Novel structure and redox chemistry of the prosthetic groups of the iron-sulfur flavoprotein sulfide dehydrogenase from Pyrococcus furiosus; evidence for a [2Fe-2S] cluster with Asp(Cys)3 ligands.

The consecutive structural genes for the iron-sulfur flavoenzyme sulfide dehydrogenase, sudB and sudA, have been identified in the genome of Pyrococcus furiosus. The translated sequences encode a heterodimeric protein with an alpha-subunit, SudA, of 52598 Da and a beta-subunit, SudB, of 30686 Da. The alpha-subunit carries a FAD, a putative nucleotide binding site for NADPH, and a [2Fe-2S]2+,+ prosthetic group. The latter exhibit EPR g-values, 2.035, 1.908, 1.786, and reduction potential, Em,8 = +80 mV, reminiscent of Rieske-type clusters; however, comparative sequence analysis indicates that this cluster is coordinated by a novel motif of one Asp and three Cys ligands. The motif is not only found in the genome of hyperthermophilic archaea and hyperthermophilic bacteria, but also in that of mesophilic Treponema pallidum. The beta-subunit of sulfide dehydrogenase contains another FAD, another putative binding site for NADPH, a [3Fe-4S]+,0 cluster, and a [4Fe-4S]2+,+ cluster. The 3Fe cluster has an unusually high reduction potential, Em,8 = +230 mV. The reduced 4Fe cluster exhibits a complex EPR signal, presumably resulting from magnetic interaction of its S = 1/2 spin with the S=2 spin of the reduced 3Fe cluster. The 4Fe cluster can be reduced with deazaflavin/EDTA/light but not with sodium dithionite; however, it is readily reduced with NADPH. SudA is highly homologous to KOD1-GO-GAT (or KOD1-GltA), a single-gene encoded protein in Pyrococcus kodakaraensis, which has been putatively identified as hyperthermophilic glutamate synthase. However, P. furiosus sulfide dehydrogenase does not have glutamate synthase activity. SudB is highly homologous to HydG, the gamma-subunit of P. furiosus NiFe hydrogenase. The latter enzyme also has sulfide dehydrogenase activity. The P. furiosus genome contains a second set of consecutive genes, sudY and sudX, with very high homology to the sudB and sudA genes, and possibly encoding a sulfide dehydrogenase isoenzyme. Each subunit of sulfide dehydrogenase is a primary structural paradigm for a different class of iron-sulfur flavoproteins.

Nitrogenase of Azotobacter vinelandii: kinetic analysis of the Fe protein redox cycle.

Nitrogenase consists of two metalloproteins (Fe protein and MoFe protein) which are assumed to associate and dissociate to transfer a single electron to the substrates. This cycle, called the Fe protein cycle, is driven by MgATP hydrolysis and is repeated until the substrates are completely reduced. The rate-limiting step of the cycle, and substrate reduction, is suggested to be the dissociation of the Fe protein-MoFe protein complex which is obligatory for the reduction of the Fe protein [Thorneley, R. N. F., and Lowe, D. J. (1983) Biochem. J. 215, 393-403]. This hypothesis is based on experiments with dithionite as the reductant. We also tested besides dithionite flavodoxin hydroquinone, a physiological reductant. Two models could describe the experimental data of the reduction by dithionite. The first model, with no reduction of Fe protein bound to MoFe protein, predicts a rate of dissociation of the protein complex of 8.1 s-1. This rate is too high to be the rate-limiting step of the Fe protein cycle (kobs = 3.0 s-1). The second model, with reduction of the Fe protein in the nitrogenase complex, predicts a rate of dissociation of the protein complex of 2.3 s-1, which in combination with reduction of the nitrogenase complex can account for the observed turnover rate of the Fe protein cycle. When flavodoxin hydroquinone (155 microM) was the reductant, the rate of reduction of oxidized Fe protein in the nitrogenase complex (kobs approximately 400 s-1) was 100 times faster than the turnover rate of the cycle with flavodoxin as the reductant (4 s-1). Pre-steady-state electron uptake experiments from flavodoxin hydroquinone indicate that before and after reduction of the nitrogenase complex relative slow reactions take place, which limits the rate of the Fe protein cycle. These results are discussed in the context of the kinetic models of the Fe protein cycle of nitrogenase.

