an H₂ atmosphere, and when the oxygen radical scavenger butylated hydroxytoluene was included in the buffer used for rupturing cells. After breaking cells, all of the O₂-dependent H₂-uptake activity was associated with a particulate membrane-containing fraction, whereas approx. 75% of the methylene blue-dependent H₂-uptake activity was sedimented. The particulate and soluble fractions containing H₂-uptake activity with methylene blue were separated by sucrose gradient centrifugation. The particulate and soluble activities behaved identically with regard to artificial electron acceptor specificity and reversible inhibition by oxygen. The hydrogenase in membranes coupled H₂ uptake with the reduction of many positive potential electron acceptors, but not with negative potential acceptors. The optimal pH for H₂ uptake with O₂ as acceptor in membranes was approx. 7.2. H₂-uptake activity in membranes was associated with an inner (lighter) membrane fraction that also contained succinate oxidase activity. H₂-reduced minus O₂-oxidized difference spectroscopy of membranes indicated the involvement of b and c-type cytochromes in the H₂-oxidation pathway, with an absorption peak at 551.5 nm and a shoulder at 560 nm. The addition of sodium dithionite to H₂-reduced membranes caused additional b-type cytochrome reduction. The methylene blue-dependent H₂-uptake activity in membranes was reversibly inhibited by brief exposure to oxygen. Recovery of full activity after oxygen exposure was achieved only after several minutes of incubation under strict anaerobic conditions.     

Effect of pH on tritium exchange and hydrogen production and uptake in free-living cells and in bacteroids of Bradyrhizobium japonicum

Soybean nodule bacteroids and Bradyrhizobium japonicum free-living cells induced for H2-uptake hydrogenase, actively catalyze the evolution of H2 in a reaction highly dependent on the pH. The optimal pHs for the evolution and uptake reactions were 4.0 and 7.5-8.0, respectively. No differences were found between free-living cells and bacteroids with respect to hydrogen acceptor specificity, although absolute rates of H2 uptake were higher for free-living cells. Both types of cells were able to evolve hydrogen from reduced methyl viologen at low pH. These intact cells also catalyzed the exchange reaction between tritium and water in the absence of oxygen. The pH profile of the exchange activity showed two peaks at values near the optimal pHs for the evolution and uptake reactions.     

Diphenylene iodonium as an inhibitor for the hydrogenase complex of Rhodobacter capsulatus. Evidence for two distinct electron donor sites

The photosynthetic bacterium Rhodobacter capsulatus synthesises a membrane-bound [NiFe] hydrogenase encoded by the H2 uptake hydrogenase (hup)SLC structural operon. The hupS and hupL genes encode the small and large subunits of hydrogenase, respectively; hupC encodes a membrane electron carrier protein which may be considered as the third subunit of the uptake hydrogenase. In Wolinella succinogenes, the hydC gene, homologous to hupC, has been shown to encode a low potential cytochrome b which mediates electron transfer from H2 to the quinone pool of the bacterial membrane. In whole cells of R. capsulatus or intact membrane preparation of the wild type strain B10, methylene blue but not benzyl viologen can be used as acceptor of the electrons donated by H2 to hydrogenase; on the other hand, membranes of B10 treated with Triton X-100 or whole cells of a HupC- mutant exhibit both benzyl viologen and methylene blue reductase activities. We report the effect of diphenylene iodonium (Ph2I), a known inhibitor of mitochondrial complex I and of various monooxygenases on R. capsulatus hydrogenase activity. With H2 as electron donor, Ph2I inhibited partially the methylene blue reductase activity in an uncompetitive manner, and totally benzyl viologen reductase activity in a competitive manner. Furthermore, with benzyl viologen as electron acceptor, Ph2I increased dramatically the observed lagtime for dye reduction. These results suggest that two different sites exist on the electron donor side of the membrane-bound [NiFe] hydrogenase of R. capsulatus, both located on the small subunit. A low redox potential site which reduces benzyl viologen, binds Ph2I and could be located on the distal [Fe4S4] cluster. A higher redox potential site which can reduce methylene blue in vitro could be connected to the high potential [Fe3S4] cluster and freely accessible from the periplasm.     

