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Production of superoxide radicals by soluble hydrogenase from Alcaligenes eutrophus H16.

ABSTRACT: The soluble hydrogenase (hydrogen-NAD+ oxidoreductase, EC 1.12.1.2) of Alcaligenes eutrophus H16 was shown to be stabilized by oxidation with oxygen and ferricyanide as long as electron donors and reducing compounds were absent. The simultaneous presence of H2, NADH and O2 in the enzyme solution, however, caused an irreversible inactivation of hydrogenase that was dependent on the O2 concentration. The half-life periods of 4 degrees C under partial pressures of 0.1, 5, 20 and 50% O2 were 11, 5, 2.5 and 1.5 h respectively. Evidence has been obtained that hydrogenase produces superoxide free radical anions (O2-.), which were detected by their ability to oxidize hydroxylamine to nitrite. The correlation between O2 concentration, nitrite formation and inactivation rates and the stabilization of hydrogenase by addition of superoxide dismutase indicated that superoxide radicals are responsible for enzyme inactivation. During short-term activity measurements (NAD+ reduction, H2 evolution from NADH), hydrogenase activity was inhibited by O2 only very slightly. In the presence of 0.7 mM-O2 an inhibition of about 20% was observed.

SUBMITTER: Schneider K

PROVIDER: S-EPMC1162581 | biostudies-other | 1981 Jan

REPOSITORIES: biostudies-other

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Positive transcriptional feedback controls hydrogenase expression in Alcaligenes eutrophus H16.

Project description:The protein HoxA is the central regulator of the Alcaligenes eutrophus H16 hox regulon, which encodes two hydrogenases, a nickel permease and several accessory proteins required for hydrogenase biosynthesis. Expression of the regulatory gene hoxA was analyzed. Screening of an 8-kb region upstream of hoxA with a promoter probe vector localized four promoter activities. One of these was found in the region immediately 5' of hoxA; the others were correlated with the nickel metabolism genes hypA1, hypB1, and hypX. All four activities were independent of HoxA and of the minor transcription factor sigma(54). Translational fusions revealed that hoxA is expressed constitutively at low levels. In contrast to these findings, immunoblotting studies revealed a clear fluctuation in the HoxA pool in response to conditions which induce the hox regulon. Quantitative transcript assays indicated elevated levels of hyp mRNA under hydrogenase-derepressing conditions. Using interposon mutagenesis, we showed that the activity of a remote promoter is required for hydrogenase expression and autotrophic growth. Site-directed mutagenesis revealed that P(MBH), which directs transcription of the structural genes of the membrane-bound hydrogenase, contributes to the expression of hoxA under hydrogenase-derepressing conditions. Thus, expression of the hox regulon is governed by a positive feedback loop mediating amplification of the regulator HoxA. These results imply the existence of an unusually large (ca. 17,000-nucleotide) transcript.

| S-EPMC94088 | biostudies-literature

Reversible and irreversible effects of nitric oxide on the soluble hydrogenase from Alcaligenes eutrophus H16.

Project description:The effects of NO on the H2-oxidizing and diaphorase activities of the soluble hydrogenase from Alcaligenes eutrophus H16 were investigated. With fully activated enzyme, NO (8-150 nM in solution) inhibited H2 oxidation in a time- and NO-concentration-dependent process. Neither H2 nor NAD+ appeared to protect the enzyme against the inhibition. Loss of activity in the absence of an electron acceptor was about 10 times slower than under turnover conditions. The inhibition was partially reversible; approx. 50% of full activity was recoverable after removal of the NO. Recovery was slower in the absence of an electron acceptor than in the presence of H2 plus an electron acceptor. The diaphorase activity of the unactivated hydrogenase was not affected by NO concentrations of up to 200 microM in solution. Exposure of the unactivated hydrogenase to NO irreversibly inhibited the ability of the enzyme to be fully activated for H2-oxidizing activity. The enzyme also lost its ability to respond to H2 during activation in the presence of NADH. The results are interpreted in terms of a complex inhibition that displays elements of (1) a reversible slow-binding inhibition of H2-oxidizing activity, (2) an irreversible effect on H2-oxidizing activity and (30 an irreversible inhibition of a regulatory component of the enzyme. Possible sites of action for NO are discussed.

| S-EPMC1135101 | biostudies-other

The Alcaligenes eutrophus H16 hoxX gene participates in hydrogenase regulation.

Project description:Nucleotide sequence analysis revealed a 1,791-bp open reading frame in the hox gene cluster of the gram-negative chemolithotroph Alcaligenes eutrophus H16. In order to investigate the biological role of this open reading frame, we generated an in-frame deletion allele via a gene replacement strategy. The resulting mutant grew significantly more slowly than the wild type under lithoautotrophic conditions (6.1 versus 4.2 h doubling time). A reduction in the level of the soluble NAD-reducing hydrogenase (60% of the wild-type activity) was shown to be the cause of the slow lithoautotrophic growth. We used plasmid-borne gene fusions to monitor the expression of the operons encoding the soluble and membrane-bound hydrogenases. The expression of both operons was lower in the mutant than in the wild-type strain. These results suggest that the newly identified gene, designated hoxX, encodes a regulatory component which, in conjunction with the transcriptional activator HoxA, controls hydrogenase synthesis.

