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R H Burris

Publications and source records attributed to R H Burris.

At least 55 records · Page 3Linked to original sources
PubMed1983

Inhibition of nitrogenase-catalyzed NH3 formation by H2.

We have investigated the inhibition by H2 (D2) of NH3 formation by nitrogenase from Klebsiella pneumoniae and have confirmed that the inhibition is competitive vs. N2. D2 inhibits NH3 formation by diverting nitrogenase from production of NH3 to production of HD (one electron per HD). By careful exclusion of N2 from the reaction mixture, we have been able to place an upper limit on N2-independent HD formation by nitrogenase, under 1 atm of D2, at 1% of the total electron flux. Formation of NH3 and formation of HD were inhibited identically by CO. We observed that as the ratio of dinitrogenase to dinitrogenase reductase is increased, the ratio of HD formed to NH3 formed rises, and D2 becomes a stronger inhibitor of N2 reduction. This may be caused in part by an accompanying increase that is observed in the Km of nitrogenase for N2. We propose a model for D2 inhibition of NH3 formation in which D2 and N2 complete for the same form of nitrogenase. According to our proposal, when N2 reacts with nitrogenase, either N2 reduction proceeds to completion if H2 (D2) is absent or, if D2 already is bound to nitrogenase, N2 reduction is aborted and two molecules of HD are produced at the net expense of one electron per HD. Key consequences of the model are that it predicts that H2 (D2) is a competitive inhibitor of NH3 formation and that the apparent Km (N2) for formation of HD and NH3 may differ.

Ammonia↗
PubMed1983

Influence of pN2 and pD2 on HD formation by various nitrogenases.

Formation of HD from D2 has been demonstrated with nitrogenase preparations from Azotobacter vinelandii, Clostridium pasteurianum, Klebsiella pneumoniae, and Azospirillum sp. We conclude that the formation of HD from D2 is a general property of nitrogenases. However, the nitrogenases differ in their Ki values for D2 (N2 fixation) and in their rates of catalyzing HD formation; among the nitrogenases tested, C. pasteurianum nitrogenase had the lowest activity for formation of HD. When contaminating N2 was removed from the atmospheres above reaction mixtures, less than 1% of the total electron flux in the system was directed to HD formation; hence, we doubt that N2-independent HD formation is significant. A working hypothesis is suggested that operates without invoking an N2-independent reaction for forming HD.

Anaerobiosis↗
PubMed1983

The role of Mg2+ and Mn2+ in the enzyme-catalysed activation of nitrogenase Fe protein from Rhodospirillum rubrum.

The activation of the Fe protein of nitrogenase (Rr2) from glutamate-grown Rhodospirillum rubrum by activating enzyme (AE) was investigated. AE is confirmed to have Mr about 20 000 and is shown to operate catalytically. There is a role in activation for metal-ion-ATP, which can be met by either MnATP or MgATP. There is also a site of action for free metal ions. This site prefers Mn2+ (apparent Kd approx. 20 microM) over Mg2+ (apparent Kd approx. 20 mM) by a factor of 1000-fold. Non-activated Rr2 does not contain this binding site. MnATP is an inhibitor of C2H2 reduction, and excess Mg2+ inhibits both AE activity and C2H2 reduction, when each is studied independently under otherwise optimal conditions. The activity of AE is increased in normal reaction mixtures (in which AE activity and nitrogenase activity occur simultaneously) by Mg2+ concentrations in excess of ATP concentrations; this occurs because the excess Mg2+ prevents ATP from chelating the free Mn2+ necessary for optimal AE activity.

Acetylene↗
PubMed1983

Adenine nucleotide levels in and nitrogen fixation by the cyanobacterium Anabaena sp. strain 7120.

Adenine nucleotide levels were determined in whole filaments of Anabaena sp. 7120 grown under different N2-fixing or non-N2-fixing conditions. These were compared with levels in isolated heterocysts, Rhodospirillum rubrum, and Azotobacter vinelandii. Adenine nucleotides in whole filaments of Anabaena sp. do not reflect the energetic expense of N2 fixation as they do in R. rubrum and A. vinelandii. However, adenine nucleotide levels in heterocysts were similar to the levels found in N2-fixing R. rubrum, i.e., an ATP:ADP ratio near 1 and an energy charge between 0.5 and 0.7. Nitrogenase activity was only 50% of optimal in permeabilized heterocysts at an exogenous ATP:ADP ratio of 3.33. Hydrogen, which increases acetylene reduction activity, also causes a transient increase (2 to 5 min) in the ATP:ADP ratio. Hydrogen has little effect on energy charge.