花生根瘤菌类菌体氢酶的分离提纯及特性

花生根瘤菌类菌体经超声波破碎,ＴｒｉｔｏｎＸ－１００溶解,正已烷－硫酸铵处理后,再经ＤＥＡＥ－纤维素和Ｓｅｐｈａｃｒｙｌ凝胶柱层析等纯化步骤,获得凝胶电泳纯的膜结合态氢酶,比活为７１．４μｍｏｌＨ２ｍｇ－１Ｐｒｏｔｍｉｎ－１,为类菌体吸Ｈ２活性的２１１倍。纯化的氢酶分子量为１１０ｋＤ。经ＳＤＳ－ＰＡＧＥ后,呈现两个蛋白带,分子量分利为６５ｋＤ和３５ｋＤ。纯酶的Ｎｉ含量为０．６２ｍｏｌＮｉ／ｍｏｌ氢酶。在磷酸缓冲液中其活性的最适ｐＨ为６．５。ＤＣＩＰ、亚甲蓝、铁氰化钾、细胞色素Ｃ均可作为氢酶的电子受体,其中以ＤＣＩＰ为最适。     

Isolation, sequence, and expression in Escherichia coli of an unusual thioredoxin gene from the cyanobacterium Anabaena sp. strain PCC 7120.

Two sequences with homology to a thioredoxin oligonucleotide probe were detected by Southern blot analysis of Anabaena sp. strain PCC 7120 genomic DNA. One of the sequences was shown to code for a protein with 37% amino acid identity to thioredoxins from Escherichia coli and Anabaena sp. strain PCC 7119. This is in contrast to the usual 50% homology observed among most procaryotic thioredoxins. One gene was identified in a library and was subcloned into a pUC vector and used to transform E. coli strains lacking functional thioredoxin. The Anabaena strain 7120 thioredoxin gene did not complement the trxA mutation in E. coli. Transformed cells were not able to use methionine sulfoxide as a methionine source or support replication of T7 bacteriophage or the filamentous viruses M13 and f1. Sequence analysis of a 720-base-pair TaqI fragment indicated an open reading frame of 115 amino acids. The Anabaena strain 7120 thioredoxin gene was expressed in E. coli, and the protein was purified by assaying for protein disulfide reductase activity, using insulin as a substrate. The Anabaena strain 7120 thioredoxin exhibited the properties of a conventional thioredoxin. It is a small heat-stable redox protein and an efficient protein disulfide reductase. It is not a substrate for E. coli thioredoxin reductase. Chemically reduced Anabaena strain 7120 thioredoxin was able to serve as reducing agent for both E. coli and Anabaena strain 7119 ribonucleotide reductases, although with less efficiency than the homologous counterparts. The Anabaena strain 7120 thioredoxin cross-reacted with polyclonal antibodies to Anabaena strain 7119 thioredoxin. However, this unusual thioredoxin was not detected in extracts of Anabaena strain 7120, and its physiological function is unknown.     

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.     

Autotrophic growth of nitrogen-fixingAzospirillum species and partial characterization of hydrogenase from strain CC

