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D J Arp

Publications and source records attributed to D J Arp.

At least 73 records · Page 4Linked to original sources
PubMed1989

Carbonyl sulfide inhibition of CO dehydrogenase from Rhodospirillum rubrum.

Carbonyl sulfide (COS) has been investigated as a rapid-equilibrium inhibitor of CO oxidation by the CO dehydrogenase purified from Rhodospirillum rubrum. The kinetic evidence suggests that the inhibition by COS is largely competitive versus CO (Ki = 2.3 microM) and uncompetitive versus methylviologen as electron acceptor (Ki = 15.8 microM). The data are compatible with a ping-pong mechanism for CO oxidation and COS inhibition. Unlike the substrate CO, COS does not reduce the iron-sulfur centers of dye-oxidized CO dehydrogenase and thus is not an alternative substrate for the enzyme. However, like CO, COS is capable of protecting CO dehydrogenase from slow-binding inhibition by cyanide. A true binding constant (KD) of 2.2 microM for COS has been derived on the basis of the saturable nature of COS protection against cyanide inhibition. The ability of CO, CO2, COS, and related CO/CO2 analogues to reverse cyanide inhibition of CO dehydrogenase is also demonstrated. The kinetic results are interpreted in terms of two binding sites for CO on CO dehydrogenase from R. rubrum.

Aldehyde Oxidoreductases↗
PubMed1989

Hydrogen inhibition of nitrogen reduction by nitrogenase in isolated soybean nodule bacteroids.

Dihydrogen, a by-product of biological nitrogen fixation, is a competitive inhibitor of N(2) reduction by nitrogenase. To evaluate the significance of H(2) inhibition in vivo, we have measured the apparent inhibition constant for H(2) inhibition of N(2) reduction in Bradyrhizobium japonicum bacteroids isolated from soybean nodules. The rate of N(2) reduction was measured as ammonia production by bacteroids incubated in a buffer containing 200 micromolar leghemoglobin and 10 millimolar succinate under 0.02 atmosphere O(2) and various concentrations of N(2) and H(2). The apparent inhibition constant for H(2) under these conditions was determined to be approximately 0.03 atmosphere. This relatively low value strengthens the proposal that H(2) inhibition of N(2) reduction may be a significant factor in lowering the efficiency of nitrogen fixation in legume nodules.

Journal Article↗
PubMed1989

Immunological relationship among hydrogenases.

We examined the immunological cross-reactions of 11 different hydrogenase antigens with 9 different hydrogenase antibodies. Included were antibodies and antigens of both subunits of the hydrogenases of Bradyrhizobium japonicum and Thiocapsa roseopersicina. The results showed a strong relationship among the Ni-Fe dimeric hydrogenases. The two subunits of Ni-Fe dimeric hydrogenases appeared immunologically distinct: specific interactions occurred only when antibodies to the 60- and 30-kilodalton subunits reacted with the 60- and 30-kilodalton-subunit antigens. The interspecies cross-reactions suggested that at least one conserved protein region exists among the large subunits of these enzymes, whereas the small subunits are less conserved. Antibodies to the Fe-only bidirectional hydrogenase of Clostridium pasteurianum reacted with the Desulfovibrio vulgaris bidirectional hydrogenase. Surprisingly, antibodies to the clostridial uptake hydrogenase did not react with any of the Fe-only bidirectional hydrogenases but did react with several of the Ni-Fe dimeric hydrogenases. The two hydrogenases from C. pasteurianum were found to be quite different immunologically. The possible relationship of these findings to the structure and catalytic functions of hydrogenase are discussed.

Antibodies↗
PubMed1989

Cyanide inactivation of hydrogenase from Azotobacter vinelandii.