Adenine Nucleotides↗
PubMed1983

Permeabilization of isolated heterocysts of Anabaena sp. strain 7120 with detergent.

Heterocysts isolated from Anabaena sp. strain 7120 with lysozyme plus sonication were permeabilized with the cationic detergent cetyltrimethylammonium bromide, and they then exhibited comparable acetylene reduction activity in the light and dark with an ATP-regenerating system plus dithionite. The detergent diminished the effect of H2 in enhancing acetylene reduction.

Acetylene↗
PubMed1981

Kinetic mechanism of the hydrogen-oxidizing hydrogenase from soybean nodule bacteroids.

The kinetic mechanism of the unidirectional H2-oxidizing hydrogenase from soybean nodule bacteroids has been investigated with highly purified enzyme. Measurements of the Km for H2 vary from 0.97 to 2.6 microM, and the Km for methylene blue varies from 6 to 17 microM. With H2 and methylene blue as substrates, the initial velocity patterns are intersecting. High levels of methylene blue are inhibitory (KI =2.4 mM): the inhibition is competitive vs. H2. CO is a competitive inhibitor of H2 (KI = 157 microM) and noncompetitive vs. methylene blue. O2 inactivates the enzyme (t 1/2 approximately 1 h) but also is a reversible inhibitor of hydrogenase activity upon short exposure to low concentrations. Inhibition by O2 is uncompetitive vs. H2 and noncompetitive vs. methylene blue. Hydrogenase was not inhibited by C2H2; preincubation under C2H2 inactivates the enzyme. Reduced methyl and benzyl viologens support low rates of H2 evolution by the hydrogenase. The Km for reduced methyl viologen is 11 microM. H2 is a potent inhibitor of H2 evolution: the inhibition is noncompetitive vs. reduced methyl viologen. The hydrogenase will catalyze a low rate of exchange in the reaction between D2 and H2O to form both HD and H2. We propose a two-site ping-pong mechanism for the enzyme in which H2 is reversibly activated at one site and e- carriers interact at the second site.

Binding, Competitive↗
PubMed1981

Light and dark reactions of the uptake hydrogenase in anabaena 7120.

Reactions of the uptake hydrogenase from Anabaena 7120 (A.T.C.C. 27893, Nostoc muscorum) were examined in whole filaments, isolated heterocysts, and membrane particles. Whole filaments or isolated heterocysts that contained nitrogenase consumed H(2) in the presence of C(2)H(2) or N(2) in a light-dependent reaction. If nitrogenase was inactivated by O(2) shock, filaments catalyzed H(2) uptake to an unidentified endogenous acceptor in the light. Addition of NO(3) (-) or NO(2) (-) enhanced these rates. Isolated heterocysts consumed H(2) in the dark in the presence of electron acceptors with positive midpoint potentials, and these reactions were not enhanced by light. With acceptors of negative midpoint potential, significant light enhancement of H(2) uptake occurred. Maximum rates of light-dependent uptake were approximately 25% of the maximum dark rates observed. Membrane particles prepared from isolated heterocysts showed similar specificity for electron acceptors. These particles catalyzed a cyanide-sensitive oxyhydrogen reaction that was inactivated by O(2) at O(2) concentrations above 2%. Light-dependent H(2) uptake to low potential acceptors by these particles was inhibited by dibromothymoquinone but was insensitive to cyanide. In the presence of O(2), light-dependent H(2) uptake occurred simultaneously with the oxyhydrogen reaction. The pH optima for both types of H(2) uptake were near 7.0. These results further clarify the role of uptake hydrogenase in donating electrons to both the photosynthetic and respiratory electron transport chains of Anabaena.

Journal Article↗
PubMed1981

Physiological reactions of the reversible hydrogenase from anabaena 7120.