Out of 15 strains ofAzospirillum spp. isolated from the roots of different plants, only 4 (CY, M, CC, and AM) were able to grow autotrophically with H₂ and CO₂. All of them showed H₂ uptake in the presence of oxygen or methylene blue and ribulose-1,5-bisphosphate carboxylase activity. Among the four strains, strain CC isolated from the roots ofCenchrus cilliaris showed maximum H₂+O₂ uptake (32.5 l/min. mg protein) as well as H₂ uptake in the presence of methylene blue (41.4 l/min·mg protein) and also the maximum activity of ribulose-1,5-bisphosphate carboxylase (17 units [U]/g protein). The doubling time of this strain under autotrophic growth conditions and at low oxygen concentration (2.5%, vol/vol) was 10 h. At the same O₂ concentration the maximal rates of H₂+O₂ uptake were reached. The distribution of hydrogenase activity among soluble and particulate protein fractions revealed that the hydrogenase ofAzospirillum strain CC is a membrane-bound enzyme. It showed cross-reaction with antibodies raised against the membrane-bound hydrogenase ofAlcaligenes eutrophus. The hydrogenase in intact cells and crude extracts reacted with methylene blue, phenazine methosulfate, and ferricyanide, but not with NAD or FMN. The specific hydrogenase activity, with methylene blue as an acceptor, was 5.71 U/mg protein in crude extract at 9.38 U/mg protein in the membrane suspension. Hydrogen evolution from reduced viologen dyes could not be demonstrated. The hydrogenase is oxygen sensitive and can be optimally stabilized by addition of dithionite to H₂-gased samples.     

Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii

Uptake hydrogenase (EC 1.12) from Azotobacter vinelandii has been purified 250-fold from membrane preparations. Purification involved selective solubilization of the enzyme from the membranes, followed by successive chromatography on DEAE-cellulose, Sephadex G-100, and hydroxylapatite. Freshly isolated hydrogenase showed a specific activity of 110 mumol of H2 uptake (min X mg of protein)-1. The purified hydrogenase still contained two minor contaminants that ran near the front on sodium dodecyl sulfate-polyacrylamide gels. The enzyme appears to be a monomer of molecular weight near 60,000 +/- 3,000. The pI of the protein is 5.8 +/- 0.2. With methylene blue or ferricyanide as the electron acceptor (dyes such as methyl or benzyl viologen with negative midpoint potentials did not function), the enzyme had pH optima at pH 9.0 or 6.0, respectively, It has a temperature optimum at 65 to 70 degrees C, and the measured half-life for irreversible inactivation at 22 degrees C by 20% O2 was 20 min. The enzyme oxidizes H2 in the presence of an electron acceptor and also catalyzes the evolution of H2 from reduced methyl viologen; at the optimal pH of 3.5, 3.4 mumol of H2 was evolved (min X mg of protein)-1. The uptake hydrogenase catalyzes a slow deuterium-water exchange in the absence of an electron acceptor, and the highest rate was observed at pH 6.0. The Km values varied widely for different electron acceptors, whereas the Km for H2 remained virtually constant near 1 to 2 microM, independent of the electron acceptors.     

Further characterization of the [Fe]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757.

The properties of the periplasmic hydrogenase from Desulfovibrio desulfuricans ATCC 7757, previously reported to be a single-subunit protein [Glick, B. R., Martin, W. G., and Martin, S. M. (1980) Can. J. Microbiol. 26, 1214-1223] were reinvestigated. The pure enzyme exhibited a molecular mass of 53.5 kDa as measured by analytical ultracentrifugation and was found to comprise two different subunits of 42.5 kDa and 11 kDa, with serine and alanine as N-terminal residues, respectively. The N-terminal amino acid sequences of its large and small subunits, determined up to 25 residues, were identical to those of the Desulfovibrio vulgaris Hildenborough [Fe]-hydrogenase. D. desulfuricans ATCC 7757 hydrogenase was free of nickel and contained 14.0 atoms of iron and 14.4 atoms of acid-labile sulfur/molecule and had E400, 52.5 mM-1.cm-1. The purified hydrogenase showed a specific activity of 62 kU/mg of protein in the H2-uptake assay, and the H2-uptake activity was higher than H2-evolution activity. The enzyme isolated under aerobic conditions required incubation under reducing conditions to express its maximum activity both in the H2-uptake and 2H2/1H2 exchange reaction. The ratio of the activity of activated to as-isolated hydrogenase was approximately 3. EPR studies allowed the identification of two ferredoxin-type [4Fe-4S]1+ clusters in hydrogenase samples reduced by hydrogen. In addition, an atypical cluster exhibiting a rhombic signal (g values 2.10, 2.038, 1.994) assigned to the H2-activating site in other [Fe]-hydrogenases was detected in partially reduced samples. Molecular properties, EPR spectroscopy, catalytic activities with different substrates and sensitivity to hydrogenase inhibitors indicated that D. desulfuricans ATCC 7757 periplasmic hydrogenase is a [Fe]-hydrogenase, similar in most respects to the well characterized [Fe]-hydrogenase from D. vulgaris Hildenborough.     