The effects of cyanide on membrane-associated and purified hydrogenase from Azotobacter vinelandii were characterized. Inactivation of hydrogenase by cyanide was dependent on the activity (oxidation) state of the enzyme. Active (reduced) hydrogenase showed no inactivation when treated with cyanide over several hours. Treatment of reversibly inactive (oxidized) states of both membrane-associated and purified hydrogenase, however, resulted in a time-dependent, irreversible loss of hydrogenase activity. The rate of cyanide inactivation was dependent on the cyanide concentration and was an apparent first-order process for purified enzyme (bimolecular rate constant, 23.1 M-1 min-1 for CN-). The rate of inactivation decreased with decreasing pH. [14C]cyanide remained associated with cyanide-inactivated hydrogenase after gel filtration chromatography, with a stoichiometry of 1.7 mol of cyanide bound per mol of inactive enzyme. The presence of saturating concentrations of CO had no effect on the rate or extent of cyanide inactivation of hydrogenases. The results indicate that cyanide can cause a time-dependent, irreversible inactivation of hydrogenase in the oxidized, activatable state but has no effect when hydrogenase is in the reduced, active state.

Azotobacter↗
PubMed1988

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.

Acetylene↗
PubMed1988

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

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.

Alcaligenes↗
PubMed1988

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

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.

Alcaligenes↗
PubMed1988

Effects of alcohols on the reactivity and stability of Azotobacter vinelandii hydrogenase.

The effects of alcohols on the reactivity of Azotobacter vinelandii hydrogenase were investigated. Hydrogenase catalyzed H2 oxidation coupled to methylene blue, benzyl viologen, or phenazine methosulfate when in the presence of solvents containing 15 or 40% ethanol or 40% methanol or 2-propanol. In general, the Km's for the electron acceptors were increased substantially by the presence of the alcohols, while the Km for H2 was not altered in a solvent containing 40% ethanol. Calculation of the apparent maximum velocities for H2 oxidation in the presence of alcohols indicated that the maximum velocity was not decreased in most cases. In contrast, the rates of both H2 evolution and isotope exchange by hydrogenase were substantially decreased when solvent containing alcohol. Hydrogenase was inactivated by 100% ethanol with a half-life of 17 s. Hydrogenase from A. vinelandii was stable when stored in alcohol/buffer solvents at 20 degrees C or below. However, the thermal stability of hydrogenase was greatly decreased by inclusion of an alcohol in the solvent. When incubated at 55 degrees C in a solvent containing 40% ethanol, activity decreased in a first-order process with a half-life of 7 min. When incubated at the same temperature in aqueous buffer, no loss of activity was observed over 30 min.

1-Propanol↗
PubMed1988

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.

Alcaligenes↗
PubMed1987

Redox-dependent subunit dissociation of Azotobacter vinelandii hydrogenase in the presence of sodium dodecyl sulfate.

Hydrogenases catalyze the reversible activation of dihydrogen. We have previously demonstrated that the purified hydrogenase from the nitrogen-fixing microorganism Azotobacter vinelandii is an alpha beta dimer (98,000 Da) with subunits of 67,000 (alpha) and 31,000 (beta) daltons and that this enzyme contains iron and nickel. The enzyme can be purified anaerobically in the presence of dithionite in a fully active state that is irreversibly inactivated by exposure to O2. Analysis of this hydrogenase by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) following boiling in SDS yields two protein staining bands corresponding to the alpha and beta subunits. However, when this enzyme was treated with SDS (25-65 degrees C) for up to 30 min under anaerobic/reductive conditions and then analyzed by anaerobic SDS-PAGE, a protein staining band corresponding to an apparent molecular mass of 58,000 Da was observed that stained for hydrogenase activity. Analysis of the 58,000-Da activity staining band by a Western immunoblot or a second aerobic SDS-polyacrylamide gel revealed that this protein actually consisted of both the alpha and beta subunits. Thus, the activity staining band (apparent 58,000 Da) represents the 98,000-Da dimer migrating abnormally on SDS-PAGE. Treatment of the anaerobically purified hydrogenase with SDS under aerobic conditions or under anaerobic conditions with electron acceptors prior to electrophoresis resulted in no activity staining band and the separated alpha and beta subunits. A. vinelandii hydrogenase was also purified under aerobic conditions in an inactive O2 stable form that can be activated by removal of oxygen followed by addition of reductant. This enzyme (as isolated), the activated form, and the reoxidized form were analyzed for their stability toward denaturation by SDS. We conclude that the dissociation of the A. vinelandii hydrogenase subunits in SDS is controlled by the redox state of the enzyme suggesting an important role of one or more redox sites in controlling the structure of this enzyme.