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A megaplasmid-borne anaerobic ribonucleotide reductase in Alcaligenes eutrophus H16.

Project description:The conjugative 450-kb megaplasmid pHG1 is essential for the anaerobic growth of Alcaligenes eutrophus H16 in the presence of nitrate as the terminal electron acceptor. We identified two megaplasmid-borne genes (nrdD and nrdG) which are indispensable under these conditions. Sequence alignment identified significant similarity of the 76.2-kDa gene product NrdD and the 30.9-kDa gene product NrdG with anaerobic class III ribonucleotide reductases and their corresponding activases. Deletion of nrdD and nrdG in A. eutrophus abolished anaerobic growth and led to the formation of nondividing filamentous cells, a typical feature of bacteria whose DNA synthesis is blocked. Enzyme activity of NrdD-like ribonucleotide reductases is dependent on a stable radical at a glycine residue in a conserved C-terminal motif. A mutant of A. eutrophus with a G650A exchange in NrdD showed the DNA-deficient phenotype as the deletion strain, suggesting that G650 forms the glycyl radical. Analysis of transcriptional and translational fusions indicate that nrdD and nrdG are cotranscribed and that the translation efficiency of nrdD is 40-fold higher than that of nrdG. Thus, the two proteins NrdD and NrdG are not synthesized at a stoichiometric level.

| S-EPMC93980 | biostudies-literature

A gene complex coding for the membrane-bound hydrogenase of Alcaligenes eutrophus H16.

Project description:One of the key enzymes in the chemolithoautotrophic metabolism of Alcaligenes eutrophus H16 is a dimeric, membrane-associated hydrogenase. The genetic determinants of this enzyme are located on the endogenous megaplasmid pHG1 (G. Eberz, C. Hogrefe, C. Kortlüke, A. Kamienski, and B. Friedrich, J. Bacteriol. 168:636-641, 1986). Complementation studies showed that the information required for the formation of active membrane-bound hydrogenase occupies more than 7.5 kb of megaplasmid DNA. We cloned and sequenced this region and identified the genes encoding the two hydrogenase subunits (hoxK and hoxG). The nucleotide sequence contains nine additional closely spaced open reading frames. Immunoelectron microscopy showed that the gene product of one of these open reading frames (hoxM) is involved in the process leading to the attachment of hydrogenase to the membrane. Other open reading frames may encode additional processing functions and components of a hydrogenase-linked electron transport chain. Analysis of Tn5-B21-mediated transcriptional fusions provided evidence that the structural genes and accessory functions belong to at least three coordinately regulated transcriptional units.

| S-EPMC207698 | biostudies-other

Role of hydrogen in the activation and regulation of hydrogen oxidation by the soluble hydrogenase from Alcaligenes eutrophus H16.

Project description:The activation kinetics of the H2-oxidizing activity of the soluble hydrogenase from Alcaligenes eutrophus H16 were investigated. Activation with Na2S2O4 plus 101 kPa H2 resulted in a rapid increase in activity over 1 h and constant activity after 3 h incubation. Less-stable activations were achieved if enzyme was incubated with Na2S2O4 under 1 kPa H2 or 101 kPa N2. The enzyme could also be partly activated either with NADH alone or with H2 alone. The level of activity obtained with both 101 kPa H2 and NADH present was greater than that obtained with either 101 kPa H2 or NADH alone. Activation with H2 plus NADH was virtually independent of NADH concentration but highly dependent on H2 concentration. The effects of various concentrations of H2 and constant concentration of NADH on the level of activation were the same whether H2 oxidation was assayed by H2-dependent Methylene Blue or NAD+ reduction. Diaphorase activity did not require activation and was little affected by the treatments that activated H2-oxidizing activity. The results suggest that H2 plays an important role in regulating the level of H2-oxidizing activity in this soluble hydrogenase.

| S-EPMC1135100 | biostudies-other

Two isofunctional nitric oxide reductases in Alcaligenes eutrophus H16.