四唑(Tetrazolium)对固氮鱼腥藻固氮和氢代谢的影响

固氮鱼腥藻(Anabaena azotica Ley)细胞能还原无色的TTC和NBT分别成为红色或蓝色的甲(月朁)(formazan)沉淀。异形胞还原TTC的速率高于营养细胞。前异形胞及异形胞附近的营养细胞对NBT的还原作用最强。而异形胞对NBT不起还原作用。无论在异形胞形成红色甲(月朁)或在营养细胞形成蓝色甲(月朁)后都抑制固氮酶活性。NBT甲(月朁)对固氮酶活性的抑制作用大于TTC甲(月朁),因为NBT氧化还原电位低于TTC。 TTC和NBT两者都明显地抑制固氮鱼腥藻完整细胞的放氢。因鱼腥藻的放氢是由固氮酶催化的结果。四唑抑制放氢推想是由于它截取了固氮酶催化系统中的电子的缘故。固氮微生物(包括蓝色细菌和根瘤菌)对四唑还原与吸氢酶之间有无相关是一个争论的问题。一些学者认为分离豆科植物体的一些根瘤菌株培养于含有TTC的琼脂培养基,如还原,便可证明这些根瘤菌株能氧化氢;换言之,应用TTC的还原可作为一些根瘤菌的菌落具有吸氢酶的验证。相反,我们发现固氮鱼腥藻还原TTC和NBT之后,都没有影响吸氢的能力。因此,我们推想固氮鱼腥藻对四唑之还原与吸氢酶是没有直接的关系。     

Unusual Gene Arrangement of the Bidirectional Hydrogenase and Functional Analysis of Its Diaphorase Subunit HoxU in Respiration of the Unicellular Cyanobacterium Anacystis nidulans

The bidirectional, NAD⁺-dependent hydrogenase from cyanobacteria is encoded by the structural genes hoxFUYH, which have been found to be clustered, though interspersed with different open reading frames (ORFs), in the heterocystous, N₂-fixing Anabaena variabilis and in the unicellular Synechocystis PCC 6803. In another unicellular, non N₂-fixing cyanobacterium, Anacystis nidulans, hoxF has now been identified as being separated by at least 16 kb from the residual structural genes hoxUYH. An ORF (termed hoxE gene) is located immediately upstream of hoxF in A. nidulans and in Synechocystis. Its deduced amino acid sequence shows similarities to the NuoE subunit of NADH dehydrogenase I of E. coli, to the homologous subunit of respiratory complex I in mitochondria, and also to the first 104 amino acids of HoxF in A. nidulans and Synechocystis. The diversity in the arrangement of hydrogenase genes in cyanobacteria is puzzling. The subunits HoxE, HoxF, and HoxU of the diaphorase part of the bidirectional hydrogenase have been discussed to be shared both by respiratory complex I and bidirectional hydrogenase in cyanobacteria. Different hoxU mutants were obtained by inserting a lacZKm^R cassette into the gene both in A. nidulans and Anacystis PCC 7942. Such mutants showed reduced H₂-evolution activities catalyzed by the bidirectional hydrogenase, but had nonimpaired respiratory O₂-uptake. A common link between respiratory complex I and the diaphorase part of the bidirectional hydrogenase in cyanobacteria may still exist, but this hypothesis could not be verified in the present study by analyzing defined mutants impaired in one of the diaphorase genes. Received: 11 August 1997 / Accepted: 23 September 1997     