2,6-Dichloroindophenol↗
PubMed1987

Quantification and removal of some contaminating gases from acetylene used to study gas-utilizing enzymes and microorganisms.

Acetylene generated from various grades of calcium carbide and obtained from commercial- and purified-grade acetylene cylinders was shown to contain high concentrations of various contaminants. Dependent on the source of acetylene, these included, at maximal values, H(2) (0.023%), O(2) (0.779%), N(2) (3.78%), PH(3) (0.06%), CH(4) (0.073%), and acetone (1 to 10%). The concentration of the contaminants in cylinder acetylene was highly dependent on the extent of cylinder discharge. Several conventional methods used to partially purify cylinder acetylene were compared. A small-scale method for extensively purifying acetylene is described. An effect of acetylene quality on acetylene reduction assays conducted with purified nitrogenase from Azotobacter vinelandii was demonstrated.

Journal Article↗
PubMed1987

Regulation of H2 oxidation activity and hydrogenase protein levels by H2, O2, and carbon substrates in Alcaligenes latus.

Regulation of H2 oxidation activity and hydrogenase protein levels in the free-living hydrogen bacterium Alcaligenes latus was investigated. Hydrogenase activity was induced when heterotrophically grown cells were transferred to chemolithoautotrophic conditions, i.e., in the presence of H2 and absence of carbon sources, with NH4Cl as the N source. Under these conditions, H2 oxidation activity was detectable after 30 min of incubation and reached near-maximal levels by 12 h. The levels of hydrogenase protein, as measured by a Western blot (immunoblot) assay of the hydrogenase large subunit, increased in parallel with activity. This increase suggested that the increased H2 oxidation activity was due to de novo synthesis of hydrogenase protein. H2 oxidation activity was controlled over a surprisingly wide range of H2 concentrations, between 0.001 and 30% in the gas phase. H2 oxidation activity was induced to high levels between 2 and 12.5% O2, and above 12.5% O2, H2 oxidation activity was inhibited. Almost all organic carbon sources studied inhibited the expression of hydrogenase, although none repressed hydrogenase synthesis completely. In all cases examined, hydrogenase protein, as detected by Western blot, paralleled the level of H2 oxidation activity, suggesting that control of hydrogenase activity was mediated through changes in hydrogenase protein levels.

Alcaligenes↗
PubMed1986

Reversible inactivation of the O2-labile hydrogenases from Azotobacter vinelandii and Rhizobium japonicum.

Hydrogenases catalyze the reversible activation of dihydrogen. The hydrogenases from the aerobic, N2-fixing microorganisms Azotobacter vinelandii and Rhizobium japonicum are nickel- and iron-containing dimers that belong to the group of O2-labile enzymes. Exposure of these hydrogenases to O2 results in an irreversible inactivation; therefore, these enzymes are purified anaerobically in a fully active state. We describe in this paper an electron acceptor-requiring and pH-dependent, reversible inactivation of these hydrogenases. These results are the first example of an anaerobic, reversible inactivation of the O2-labile hydrogenases. The reversible inactivation required the presence of an electron acceptor. The rate of inactivation was first-order, with similar rates observed for methylene blue, benzyl viologen, and phenazine-methosulfate. The rate of inactivation was also dependent on the pH. However, increasing the pH of the enzyme in the absence of an electron acceptor did not result in inactivation. Thus, the reversible inactivation was not a result of high pH alone. The inactive enzyme could not be reactivated by H2 or other reductants at high pH. Titration of enzyme inactivated at high pH back to low pH was also ineffective at reactivating the enzyme. However, if reductants were present during this titration, the enzyme could be fully reactivated. The temperature dependence of inactivation yielded an activation energy of 44 kJ X mol-1. Gel filtration chromatography of active and inactive hydrogenase indicated that neither dissociation nor aggregation of the dimer hydrogenase was responsible for this reversible inactivation. We propose a four-state model to describe this reversible inactivation.

Azotobacter↗
PubMed1986

Purification to homogeneity of Azotobacter vinelandii hydrogenase: a nickel and iron containing alpha beta dimer.