Project description:Two genes, norB and norZ, encoding two independent nitric oxide reductases have been identified in Alcaligenes eutrophus H16. norB and norZ predict polypeptides of 84.5 kDa with amino acid sequence identity of 90%. While norB resides on the megaplasmid pHG1, the norZ gene is located on a chromosomal DNA fragment. Amino acid sequence analysis suggests that norB and norZ encode integral membrane proteins composed of 14 membrane-spanning helices. The region encompassing helices 3 to 14 shows similarity to the NorB subunit of common bacterial nitric oxide reductases, including the positions of six strictly conserved histidine residues. Unlike the Nor enzymes characterized so far from denitrifying bacteria, NorB and NorZ of A. eutrophus contain an amino-terminal extension which may form two additional helices connected by a hydrophilic loop of 203 amino acids. The presence of a NorB/NorZ-like protein was predicted from the genome sequence of the cyanobacterium Synechocystis sp. strain PCC6803. While the common NorB of denitrifying bacteria is associated with a second cytochrome c subunit, encoded by the neighboring gene norC, the nor loci of A. eutrophus and Synechocystis lack adjacent norC homologs. The physiological roles of norB and norZ in A. eutrophus were investigated with mutants disrupted in the two genes. Mutants bearing single-site deletions in norB or norZ were affected neither in aerobic nor in anaerobic growth with nitrate or nitrite as the terminal electron acceptor. Inactivation of both norB and norZ was lethal to the cells under anaerobic growth conditions. Anaerobic growth was restored in the double mutant by introducing either norB or norZ on a broad-host-range plasmid. These results show that the norB and norZ gene products are isofunctional and instrumental in denitrification.

| S-EPMC179608 | biostudies-other

Structure and function of a periplasmic nitrate reductase in Alcaligenes eutrophus H16.

Project description:Alcaligenes eutrophus H16 shows three distinct nitrate reductase activities (U. Warnecke-Eberz and B. Friedrich, Arch. Microbiol. 159:405-409, 1993). The periplasmic enzyme, designated NAP (nitrate reductase, periplasmic), has been isolated. The 80-fold-purified heterodimeric enzyme catalyzed nitrate reduction with reduced viologen dyes as electron donors. The nap genes were identified in a library of A. eutrophus H16 megaplasmid DNA by using oligonucleotide probes based on the amino-terminal polypeptide sequences of the two NAP subunits. The two structural genes, designated napA and napB, code for polypeptides of 93 and 18.9 kDa, respectively. Sequence comparisons indicate that the putative gene products are translated with signal peptides of 28 and 35 amino acids, respectively. This is compatible with the fact that NAP activity was found in the soluble fraction of cell extracts and suggests that the mature enzyme is located in the periplasm. The deduced sequence of the large subunit, NAPA, contained two conserved amino-terminal stretches of amino acids found in molybdenum-dependent proteins such as nitrate reductases and formate dehydrogenases, suggesting that NAPA contains the catalytic site. The predicted sequence of the small subunit, NAPB, revealed two potential heme c-binding sites, indicating its involvement in the transfer of electrons. An insertion in the napA gene led to a complete loss of NAP activity but did not abolish the ability of A. eutrophus to use nitrate as a nitrogen source or as an electron acceptor in anaerobic respiration. Nevertheless, the NAP-deficient mutant showed delayed growth after transition from aerobic to anaerobic respiration, suggesting a role for NAP in the adaptation to anaerobic metabolism.

| S-EPMC206666 | biostudies-other

Three trans-acting regulatory functions control hydrogenase synthesis in Alcaligenes eutrophus.

Project description:Random Tn5 mutagenesis of the regulatory region of megaplasmid pHG1 of Alcaligenes eutrophus led to the identification of three distinct loci designated hoxA, hoxD, and hoxE. Sequencing of the hoxA locus revealed an open reading frame which could code for a polypeptide of 482 amino acids with a molecular mass of 53.5 kDa. A protein of comparable apparent molecular mass was detected in heterologous expression studies with a plasmid-borne copy of the hoxA gene. Amino acid alignments revealed striking homologies between HoxA and the transcriptional activators NifA and NtrC of Klebsiella pneumoniae and HydG of Escherichia coli. HoxA- mutants of A. eutrophus lacked both NAD-reducing soluble hydrogenase and membrane-bound hydrogenase. In HoxA- mutants, the synthesis of beta-galactosidase from a hoxS'-'lacZ operon fusion was drastically reduced, indicating that HoxA is essential for the transcription of hydrogenase genes. Mutants defective in hoxD and hoxE also lacked the catalytic activities of the two hydrogenases; however, in contrast to HoxA- mutants, they contained immunologically detectable NAD-reducing soluble hydrogenase and membrane-bound hydrogenase proteins, although at a reduced level. The low hydrogenase content in the HoxD- and HoxE- mutants correlated with a decrease in beta-galactosidase synthesized under the direction of a hoxS'-'lacZ operon fusion. Thus, hoxD and hoxE apparently intervene both in the regulation of hydrogenase synthesis and in subsequent steps leading to the formation of catalytically active enzymes.

| S-EPMC207712 | biostudies-other

Structural gene (nirS) for the cytochrome cd1 nitrite reductase of Alcaligenes eutrophus H16.

Project description:Denitrification by Alcaligenes eutrophus H16 is genetically linked to megaplasmid pHG1. Unexpectedly, the gene encoding the nitrite reductase (nirS) was identified on chromosomal DNA. The nirS product showed extensive homology with periplasmic nitrite reductases of the heme cd1-type. Disruption of nirS abolished nitrite-reducing ability, indicating that NirS is the enzyme essential for denitrification in A.eutrophus.

| S-EPMC168373 | biostudies-other

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