Intracellular localization of the hydrogenase in Desulfotomaculum orientis

Abstract Cell extracts of Desulfotomaculum orientis , grown with H₂ plus sulfate as sole energy source, revealed hydrogenase activities between 0.3 and 2 μmol H₂ per min and mg protein when methyl viologen was used as electron acceptor. With benzyl viologen, methylene blue, FAD or FMN, lower activities were found; NAD was not reduced. The hydrogenase activity was strongly inhibited by CuCl₂; however, copper inhibition was not observed with whole cells, indicating that the hydrogenase is located intracellularly. After high-speed centrifugation of cell-free extracts, varying proportions, between 11 and 90%, of the hydrogenase were detected in the soluble fraction, the rest being associated with the membrane fraction.     

Aerobic hydrogenase activity in Anacystis nidulans. The oxyhydrogen reaction.

1. The oxyhydrogen reaction of Anacystis nidulans was studied manometrically and polarographically in whole cells and in cell-free preparations; the activity was found to be associated with the particulate fraction. 2. Besides O2, the isolated membranes reduced artificial electron acceptors of positive redox potential; the reactions were unaffected by O2 levels less than 10--15%; aerobically the artificial acceptors were reduced simultaneously with O2. 3. H2-supported O2 uptake was inhibited by CO, KCN and 2-n-heptyl-8-hydroxyquinoline-N-oxide. Inhibition by CO was partly reversed by strong light. Uncouplers stimulated the oxyhydrogen reaction. 4. The kinetic properties of O2 uptake by isolated membranes were the same in presence of H2 and of other respiratory substrates. 5. Low rates of H2 evolution by the membrane preparations were found in presence of dithionite; methyl viologen stimulated the reaction. 6. The results indicate that under certain growth conditions Anacystis synthesizes a membrane-bound hydrogenase which appears to be involved in phosphorylative electron flow from H2 to O2 through the respiratory chain.     

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.     

Purification and characterization of the hydrogen uptake hydrogenase from the hyperthermophilic archaebacterium Pyrodictium brockii.

Pyrodictium brockii is a hyperthermophilic archaebacterium with an optimal growth temperature of 105 degrees C. P. brockii is also a chemolithotroph, requiring H2 and CO2 for growth. We have purified the hydrogen uptake hydrogenase from membranes of P. brockii by reactive red affinity chromatography and sucrose gradient centrifugation. The molecular mass of the holoenzyme was 118,000 +/- 19,000 Da in sucrose gradients. The holoenzyme consisted of two subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large subunit had a molecular mass of 66,000 Da, and the small subunit had a molecular mass of 45,000 Da. Colorometric analysis of Fe and S content in reactive red-purified hydrogenase revealed 8.7 +/- 0.6 mol of Fe and 6.2 +/- 1.2 mol of S per mol of hydrogenase. Growth of cells in 63NiCl2 resulted in label incorporation into reactive red-purified hydrogenase. Growth of cells in 63NiCl2 resulted in label incorporation into reactive red-purified hydrogenase. Temperature stability studies indicated that the membrane-bound form of the enzyme was more stable than the solubilized purified form over a period of minutes with respect to temperature. However, the membranes were not able to protect the enzyme from thermal inactivation over a period of hours. The artificial electron acceptor specificity of the pure enzyme was similar to that of the membrane-bound form, but the purified enzyme was able to evolve H2 in the presence of reduced methyl viologen. The Km of membrane-bound hydrogenase for H2 was approximately 19 microM with methylene blue as the electron acceptor, whereas the purified enzyme had a higher Km value.     