Azotobacter vinelandii hydrogenase has been purified to homogeneity from membranes. The enzyme was solubilized with Triton X-100 followed by ammonium sulfate-hexane extractions to remove lipids and detergent. The enzyme was then purified by carboxymethyl-Sepharose and octyl-Sepharose column chromatography. All purification steps were performed under anaerobic conditions in the presence of dithionite and dithiothreitol. The enzyme was purified 143-fold from membranes to a specific activity of 124 mumol of H2 uptake . min-1 . mg protein-1. Nondenaturing polyacrylamide gel electrophoresis of the hydrogenase revealed a single band which stained for both activity and protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands corresponding to peptides of 67,000 and 31,000 daltons. Densitometric scans of the SDS-gel indicated a molar ratio of the two bands of 1.07 +/- 0.05. The molecular weight of the native enzyme was determined by three different methods. While gel permeation gave a molecular weight of 53,000, sucrose density gradient centrifugation and native polyacrylamide gel electrophoresis gave molecular weights of 98,600 +/- 10,000 and 98,600 +/- 2,000, respectively. We conclude that the A. vinelandii hydrogenase is an alpha beta dimer (98,000 daltons) with subunits of 67,000 and 31,000 daltons. Analyses for nickel and iron indicated 0.68 +/- 0.01 mol Ni/mol hydrogenase and 6.6 +/- 0.5 mol Fe/mol hydrogenase. The isoelectric point of the enzyme was 6.1 +/- 0.01. In addition, several catalytic properties of the enzyme have been examined. The Km for H2 was 0.86 microM, and H2 evolution was observed in the presence of reduced methyl viologen. The pH profile of enzyme activity with methylene blue as the electron acceptor has been determined, along with the Km and Vmax for various electron acceptors.

Azotobacter↗
PubMed1986

Investigation of the H2-oxidizing activities of Alcaligenes eutrophus H16 membranes with artificial electron acceptors, respiratory inhibitors and redox-spectroscopic procedures.

Membrane particles, prepared from cells of Alcaligenes eutrophus H16 by lysozyme treatment and 100 000 X g centrifugation, catalyzed a H2-dependent reduction of methylene blue, menadione, 2,6-dichlorophenol-indophenol (DCPIP), and O2. While the reaction with methylene blue was not altered by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), the H2-dependent reductions of menadione, DCPIP and O2 were strongly inhibited, indicating that in these reaction components of the respiratory chain other than the membrane-bound hydrogenase were involved. The effect of pentane extraction of membranes on the H2-dependent reductions of methylene blue and menadione were different from those of DCPIP and O2. This suggested that ubiquinone might not be involved in the pathway of the electrons from H2 to methylene blue or menadione, while it might be involved in the pathway to DCPIP and O2. Because the H2-dependent reduction of menadione is sensitive to HQNO, it follows that HQNO might bind to a site upstream of ubiquinone. Further evidence for this hypothesis came from a new technique to record UV and visible redox-difference spectra of membranes under the conditions of a steady-state electron flow. HQNO did not increase the reduction level of ubiquinone relative to the cytochromes. Neither HQNO nor menadione had any influence on the redox difference patterns of the cytochromes as determined with low temperature and room temperature spectroscopy.

2,6-Dichloroindophenol↗
PubMed1985

Difference spectroscopy with respiratory membranes of an H2-oxidizing microorganism layered between two gas-permeable, plastic foils: a procedure that allows measurements in the ultraviolet range.

A method for recording redox difference spectra of respiratory membranes which minimizes light scattering and results in increased sensitivity and spectral range when compared to the traditional technique of recording spectra of respiratory particles in suspension is described. Membranes were sandwiched between two gas-permeable, plastic foils, placed in a sealed cuvette, and gassed with H2 as reductant or O2 as oxidant. The membranes of the H2-oxidizing microorganism (Alcaligenes eutrophus H16) used in this work contain a hydrogenase which is coupled to the electron transport chain, thereby allowing H2 to serve as a source of electrons. With this procedure, spectra of both cytochromes and quinones were recorded. By using mixtures of H2 and O2, spectra of cytochromes between the fully oxidized and fully reduced states were recorded.

Alcaligenes↗