Redox properties and electron paramagnetic resonance spectroscopy of the transition state complex of Azotobacter vinelandii nitrogenase.

Nitrogenase is a two-component metalloenzyme that catalyzes a MgATP hydrolysis driven reduction of substrates. Aluminum fluoride plus MgADP inhibits nitrogenase by stabilizing an intermediate of the on-enzyme MgATP hydrolysis reaction. We report here the redox properties and electron paramagnetic resonance (EPR) signals of the aluminum fluoride-MgADP stabilized nitrogenase complex of Azotobacter vinelandii. Complex formation lowers the midpoint potential of the [4Fe-4S] cluster in the Fe protein. Also, the two-electron reaction of the unique [8Fe-7S] cluster in the MoFe protein is split in two one-electron reactions both with lower midpoint potentials. Furthermore, a change in spin-state of the two-electron oxidized [8Fe-7S] cluster is observed. The implications of these findings for the mechanism of MgATP hydrolysis driven electron transport within the nitrogenase protein complex are discussed.

Pre-steady-state kinetics of nitrogenase from Azotobacter vinelandii. Evidence for an ATP-induced conformational change of the nitrogenase complex as part of the reaction mechanism.

The pre-steady-state electron transfer reactions of nitrogenase from Azotobacter vinelandii have been studied by stopped-flow spectrophotometry. With reduced nitrogenase proteins after the initial absorbance increase at 430 nm (which is associated with electron transfer from the Fe protein to the MoFe protein and is complete in 50 ms) the absorbance decreases, which, dependent on the ratio [Av2]/[Av1], is followed by an increase of the absorbance. The mixing of reductant-free nitrogenase proteins with MgATP leads after 20 ms to a decrease of the absorbance, which could be fitted (from 0. 05 to 1 s) with a single exponential decay with a rate constant kobs = 6.6 +/- 0.8 s-1. This reaction of nitrogenase was measured at different wavelengths. The data indicate the formation of a species with a blue shift of the absorbance of metal-sulfur clusters of nitrogenase from 430 to 360 nm. The absorbance decrease at 430 nm observed (after 50 ms) in the case of the reduced nitrogenase proteins could only be simulated well if, after the initial electron transfer from the Fe protein to the MoFe protein and before dissociation of the nitrogenase complex, an additional reaction was assumed. The rate constant of this reaction was of the same order as the rate constant of the MgATP-dependent pre-steady-state proton production by nitrogenase from A. vinelandii: kobs = 14 +/- 4 s-1 with reduced nitrogenase proteins and kobs = 6 +/- 2 s-1 with dithionite-free nitrogenase proteins (Duyvis, M. G., Wassink, H., and Haaker, H. (1994) Eur. J. Biochem. 225, 881-890). It is proposed that in the presence and absence of reductant, the observed absorbance decrease at 430 nm of nitrogenase is caused by a change of the conformation of the nitrogenase complex, as a consequence of hydrolysis of MgATP.

Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids.

The relationship between the O2 input rate into a suspension of Rhizobium leguminosarum bacteroids, the cellular ATP and ADP pools, and the whole-cell nitrogenase activity during L-malate oxidation has been studied. It was observed that inhibition of nitrogenase by excess O2 coincided with an increase of the cellular ATP/ADP ratio. When under this condition the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, the cellular ATP/ADP ratio was lowered while nitrogenase regained activity. To explain these observations, the effects of nitrogenase activity and CCCP on the O2 consumption rate of R. leguminosarum bacteroids were determined. From 100 to 5 microM O2, a decline in the O2 consumption rate was observed to 50 to 70% of the maximal O2 consumption rate. A determination of the redox state of the cytochromes during an O2 consumption experiment indicated that at O2 concentrations above 5 microM, electron transport to the cytochromes was rate-limiting oxidation and not the reaction of reduced cytochromes with oxygen. The kinetic properties of the respiratory chain were determined from the deoxygenation of oxyglobins. In intact cells the maximal deoxygenation activity was stimulated by nitrogenase activity or CCCP. In isolated cytoplasmic membranes NADH oxidation was inhibited by respiratory control. The dehydrogenase activities of the respiratory chain were rate-limiting oxidation at O2 concentrations (if >300 nM. Below 300 nM the terminal oxidase system followed Michaelis-Menten kinetics (Km of 45 +/- 8 nM). We conclude that (i) respiration in R. leguminosarum bacteroids takes place via a respiratory chain terminating at a high-affinity oxidase system, (ii) the activity of the respiratory chain is inhibited by the proton motive force, and (iii) ATP hydrolysis by nitrogenase can partly relieve the inhibition of respiration by the proton motive force and thus stimulate respiration at nanomolar concentrations of O2.

Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase.

A stable complex is formed between the nitrogenase proteins of Azotobacter vinelandii, aluminium fluoride and MgADP. All nitrogenase activities are inhibited. The complex formation was found to be reversible. An incubation at 50 degrees C recovers nitrogenase activity. The complex has been characterized with respect to protein and nucleotide composition and redox state of the metal-sulfur clusters. Based on the inhibition by aluminium fluoride together with MgADP, it is proposed that a stable transition state complex with nitrogenase is isolated.

Pre-steady-state MgATP-dependent proton production and electron transfer by nitrogenase from Azotobacter vinelandii.

MgATP-dependent pre-steady-state proton production by nitrogenase from Azotobacter vinelandii was studied by monitoring the absorbance changes at 572 nm of the pH indicator o-cresolsulphonphtalein in a weakly buffered solution. The absorbance changes are characterized by a constant phase, a single exponential decrease and a linear decrease. The observed rate constant for the single exponential MgATP-dependent proton production by reduced nitrogenase proteins at 20.0 degrees C is 14 +/- 4 s-1. No proton production with a rate constant comparable to the observed rate constant of electron transfer (kobs approximately 100 s-1) was detected. The extent of the observed MgATP-dependent proton production is determined by the redox state of the nitrogenase proteins before mixing with MgATP; less protons are produced when more electrons are transferred from the Fe protein to the MoFe protein. Values of 2.7 +/- 0.3 mol H+produced/mol MoFe protein with oxidized Fe protein, and 1.1 +/- 0.1 mol H+produced/mol MoFe protein with reduced Fe protein, were found. The data are interpreted to mean that protons are taken up after electron transfer from the Fe protein to the MoFe protein; the ratio electrons(transferred)/H-uptake was calculated to be 1.2 +/- 0.2. After mixing the nitrogenase proteins with MgADP, proton production takes place as well. The proton-production curve did not have a constant phase and the observed rate constant of the single exponential reaction is higher, compared to MgATP-dependent proton production (kobs approximately 35 s-1). The amount of protons produced depends also on the redox state of the Fe protein; no proton production was observed with the oxidized Fe protein; with dithionite-reduced Fe protein a value of 3.1 +/- 0.4 mol H+produced/mol MoFe protein was found (or 0.5 +/- 0.1 mol H+/mol Fe protein). Similar results were obtained when only the Fe protein was mixed with MgADP, but the observed absorbance changes were smaller; mixing of dithionite-reduced Fe protein with MgADP resulted in the production of 0.17 +/- 0.05 mol H+/mol Fe protein. All reported absorbance changes were absent when the experiments were performed in a buffered solution. The series of events that occur after mixing of the nitrogenase proteins with MgATP will be presented and discussed. In the case of the reduced Fe protein, electron transfer takes place at a rate of 100 s-1, which is followed by H+ production (kobs approximately 14 s-1). When there is no electron transfer (oxidized Fe protein) the rate constant of the MgATP-induced proton production decreases.(ABSTRACT TRUNCATED AT 400 WORDS)

Temperature effects on the MgATP-induced electron transfer between the nitrogenase proteins from Azotobacter vinelandii.