Isolation and characterization of thioredoxin f from the filamentous cyanobacterium, Anabaena sp. 7119

Two thioredoxin fractions had previously been reported to occur in Anabaena 7119 by Buchanan and co-workers (Yee, B. C., dela Torre, A., Crawford, N. A., Lara, C., Carlson, D. E., and Buchanan, B. B. (1981) Arch. Microbiol. 130, 14-18). These proteins were detected by their ability to activate spinach fructose-1,6-bisphosphatase (Fru-P2-ase). The partially purified proteins resembled similar thioredoxins found in spinach chloroplasts and were designated thioredoxin f (Tf) for the fraction most effective in activating spinach Fru-P2-ase and thioredoxin m (Tm) for the fraction most effective in activating spinach NADPH-malate dehydrogenase. Using the assay system of Yee and co-workers, we were able to separate and purify to homogeneity two thioredoxin fractions from Anabaena extracts. Tm corresponded to the thioredoxin fraction we had isolated and studied previously (Gleason, F. K., and Holmgren, A. (1981) J. Biol. Chem. 256, 8301-8309). The other fraction, Tf, was characterized further. Unlike the thioredoxins found in higher plants, the cyanobacterial thioredoxins do not appear to be related. Anabaena thioredoxin f has a Mr = 25,500 as compared to the more usual Mr = 12,000 for Tm. From a comparison of the amino acid composition, Tf is not obviously a dimer or otherwise related to Tm. Tf has one active center cystine disulfide. Anabaena Tf activates spinach Fru-P2-ase very efficiently but has very little activity with spinach malate dehydrogenase. Anabaena Tf, unlike Tm, does not reduce the homologous ribonucleotide reductase. Anabaena Tf also does not activate a partially purified preparation of Anabaena Fru-P2-ase. We conclude that the cyanobacterial Tf is a unique protein with no structural or functional properties in common with other thioredoxins.     

Comparison of the membrane-bound hydrogenases from Alcaligenes eutrophus H16 and Alcaligenes eutrophus type strain

Whereas the membrane-bound hydrogenase from Alcaligenes eutrophus H16 is an integral membrane protein and can only be solubilized by detergent treatment, the membrane-bound hydrogenase of Alcaligenes eutrophus type strain was found to be present in a soluble form after cell disruption. For the enzyme of A. eutrophus H16 a new, highly effective purification procedure was developed including phase separation with Triton X-114 and triazine dye chromatography on Procion Blue H-ERD-Sepharose. The purification led to an homogeneous hydrogenase preparation with a specific activity of 269 U/mg protein (methylene blue reduction) and a yield of 45%. During purification and storage the enzyme was optimally stabilized by the presence of 0.2 mM MnCl2. The hydrogenase of A. eutrophus type strain was purified from the soluble extract by a similar procedure, however, with less specific activity and activity yield. Comparison of the two purified enzymes revealed no significant differences: They have the same molecular weight, both consist of two different subunits (Mr = 62,000, 31,000) and both have an isoelectric point near pH 7.0. They have the same electron acceptor specificity reacting with similar high rates and similar Km values. The acceptors reduced include viologen dyes, flavins, quinones, cytochrome c, methylene blue, 2,6-dichlorophenolindophenol, phenazine methosulfate and ferricyanide. Ubiquinones and NAD were not reduced. The two hydrogenases were shown to be immunologically identical and both have identical electrophoretic mobility. For the membrane-bound hydrogenase of A. eutrophus H16 it was demonstrated that this type of hydrogenase in its solubilized, purified state is able to catalyze also the reverse reaction, the H2 evolution from reduced methyl viologen.     

Effect of enzyme concentration on apparent specific activity of hydrogenase

The effect of enzyme concentration on the H2-uptake and H2-evolving activities of the reversible hydrogenase from Thiocapsa roseopersicina was examined. In the activity range assayed by a spectrophotometric technique the apparent H2-uptake specific activity varied greatly with hydrogenase concentration. Study of H2-evolving activity measured by the H2 electrode method and compared with a gas chromatographic assay also indicated that specific activity was highly dependent on enzyme concentration. The results indicate that the widely applied hydrogenase assays give systematically erroneous specific activity values. These assays should be used only for relative measurements and the hydrogenase concentration in the reaction mixture should be kept constant. To make the data from various laboratories comparable the assay parameters should be standardized.