Purification of hydrogenases by affinity chromatography on Procion Red-agarose.
ABSTRACT: The agarose-coupled triazine dye Procion Red HE-3B has been demonstrated to be applicable as an affinity gel for the purification of five diverse hydrogenases, namely the soluble, NAD-specific and the membrane-bound hydrogenase of Alcaligenes eutrophus, the membrane-bound hydrogenase of the N2-fixing Alcaligenes latus, the reversible H2-evolving and the unidirectional H2-oxidizing hydrogenase of Clostridium pasteurianum. In the case of the soluble hydrogenase of A. eutrophus, chromatography on Procion Red-agarose even permitted the separation of inactive from active enzyme, thus yielding a 2-3-fold increase in specific activity. For the homogeneous enzyme preparation obtained after two column steps (Procion Red-agarose, DEAE-Sephacel), a specific activity of 121 mumol of H2 oxidized/min per mg of protein was determined. Kinetic studies with free Procion Red provided evidence that the diverse hydrogenases are competitively inhibited by the dye, each with respect to the electron carrier (NAD, Methylene Blue, Methyl Viologen), indicating a specific interaction between Procion Red and the catalytic centres of the enzymes. For the highly purified preparations of the soluble and the membrane-bound hydrogenase of A. eutrophus, in 50 mM-potassium phosphate, pH 7.0, Ki values for Procion Red of 103 and 19 microM have been determined.
Project description:Heterologous complementation studies using Alcaligenes eutrophus H16 as a recipient identified a hydrogenase-specific regulatory DNA region on megaplasmid pHG21-a of the related species Alcaligenes hydrogenophilus. Nucleotide sequence analysis revealed four open reading frames on the subcloned DNA, designated hoxA, hoxB, hoxC, and hoxJ. The product of hoxA is homologous to a transcriptional activator of the family of two-component regulatory systems present in a number of H2-oxidizing bacteria. hoxB and hoxC predict polypeptides of 34.5 and 52.5 kDa, respectively, which resemble the small and the large subunits of [NiFe] hydrogenases and correlate with putative regulatory proteins of Bradyrhizobium japonicum (HupU and HupV) and Rhodobacter capsulatus (HupU). hoxJ encodes a protein with typical consensus motifs of histidine protein kinases. Introduction of the complete set of genes on a broad-host-range plasmid into A. eutrophus H16 caused severe repression of soluble and membrane-bound hydrogenase (SH and MBH, respectively) synthesis in the absence of H2. This repression was released by truncation of hoxJ. H2-dependent hydrogenase gene transcription is a typical feature of A. hydrogenophilus and differs from the energy and carbon source-responding, H2-independent mode of control characteristic of A. eutrophus H16. Disruption of the A. hydrogenophilus hoxJ gene by an in-frame deletion on megaplasmid pHG21-a led to conversion of the regulatory phenotype: SH and MBH of the mutant were expressed in the absence of H2 in response to the availability of the carbon and energy source. RNA dot blot analysis showed that HoxJ functions on the transcriptional level. These results suggest that the putative histidine protein kinase HoxJ is involved in sensing molecular hydrogen, possibly in conjunction with the hydrogenase-like polypeptides HoxB and HoxC.
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.
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.
Project description:Oxidation of molecular hydrogen catalyzed by [NiFe] hydrogenases is a widespread mechanism of energy generation among prokaryotes. Biosynthesis of the H2-oxidizing enzymes is a complex process subject to positive control by H2 and negative control by organic energy sources. In this report we describe a novel signal transduction system regulating hydrogenase gene (hox) expression in the proteobacterium Alcaligenes eutrophus. This multicomponent system consists of the proteins HoxB, HoxC, HoxJ*, and HoxA. HoxB and HoxC share characteristic features of dimeric [NiFe] hydrogenases and form the putative H2 receptor that interacts directly or indirectly with the histidine protein kinase HoxJ*. A single amino acid substitution (HoxJ*G422S) in a conserved C-terminal glycine-rich motif of HoxJ* resulted in a loss of H2-dependent signal transduction and a concomitant block in autophosphorylating activity, suggesting that autokinase activity is essential for the response to H2. Whereas deletions in hoxB or hoxC abolished hydrogenase synthesis almost completely, the autokinase-deficient strain maintained high-level hox gene expression, indicating that the active sensor kinase exerts a negative effect on hox gene expression in the absence of H2. Substitutions of the conserved phosphoryl acceptor residue Asp55 in the response regulator HoxA (HoxAD55E and HoxAD55N) disrupted the H2 signal-transduction chain. Unlike other NtrC-like regulators, the altered HoxA proteins still allowed high-level transcriptional activation. The data presented here suggest a model in which the nonphosphorylated form of HoxA stimulates transcription in concert with a yet unknown global energy-responsive factor.