The temperature dependence of the pre-steady-state MgATP-dependent electron transfer from the MoFe protein to the Fe protein of the nitrogenase from Azotobacter vinelandii has been investigated between 6 degrees C and 31 degrees C by stopped-flow spectrophotometry. Below 14 degrees C, the data are consistent with a model in which interaction of MgATP with nitrogenase is fast and irreversible, and is followed by reversible electron transfer. From the extent and from the rate of the absorbance change, the rate constants for electron transfer from Fe protein to MoFe protein and of the reverse reaction were calculated. The direct rate constant increases with temperature (6-14 degrees C) from about 1 s-1 to about 26 s-1. The rate constant for the reverse reaction was found to be approximately 4 s-1 and invariant with the reaction temperature. Analysis of the data obtained in the temperature range between 6 degrees C and 12 degrees C within the framework of the transition-state theory show that electron transfer from the Fe protein to the MoFe protein occurs via a highly disordered transition state with activation parameters delta H(0) ++ = 289 kJ.mol-1 and delta S(0) ++ = 792 J.K-1.mol-1. The Eyring plot of the stopped-flow data displays an inflection point around 14 degrees C. From the stopped-flow data obtained between 18 degrees and 27 degrees C the activation parameters delta H(0) ++ and delta S(0) ++ for the reduction of the MoFe protein by Fe protein are calculated to be 90 kJ.mol-1 and 99 J.K-1.mol-1 respectively. A second inflection point in the Eyring plot could exist around 28 degrees C.

A reinvestigation of the pre-steady-state ATPase activity of the nitrogenase from Azotobacter vinelandii.

The pre-steady-state ATPase activity of nitrogenase has been reinvestigated. The exceptionally high burst in the hydrolysis of MgATP by the nitrogenase from Azotobacter vinelandii communicated by Cordewener et al. (1987) [Cordewener J., ten Asbroek A., Wassink H., Eady R. R., Haaker H. & Veeger C. (1987) Eur. J. Biochem. 162, 265-270] was found to be caused by an apparatus artefact. A second possible artefact in the determination of the stoichiometry of the pre-steady-state ATPase activity of nitrogenase was observed. Acid-quenched mixtures of dithionite-reduced MoFe or Fe protein of Azotobacter vinelandii nitrogenase and MgATP contained phosphate above the background level. It is proposed that due to this reaction, quenched reaction mixtures of nitrogenase and MgATP may contain phosphate in addition to the phosphate released by the ATPase activity of the nitrogenase complex. It was feasible to monitor MgATP-dependent pre-steady-state proton production by the absorbance change at 572 nm of the pH indicator o-cresolsulfonaphthalein in a weakly buffered solution. At 5.6 degrees C, a pre-steady-state phase of H+ production was observed, with a first-order rate constant of 2.2 s-1, whereas electron transfer occurred with a first-order rate constant of 4.9 s-1. At 20.0 degrees C, MgATP-dependent H+ production and electron transfer in the pre-steady-state phase were characterized by observed rate constants of 9.4 s-1 and 104 s-1, respectively. The stopped-flow technique failed to detect a burst in the release of protons by the dye-oxidized nitrogenase complex. It is concluded that the hydrolysis rate of MgATP, as judged by proton release, is lower than the rate of electron transfer from the Fe protein to the MoFe protein.

The role of MgATP hydrolysis in nitrogenase catalysis.

Kinetic studies on MgATP hydrolysis by nitrogenase of Azotobacter vinelandii were performed in the presence and in the absence of reducing equivalents. By measuring the ATPase activity of dye-oxidized nitrogenase proteins it can be excluded that reductant-independent ATPase activity is the result of futile cycling of electrons. The turnover rates of MoFe protein during reductant-dependent and reductant-independent ATPase activity, when measured with excess Fe protein, have approximately the same value, i.e. 5 s-1 at pH 7.4 and 22 degrees C, assuming the hydrolysis of four molecules of MgATP per turnover of MoFe protein. For Fe protein on the other hand, the maximum turnover rate during reductant-independent ATPase activity is only about 6% of that of reductant-dependent ATPase activity. While the reductant-dependent ATPase activity shows a sigmoidal dependence on the concentration of MgATP, the reductant-independent ATPase activity yields hyperbolic saturation curves. To account for these results it is proposed that the rate-limiting step during MgATP hydrolysis by oxidized nitrogenase is the rate of regeneration of active Fe protein. In the presence of reductant, the regeneration of active Fe protein is stimulated, explaining the higher ATPase activity of nitrogenase during substrate reduction.