The reversible hydrogenase from Anabaena 7120 appeared when O(2) was continuously removed from a growing culture. Activity increased further when cells were incubated under argon in the dark or in the light plus 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Hydrogenase existed in an inactive state during periods of O(2) evolution. It could be reductively activated by exposure to reduced methyl viologen or by dark, anaerobic incubation. Hydrogenase-containing cells evolved H(2) slowly during dark anaerobic incubations, and the rate of H(2) evolution was increased by illumination with low intensity light. Light enhancement of H(2) evolution was of short duration and was eliminated by the ferredoxin antagonist disalicylidene diaminopropane. Physiological acceptors that supported H(2) uptake included NO(3) (-), NO(2) (-), and HSO(3) (-), and light had a slight influence on the rate of H(2) uptake with these acceptors. Low levels of O(2) supported H(2) uptake, but higher concentrations of O(2) inactivated the hydrogenase. Hydrogen uptake with HCO(3) (-) as acceptor was the most rapid reaction measured, and it was strictly light-dependent. It occurred only at low light intensities, and higher light intensities restored normal O(2)-evolving photosynthesis. It is suggested that hydrogenase is present to capture exogenous H(2) as a source of reducing equivalents during growth in anaerobic environments.

Journal Article↗
PubMed1981

Organic Acid contents of soybean: age and source of nitrogen.

The organic acid content of soybean (Glycine max v. Hodgson) root, stem, and leaf tissue was followed for 33 days after germination. Malonate was the predominant acid in leaf and root tissue, whereas fumarate was predominant in the stem. The malonate concentrations of the stem and root showed similar variations with time, but the leaf response was quite different. In nodules from 33-day-old plants, malonate was the predominant acid. Malonate levels in root and nodule tissue of 33-day-old plants were depressed in response to the addition of either nitrate or ammonia. Nodule tissue had a higher malonate concentration on nitrate nitrogen than it did on ammonium nitrogen, whereas root tissue had the higher malonate concentration on ammonium nitrogen. Analysis of organic acid concentrations of roots as a function of age and distance from the root tip in young soybean seedlings revealed a zone consistently high in malate. The malonate level in the entire root rose dramatically in tissue of age 96 to 120 hours.

Journal Article↗
PubMed1981

Biosynthesis of malonate in roots of soybean seedlings.

Many plants accumulate malonate, but it was shown earlier that malonate does not accumulate as a deadend product of metabolism in soybean (Glycine max v. Hodgson tissues. The metabolism of malonate in the soybean plant at the whole tissue and enzymic level was followed, and the pathway of malonate biosynthesis in young soybean root tissue was shown to be via acetyl-coenzyme A carboxylase.

Journal Article↗
PubMed1981

Inhibition of nitrogenase activity by NH+4 in Rhodospirillum rubrum.

Nitrogenase activities and the patterns of in vivo inhibition of nitrogenase by NH+4 were compared in Rhodospirillum rubrum grown under several conditions of nitrogen availability. In cells grown on N2 or glutamate plus N2, nitrogenase activity was relatively low and was totally inhibited by added NH+4 in 15 to 20 min. In contrast, cells grown on glutamate alone displayed higher nitrogenase activity, and NH+4 had very little effect. Cells grown on limiting amounts of NH+4 had lower nitrogenase activity, but NH+4 produced little inhibitory effect. Uptake of NH+4 could be demonstrated under all of these conditions, and this uptake was blocked by DL-methionine-dl-sulfoximine. The data indicated that cells not recently exposed to NH+4 had no mechanism for rapidly turning off nitrogenase activity in response to sudden additions of NH+4. In contrast, cells grown in the presence of N2, which form NH+4 internally, inhibited nitrogenase activity relatively quickly in response to added NH+4.

Ammonia↗
PubMed1981

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.

Cyanobacteria↗
PubMed1981

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.

Cyanobacteria↗
PubMed1981

Effect of oxygen on acetylene reduction by photosynthetic bacteria.

The effect of dissolved oxygen concentration on nitrogenase activity was studied in three species of photosynthetic bacteria. The O2 concentration in the cell suspension was measured with an O2 electrode inserted into the reaction vessel. Acetylene reduction by whole cells of Rhodopseudomonas capsulata, Rhodospirillum rubrum, and Chromatium vinosum strain D was inhibited 50% by 0.73, 0.32, and 0.26 microM O2, respectively. The inhibition of the activity by O2 in R. capsulata usually was reversed completely by reestablishing anaerobic conditions. In R. rubrum and C. vinosum the inhibition was only partially reversible. The respiration rate of R. capsulata was the highest of the three, that of R. rubrum was intermediate, and that of C. vinosum was lowest. R. capsulata and R. rubrum cells were broken after their acetylene reduction activity in vivo had been completely inhibited by O2, and nitrogenase was found to be active in vitro. A concentration of cyanide that did not affect acetylene reduction activity, but which inhibited 75 to 90% of the O2 uptake by whole cells of R. capsulata, shifted the O2 concentration causing 50% inhibition of nitrogenase activity from 0.73 microM to 2.03 microM. These results are in accordance with the assumption that within a limited range of O2 concentrations, the respiratory activity of the cells is enough to scavenge the O2 and to keep the interior of the cells essentially anaerobic. It is suggested that O2 inhibits nitrogenase activity by competing for a limited supply of electrons. When cyanide is present, respiration is slower but is adequate to keep the nitrogenase environment in the cell anaerobic. The lower respiration rate may allow a greater proportion of the electrons to be used for acetylene reduction.