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.
Project description:The soluble hydrogenase (hydrogen-NAD+ oxidoreductase, EC 126.96.36.199) 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.
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.
Project description:Nickel is an essential component of all H2-uptake hydrogenases. A fragment of DNA that complements a H2-uptake-deficient but nickel-cured mutant strain (JHK7) of Bradyrhizobium japonicum was isolated and sequenced. This 4.5-kb DNA fragment contains four open reading frames designated as ORF1, hupN, hupO, and hupP, which encode polypeptides with predicted masses of 17, 40, 19, and 63.5 kDa, respectively. The last three open reading frames (hupNOP) are most likely organized as an operon with a putative sigma 54-type promoter. Based on its hydropathy profile, HupN is predicted to be a transmembrane protein. It has 56% identity to the previously described HoxN (high-affinity nickel transport protein) of Alcaligenes eutrophus. A subclone (pJF23) containing the hupNOP genes excluding ORF1 completely complemented (in trans) strain JHK7 for hydrogenase activity in low nickel conditions. pJF26 containing only a functional hupN complemented the hydrogenase activity of mutant strain JHK7 to 30-55% of the wild-type level. Mutant strain JHK70, with a chromosomal deletion in hupP but with an intact hupNO, showed greater activities than pJF26-complemented JHK7 but still had lower activities than the wild type at all nickel levels tested. pJF25, containing the entire hupO and hupP, but without hupN (a portion of hupN was deleted), did not complement hydrogenase activity of mutant strain JHK7. The results suggest that the products of the hupNOP operon are all involved in nickel incorporation/metabolism into the hydrogenase apoprotein. Based on (previous) nickel transport studies of strain JHK7, the hupNOP genes appear not to be involved in nickel transport by whole cells. The deleterious effects on hydrogenase expression are most pronounced by lack of the HupN product.
Project description:Azotobacter vinelandii contains a heterodimeric, membrane-bound [NiFe]hydrogenase capable of catalyzing the reversible oxidation of H2. The beta and alpha subunits of the enzyme are encoded by the structural genes hoxK and hoxG, respectively, which appear to form part of an operon that contains at least one further potential gene (open reading frame 3 [ORF3]). In this study, determination of the nucleotide sequence of a region of 2,344 bp downstream of ORF3 revealed four additional closely spaced or overlapping ORFs. These ORFs, ORF4 through ORF7, potentially encode polypeptides with predicted masses of 22.8, 11.4, 16.3, and 31 kDa, respectively. Mutagenesis of the chromosome of A. vinelandii in the area sequenced was carried out by introduction of antibiotic resistance gene cassettes. Disruption of hoxK and hoxG by a kanamycin resistance gene abolished whole-cell hydrogenase activity coupled to O2 and led to loss of the hydrogenase alpha subunit. Insertional mutagenesis of ORF3 through ORF7 with a promoterless lacZ-Kmr cassette established that the region is transcriptionally active and involved in H2 oxidation. We propose to call ORF3 through ORF7 hoxZ, hoxM, hoxL, hoxO, and hoxQ, respectively. The predicted hox gene products resemble those encoded by genes from hydrogenase-related operons in other bacteria, including Escherichia coli and Alcaligenes eutrophus.
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.