Binding of ADP and orthophosphate during the ATPase reaction of nitrogenase.

The pre-steady-state ATPase activity of nitrogenase from Azotobacter vinelandii was investigated. By using a rapid-quench technique, it has been demonstrated that with the oxidized nitrogenase complex the same burst reaction of MgATP hydrolysis occurs as observed with the reduced complex, namely 6-8 mol orthophosphate released/mol MoFe protein. It is concluded that the pre-steady-state ATPase activity is independent of electron transfer from Fe protein to MoFe protein. Results obtained from gel centrifugation experiments showed that during the steady state of reductant-independent ATP hydrolysis there is a slow dissociation of one molecule of MgADP from the nitrogenase proteins (koff less than or equal to 0.2 s-1); the second MgADP molecule dissociates much faster (koff greater than or equal to 0.6 s-1). Under the same conditions orthophosphate was found to be associated with the nitrogenase proteins. The rate of dissociation of orthophosphate from the nitrogenase complex, as estimated from the gel centrifugation experiments, is in the same order of magnitude as the steady-state turnover rate of the reductant-independent ATPase activity (0.6 mol Pi formed X s-1 X mol Av2(-1) at 22 degrees C). These data are consistent with dissociation of orthophosphate or MgADP being rate-limiting during nitrogenase-catalyzed reductant-independent ATP hydrolysis.

Studies on the mechanism of electron transport to nitrogenase in Azotobacter vinelandii.

The involvement of the cytoplasmic membrane in electron transport to nitrogenase has been studied. Evidence shows that nitrogenase activity in Azotobacter vinelandii is coupled to the flux of electrons through the respiratory chain. To obtain information about proteins involved, the changes occurring in A. vinelandii cells transferred to nitrogen-free medium after growth on NH4Cl (depression of nitrogenase activity) were studied. Synthesis of the nitrogenase polypeptides was detectable 5 min after transfer to nitrogen-free medium. No nitrogenase activity could be detected until t = 20 min, whereupon a linear increase of nitrogenase activity with time was observed. Synthesis of nitrogenase was accompanied by synthesis of flavodoxin II and two membrane-bound polypeptides of Mr 29,000 and 30,000. Analysis with respect to changes in membrane-bound NAD(P)H dehydrogenase activities revealed the induction of an NADPH dehydrogenase activity, which was not detectable in membranes isolated from cells grown in the presence of NH4OAc. This induced activity was associated with the appearance of a polypeptide of Mr 29,000 in the NADPH dehydrogenase complex.

A novel S = 3/2 EPR signal associated with native Fe-proteins of nitrogenase.

In addition to their g = 1.94 EPR signal, nitrogenase Fe-proteins from Azotobacter vinelandii, Azotobacter chroococcum and Klebsiella pneumoniae exhibit a weak EPR signal with g approximately equal to 5. Temperature dependence of the signal was consistent with an S = 3/2 system with negative zero-field splitting, D = -5 +/- 0.7 cm-1. The ms = +/- 3/2 ground state doublet gives rise to a transition with geff = 5.90 and the transition within the excited ms = +/- 1/2 doublet has a split geff = 4.8, 3.4. Quantitation gave 0.6 to 0.8 spin . mol-1 which summed with the spin intensity of the S = 1/2 g = 1.94 line to roughly 1 spin/mol. MgATP and MgADP decreased the intensity of the S = 3/2 signal with no concomitant changes in intensity of the S = 1/2 signal.

Properties of the MgATP and MgADP binding sites on the Fe protein of nitrogenase from Azotobacter vinelandii.

Flow dialysis was used to study the binding of MgATP and MgADP to the nitrogenase proteins of Azotobacter vinelandii. Both reduced and oxidized Av2 bind two molecules of MgADP, with the following dissociation constants: reduced Av2, K1 = 0.091 +/- 0.021 mM and K2 = 0.044 +/- 0.009 mM; oxidized Av2, K1 = 0.024 +/- 0.015 mM and K2 = 0.039 +/- 0.022 mM. Binding of MgADP to reduced Av2 shows positive co-operativity. Oxidized Av2 binds two molecules of MgATP with dissociation constants K1 = 0.049 +/- 0.016 mM and K2 = 0.18 +/- 0.05 mM. Binding data of MgATP to reduced Av2 can be fitted by assuming one binding site, but a better fit was obtained by assuming two binding sites on the protein with negative co-operativity and with dissociation constants K1 = 0.22 +/- 0.03 mM and K2 = 1.71 +/- 0.50 mM. It was found that results concerning the number of binding sites and the dissociation constants of MgATP-Av2 and MgADP-Av2 complexes depend to a great extent on the specific activity of the Av2 preparation used, and that it is difficult to correct binding data for inactive protein. No binding of MgADP to Av1 could be demonstrated. Binding studies of MgADP to a mixture of Av1 and Av2 showed that Av1 did not affect the binding of MgADP to either oxidized or reduced Av2. Inhibition studies were performed to investigate the interaction of MgATP and MgADP binding to oxidized and reduced Av2. All the experimental data can be explained by the minimum hypothesis, i.e. the presence of two adenine nucleotide binding sites on Av2. MgATP and MgADP compete for these two binding sites on the Fe protein.

Electron allocation to H+ and N2 by nitrogenase in Rhizobium leguminosarum bacteroids.

Electron allocation to H+ and N2 by nitrogenase in intact Rhizobium leguminosarum bacteroids has been studied. Nitrogenase activity was measured in intact cells with succinate and oxygen substrates. When whole cell nitrogenase activity was inhibited by oxygen-limitation or by the addition of the H+-conducting ionophore carbonylcyanide m-chlorophenylhydrazone, both inducing a low intracellular ATP/ADP ratio, the electron allocation to H+ was favoured over that to N2. When whole cell nitrogenase activity was inhibited by excess oxygen or by the addition of the K+-conducting ionophore valinomycin, both inhibiting electron transport to nitrogenase without affecting the intracellular ATP/ADP ratio, no effect upon the electron allocation to H+ and N2 was observed. The whole cell experiments could be confirmed by experiments with bacteroids treated with hexadecyltrimethylammonium bromide. Nitrogenase is highly active in these preparations with Na2S2O4 and MgATP as substrates. No effect was observed upon electron allocation to H+ and N2 when nitrogenase was inhibited by limitation of reductant (Na2S2O4) or MgATP. Only when nitrogenase was inhibited by MgADP, electron allocation to H+ was favoured. The amount of nitrogenase component 1 and 2 in bacteroids was estimated with protein blotting, followed by an immunological detection. It was found that 17% +/- 3% of total bacteroid protein is component 1 and 12% +/- 2% is component 2. The specific nitrogenase activity of bacteroids treated with hexadecyltrimethylammonium bromide is 178 +/- 62 nmol C2H4 formed X min-1 X mg total protein-1. Despite the high protein concentrations, nitrogenase is not inhibited. With cell-free extracts or with purified nitrogenase components isolated from R. leguminosarum bacteroids, electron allocation to H+ was favoured over that to N2, independently of the mechanism of inhibition. The discrepancies between the whole cell studies and those with isolated enzyme will be discussed with respect to the present mechanism of action of nitrogenase.

Fully active Fe-protein of the nitrogenase from Azotobacter vinelandii contains at least eight iron atoms and eight sulphide atoms per molecule.

The Fe-protein of the Azotobacter vinelandii nitrogenase enzyme complex contains a variable iron and sulphide content. The iron and sulphide content of the protein is dependent upon the specific activity. Up to a specific activity of 1000 nmol C2H4 produced X min-1 X mg Av-1(2), three iron and three sulphide atoms per molecule Av2 are found. At specific activities above 1000 nmol C2H4 produced X min-1 X mg Av-1(2), a linear relationship between specific activity and iron and sulphide content of Av2 is found. The maximum values found are 8.8 iron atoms and 8.6 sulphide atoms/molecule at a specific activity of 2250 nmol C2H4 produced X min-1 X mg Av-1(2). Also the experimental molar absorption coefficients at 430 nm of the oxidized and reduced forms depend on the specific activity. The highest values found are 15.9 mM-1 cm-1 and 9.1 mM-1 cm-1, respectively. Since occasionally the preparations with specific activities around 3000 nmol X min-1 X mg-1 are isolated which contain more than 10 iron atoms and 11 sulphide atoms per molecule, it cannot be excluded that under certain physiological conditions Av2 contains even more than two [4 Fe-4 S] clusters. The addition of MgATP induces a conformational change in the Fe-protein which results in a higher reactivity with iron chelators. But irrespective of the specific activity, the amount of iron extracted from the protein after addition of MgATP never exceeds four atoms/molecule. The results are discussed with respect to the present molecular model of the Fe-protein.

Binding of MgATP to the nitrogenase proteins from Azotobacter vinelandii.

Binding of MgATP to the MoFe and Fe proteins from Azotobacter vinelandii has been studied. By means of the flow dialysis technique it was demonstrated that one molecule of reduced Fe protein binds one molecule of MgATP, with a dissociation constant of 0.56 +/- 0.11 mM. The oxidized Fe protein binds two molecules of MgATP, with identical intrinsic dissociation constants of 0.29 +/- 0.05 mM. The binding of MgATP to the Fe protein was also studied by equilibrium dialysis. It was found that during dialysis of reduced Fe protein in the presence of MgATP, dithionite was oxidized. Moreover, in the presence of MgATP both reduced and oxidized Fe protein were inactivated during the dialysis. These observations demonstrate that binding of MgATP to the Fe protein can only be measured by a relatively fast method. With the same methods as used for the Fe protein, no binding of MgATP to the MoFe protein of A. vinelandii could be demonstrated. The redox properties of the Fe protein in the presence and absence of MgATP are discussed with respect to the observed binding properties of MgATP for the Fe protein. The implications of these results are discussed with respect to the present models for the interactions between the Fe and MoFe proteins of nitrogenase.

Short-term regulation of the nitrogenase activity in Rhodopseudomonas sphaeroides.

The nitrogenase activity in whole cells of Rhodopseudomonas sphaeroides could be inhibited by lowering the electrical potential across the cytoplasmic membrane. The membrane potential was partly dissipated either by lowering the light intensity or by the addition of a lipophilic cation, tetraphenylphosphonium. Under these circumstances, it was shown that the intracellular ATP/ADP ratio was not affected and that the inhibition of the whole cell nitrogenase activity was not due to an inactivation of the nitrogenase enzyme. From these results it is concluded that electron transport to nitrogenase in Rps. sphaeroides is dependent on a high membrane potential. The nitrogenase enzyme in whole cells could be inactivated by lowering the membrane potential across the cytoplasmic membrane by incubating the cells in the dark or in the light in the presence of uncouplers. Nitrogenase could be reactivated in the light in the absence of uncouplers. Some possible mechanisms of action of NH+4 inhibition of whole cell nitrogenase activity could be excluded. Inhibition by NH4Cl of whole cell nitrogenase activity in Rps. sphaeroides could neither be explained by a rapid inactivation of the nitrogenase enzyme, nor by an effect on the intracellular ATP/ADP ratio or the membrane potential. NH+4 inhibits whole cell nitrogenase activity not directly but probably after being assimilated by glutamine synthetase. The role of glutamine, glutamate and 2-oxoglutarate on the regulation of electron transport to nitrogenase will be discussed.

Purification and reconstitution of the Ca2+-ATPase from plasma membrane of pig erythrocytes.

The Ca2+-ATPase from plasma membranes of pig erythrocytes was purified by mixed micelle gel chromatography (Wolf, H.U., Diekvoss, G., and Lichtner, R. (1977) Acta Biol. Med. Germ. 36, 847-858). The enzyme was activated at high concentrations of Tween 20 (10 mg/ml) or by appropriate mixtures of Triton X-100 and phospholipids. It was highly unstable in the absence of Ca2+ and activator protein. The Ca2+-ATPase was incorporated into liposomes by freeze-thaw sonication. After removal of non-ionic detergent by passage through a phenyl Sepharose 4B column, the reconstituted vesicles catalyzed a rapid ATP-dependent uptake of Ca2+. