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R H Burris

Publications and source records attributed to R H Burris.

At least 73 records · Page 4Linked to original sources
PubMed1979

Changes in the EPR signal of dinitrogenase from Azotobacter vinelandii during the lag period before hydrogen evolution begins.

During the lag period before H2 is evolved by the nitrogenase system, the EPR signal of dinitrogenase decreases steadily, indicating transfer of electrons into dinitrogenase. The rate constant for the decrease in amplitude of the EPR signal, the steady state rate of H2 evolution from nitrogenase, and the length of the lag period have been measured. The data suggest that H2 is evolved only after dinitrogenase has been reduced by 2 electrons/molybdenum. The electrons that have been transfered into dinitrogenase during the lag period are not evolved as H2 upon denaturation of dinitrogenase. The existence of a lag indicates that the two nitrogenase proteins dissociate after every electron transfer. The lag occurs and the nitrogenase proteins dissociate under a variety of conditions of pH and temperature.

Azotobacter↗
PubMed1979

Iron-sulfur clusters in the molybdenum-iron protein component of nitrogenase. Electron paramagnetic resonance of the carbon monoxide inhibited state.

Carbon monoxide inhibits reduction of dinitrogen (N2) by purified nitrogenase from Azotobacter vinelandii and Clostridium pasteurianum in a noncompetitive manner (Kii and Kis = 1.4 X 10(-4) and 4.5 X 10(-4) and 7 X 10(-4) atm and 14 X 10(-4) atm for the two enzymes, respectively). The onset of inhibition is within the turnover time of the enzyme, and CO does not affect the electron flux to the H2-evolving site. The kinetics of CO inhibition of N2 reduction are simple, but CO inhibition of acetylene reduction is complicated by substrate inhibition effects. When low-temperature (approximately 13 K) electron paramagnetic resonance (EPR) spectra of CO-inhibited nitrogenase are examined, it is found that low concentrations of CO ([CO] = [enzyme]) induce the appearance of a signal with g values near 2.1, 1.98, and 1.92 with t1/2 approximately 4 s, while higher concentrations of CO lead to the appearance of a signal with g values near 2.17, 2.1, and 2.05 with a similar time course. The MoFe proteins from Rhizobium japonicum and Rhodospirillum rubrum, reduced with Azotobacter Fe protein in the presence of CO, give similar results. Under conditions which promote the accumulation of H2 in the absence of CO, an additional EPR signal with g values near 2.1, 2.0, and 1.98 is observed. The use of Azotobacter nitogenase components enriched selectively with 57Fe or 95Mo, as well as the use of 13CO, permitted the assignment of the center(s) responsible for the induced signals. Only 57Fe, when present in the MoFe protein, yielded broadened EPR signals. It is suggested that the MoFe protein of nitrogenase contains one or more iron-sulfur clusters of the type found in the simple ferrodoxins. It is further proposed that the CO-induced signals arise from states of the MoFe protein in which CO inhibits electron flow to the N2-reducing site so that the iron-sulfur cluster achieves steady-state net charges of -1 (high CO complex) and -3 (low CO complex) in analogy to the normal paramagnetic states of high-potential iron-sulfur proteins and ferredoxins, respectively. The "no-CO" signal may be either an additional center or the N2-reducing site with H2 bound competitively.

Azotobacter↗
PubMed1979

Purification and properties of the particulate hydrogenase from the bacteroids of soybean root nodules.

The uptake hydrogenase (hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1) from the bacteroids of soybean root nodules infected with Rhizobium japonicum 110 has been purified and characterized. Bacteroids were prepared, then broken by sonication. The particulate enzyme was solubilized by treatment with Triton X-100 and further purified by polyethylene glycol fractionation, DEAE-cellulose and Sephadex G-100 chromatography. The specific activity has been increased 196-fold to 19.6 units/mg protein. The molecular weight is 63 300 as determined by gel filtration and 65 300 as determined by SDS-polyacrylamide gel electrophoresis, indicating that the enzyme is a monomer. The enzyme is O2 sensitive, with a half-life of 70 min when exposed to air. The pH optimum of the solubilized enzyme is near 5.5; the Km for H2 is 1.4 microM. Suitable electron acceptors are methylene blue, ferricyanide, 2,6-dichlorophenolindophenol, and cytochrome c. Benzyl viologen is reduced slowly; methyl viologen, NAD(P)+, FAD, FMN, and O2 are not reduced. The optimum temperature for activity is 65-70 degrees C with an activation energy of 9.2 kcal. H2 evolution by the enzyme has been demonstrated. The hydrogenase is well-suited to function in an environment where all the available H2 is generated in situ.

Cytochrome c Group↗
PubMed1979

Removal of an adenine-like molecule during activation of dinitrogenase reductase from Rhodospirillum rubrum.

During the activation of the inactive dinitrogenase reductase from Rhodospirillum rubrum, an adenine-like molecules is lost and phosphate is found on both active and inactive forms of the protein. ATP and divalent metals are required for activation of the reduced protein, but ATP is not required for activation of phenazine methosulfate-oxidized dinitrogenase reductase. Snake venom diesterase and spleen diesterase have no effect on the inactive protein; alkaline phosphatase removes phosphate from the activated protein but not from the inactive protein. ATP binds to both active and inactive forms of the protein.

Adenine↗
PubMed1978

Nitrogenase: properties of the catalytically inactive complex between the Azotobacter vinelandii MoFe protein and the Clostridium pasteurianum Fe protein.

The catalytically inactive complex generated by the combination of the Azotobacter vinelandii MoFe protein (Av1) and the Clostridium pasteurianum Fe protein (Cp2) inhibits N2 reduction, C2H3 reduction, H+ reduction and ATP hydrolysis catalyzed by the homologous nitrogenases. Kinetic data indicate that the inactive complex consists of two molecules of Cp2 to one molecule of Av1, with values for the inhibitor constant in the range of 1--10 nM. Inhibition of C. pasteurianum nitrogenase by Av1 produces a lag phase in acetylene reduction that increases with increasing Av1. The lag phase is found only at levels of Av1 sufficient to keep the ratio of Cp2 : Cp1 lower than 2. Gel filtration of a mixture of Av1 and Cp2 provides evidence for complex formation and indicates that each Av1 molecule binds more than one Cp2 molecule. The Av1-Cp2 complex binds two molecules of MgATP per molecule of Cp2. MgATP is not required for complex formation, but complex formation lowers the MgATP-Cp2 dissociation constant approx. 3-fold. Av1 protects the iron-sulfur center in Cp2 completely against the MgATP-induced reaction with chelators. This provides additional evidence for formation of the Av1-Cp2 complex and together with the results of the MgATP-binding studies suggests that the two binding sites for MgATP are some distance away from the iron-sulfur site on Cp2.

Adenosine Triphosphate↗
PubMed1978

Kinetic studies on electron transfer and interaction between nitrogenase components from Azotobacter vinelandii.

Kinetic properties of electron transfer by nitrogenase of Azotobacter vinelandii are dependent on the concentration of the two components of nitrogenase. An excess of the MoFe protein inhibits electron transfer in a distinctive manner, and the inhibition is reversed by increasing levels of reductant. The saturation curve for Fe protein is hyperbolic, indicating that only one Fe protein molecule per MoFe protein is required for full activity in ATP hydrolysis and electron transfer. These results can be interpreted on the basis of a complex between the Fe protein and the MoFe protein that dissociates rapidly during turnover. Both 2:1 and 1:1 complexes (Fe-MoFe) are active. Dithionite appears to be a relatively poor reductant for nitrogenase from Azotobacter vinelandii, whereas azotobacter flavodoxin is much better. In the presence of the flavodoxin it is possible to increase the specific activity of the Fe protein more than 50% relative to its activity with dithionite alone as a reductant; specific activities greater than 3000 nmol of C2H4 formed min(-1) (mg of Fe protein)(-1) have been observed.

Adenosine Triphosphate↗
PubMed1978

Purification and properties of nitrogenase from Rhodospirillum rubrum, and evidence for phosphate, ribose and an adenine-like unit covalently bound to the iron protein.

1. The molybdenum-iron (Mo-Fe) protein, iron (Fe) protein and the activating factor of nitrogenase from Rhodospirillum rubrum were purified. 2. The Mo-Fe protein has properties similar to those of the Mo-Fe proteins of other nitrogen-fixing organisms. 3. The Fe protein is similar to other Fe proteins with respect to its molecular weight, metal composition and e.p.r. signal. 4. The Fe protein is different from other Fe proteins in that it apparently has two types of subunits rather than one, its u.v. spectrum has an extra peak, and phosphate, ribose and an adenine-like unit are covalently bound to the protein. The presence of these non-protein groups on the protein may explain the requirement for activation of R. rubrum Fe protein.

Adenine↗
PubMed1978

Nitrogenase from Bacillus polymyxa. Purification and properties of the component proteins.

A purification procedure is described for the components of Bacillus polymyxa nitrogenase. The procedure requires the removal of interfering mucopolysaccharides before the two nitrogenase proteins can be purified by the methods used with other nitrogenase components. The highest specific activities obtained were 2750 nmol C2H4 formed . min-1 . mg-1 MoFe protein and 2521 nmol C2H4 formed . min-1 . mg-1 Fe protein. The MoFe protein has a molecular weight of 215 000 and contains 2 molybdenum atoms, 33 iron atoms and 21 atoms of acid-labile sulfur per protein molecule. The Fe protein contains 3.2 iron atoms and 3.6 acid-labile sulfur atoms per molecule of 55 500 molecular weight. Each Fe protein binds two ATP molecules. The EPR spectra are similar to those of other nitrogenase proteins. MgATP changes the EPR of the Fe protein from a rhombic to an axial-type signal.

Adenosine Triphosphate↗
PubMed1978

The nitrogenase system of Spirillum lipoferum.

The nitrogenase system of Spirillum lipoferum was separated into three components, the normal Mo-Fe and Fe proteins as well as an activating factor for the Fe protein. The rate of activation is increased by Mn2+ or by an excess of Mg2+, and the process requires ATP.

Acetylene↗
PubMed1978

The kinetics of methyl viologen oxidation and reduction by the hydrogenase from Clostridium pasteurianum.

A mechanism for the reduction and oxidation of methyl viologen by Clostridium pasteurianum hydrogenase (hydrogen:ferredoxin oxidoreductase, EC 1.12.7.1) is proposed. Double reciprocal plots for methyl viologen reduction and oxidation at pH values 7.0-9.85 are linear, and the plots for reduction and oxidation are intersecting. Such data are consistent with a mechanism in which the H2 and one methyl viologen bind (either in order or randomly) with subsequent reduction and release of the methyl viologen. A second methyl viologen then is bound, reduced and released. Comparison of the calculated Keq' with the Haldane expression in which both methyl viologens react at the same rate show a large difference. This difference indicates that the two methyl viologens react at different rates. Addition of oxidized electron carriers inhibits the hydrogen-deuterium exchange reaction (i.e., the exchange of protons between H2 and 2H2O). CO reversibly inhibits methyl viologen reduction and is competitive vs. H2. O2 acts as an irreversible inhibitor.

Carbon Monoxide↗
PubMed1978

Nitrogenase: the reaction between the Fe protein and bathophenanthrolinedisulfonate as a probe for interactions with MgATP.

The reaction between the Fe(II) chelating agent, bathophenanthrolinedisulfonate, and the iron-sulfur cluster in the Fe protein of nitrogenase from Clostridium pasteurianum has been studied. This reaction is greatly accelerated by the presence of MgATP. Analysis of the relationship between reaction rate and concentration of MgATP supports a model in which both of two binding sites for MgATP on the Fe protein must be occupied before the protein undergoes a conformational change, allowing the iron-sulfur site to react rapidly with chelator. This model is also consistent with presently available data on equilibrium binding of MgATP to the Fe protein. MgADP inhibits the effect of MgATP on the chelator reaction in a manner which suggests that MgADP binds strongly to one of the MgATP sites and more weakly to the other. Loss of enzymic activity due to exposure to O2 or 0 degrees C is accompanied by a decrease in the ATP-specific chelator reaction. Hence, this reaction was used to estimate the concentration of active iron-sulfur centers for the purpose of computing the extinction coefficient of the Fe protein, giving the value delta epsilon 430nm(ox-red) = 6600 M-1 cm-1.

Adenosine Triphosphate↗
PubMed1978

Modes of reduction of nitrogen in heterocysts isolated from Anabaena species.

N2 fixation (acetylene reduction) has been studied with heterocysts isolated from Anabaena cylindrica and Anabaena 7120. In the presence of ATP and at very low concentrations of sodium dithionite, reducing equivalents for activity of nitrogenase in these cells can be derived from several compounds. In the dark, D-glucose 6-phosphate, 6-phosphogluconate and DL-isocitrate support acetylene reduction via NADPH. In the light, reductant can be generated by Photosystem I.

Acetylene↗
PubMed1978

Nitrogenase and nitrogenase reductase associate and dissociate with each catalytic cycle.

Nitrogenase and nitrogenase reductase dissociate after each electron is transferred between them, as shown by the occurrence of a lag phase approximately as long as the average turnover time of nitrogenase before hydrogen evolution occurs. Because nitrogenase was present in the reaction mixture in large excess over nitrogenase reductase, the electrons donated by nitrogenase reductase must have been distributed randomly over all of the nitrogenase present. This is accomplished by nitrogenase reductase molecules associating randomly with nitrogenase molecules for each cycle of electrons transferred. The fact that ATP is hydrolyzed without a lag indicates both that electron transfer occurs during the lag and the ATP hydrolysis is coupled to electron transfer from nitrogenase reductase to nitrogenase and not to substrate reduction. The observations support the suggestion that it now is desirable to alter nomenclature to designate the MoFe protein as nitrogenase and the Fe protein as nitrogenase reductase.

Adenosine Triphosphate↗
PubMed1978

Complementary functioning of the component proteins of nitrogenase from several bacteria.

The nitrogenase proteins from eight organisms have been highly purified, and a survey of their cross-reactions shows that the nitrogenase proteins from a wide variety of organisms can interact with one another. An active cross-reaction is the complementary functioning of the MoFe protein and the Fe protein from different organisms. Of 64 possible combinations of component proteins, 8 yielded homologous nitrogenases (components from the same organism); 45 of the 56 possible heterologous crosses generated active hybrid nitrogenases; 4 heterologous crosses yielded no measurable nitrogenase activity but did form inactive tight-binding complexes; 6 crosses did not give measurable activity; and 1 cross was not made. All these crosses were assayed for acetylene reduction, and some also were assayed for ammonia formation, hydrogen evolution, and ATP hydrolysis activity. The activity generated by combining two complementary heterologous nitrogenase components depended on pH, component ratio, and protein concentration, the same factors that determine the activity of homologous nitrogenases. However, several crosses showed an unusual dependency on component ratio and protein concentration, and some cross-reactions showed interesting ATP hydrolysis activity.

Acetylene↗