Acetylene↗
PubMed1980

Electron allocation to alternative substrates of Azotobacter nitrogenase is controlled by the electron flux through dinitrogenase.

The electron flux through dinitrogenase (MoFe protein, protein containing Mo and Fe) from Azotobacter vinelandii controls the relative effectiveness of alternative substrates as electron acceptors in the nitrogenase system. The electron flux through dinitrogenase reductase (Fe protein) or the concentration of MgATP do not directly control electron allocation but rather control it via their influence on the electron flux through dinitrogenase. Kinetic properties of substrate reduction were studied as a function of the electron flux through dinitrogenase. N2 was most effective at high electron fluxes, whereas H+ was the most effective acceptor at very low rates of electron flow through dinitrogenase. The Km for acetylene was dependent on the electron flux through dinitrogenase, whereas the Km for N2 was much less sensitive to this electron flux. The lag period before the onset of acetylene reduction was proportional to the turnover time of dinitrogenase, and was approx. 12 times greater than the dinitrogenase turnover time. pH has effects on the electron allocation to substrates beyond that expected from the effect of pH on the electron flux; thus, pH may alter the relative ability of the nitrogenase enzyme system to reduce alternative substrates.

Azotobacter↗
PubMed1980

Role of magnesium adenosine 5'-triphosphate in the hydrogen evolution reaction catalyzed by nitrogenase from Azotobacter vinelandii.

We have investigated the role of MgATP in the reaction catalyzed by nitrogenase from Azotobacter vinelandii. There is a rapid burst of ATP hydrolysis in the pre-steady-state reaction that occurs on the same time scale as the electron transfer from dinitrogenase reductase to dinitrogenase. This burst corresponds to two ATP's hydrolyzed per electron transferred between the two proteins. Two MgATP molecules are bound to dinitrogenase reductase with dissociation constants of 430 microM and 220 microM. Investigation of the effect of MgATP concentration on the pre-steady-state kinetics of electron transfer from dinitrogenase reductase to dinitrogenase showed that there are two MgATP's required for this reaction, and the Km values are 220 microM and 970 microM. These values are similar to the dissociation constants for MgATP from dinitrogenase reductase and indicate that electron transfer between the two proteins is substantially slower than the binding and dissociation of MgATP from dinitrogenase reductase. The Km values for MgATP in steady-state H2 evolution were 390 microM and 30 microM. The decrease in the value of the second Km indicates that a slow, irreversible step occurs after the electron transfer from dinitrogenase reductase to dinitrogenase. It is possible to predict quantitatively the steady-state kinetics from the pre-steady-state kinetics, and this shows that the MgATP dependence of electron transfer is sufficient to account for effects of MgATP concentration on the steady-state H2 evolution catalyzed by nitrogenase. The hydrolysis of two ATP molecules when an electron is transferred between the two proteins of the nitrogenase system is sufficient to account for all of the ATP hydrolysis occurring in the steady-state reaction. The simplified scheme proposed to account for the MgATP dependency of the nitrogenase reaction indicates that the only role of MgATP is in support of the electron transfer from dinitrogenase reductase to dinitrogenase.

Adenosine Triphosphate↗
PubMed1980

Hydrogenase in actinorhizal root nodules and root nodule homogenates.

Hydrogenases were measured in intact actinorhizal root nodules and from disrupted nodules of Alnus glutinosa, Alnus rhombifolia, Alnus rubra, and Myrica pensylvanica. Whole nodules took up H2 in an O2-dependent reaction. Endophyte preparations oxidized H2 through the oxyhydrogen reaction, but rates were enhanced when hydrogen uptake was coupled to artificial electron acceptors. Oxygen inhibited artifical acceptor-dependent H2 uptake. The hydrogenase system from M. pensylvanica had a different pattern of coupling to various electron acceptors than the hydrogenase systems from the alders; only the bayberry system evolved H2 from reduced viologen dyes.

Actinomycetales↗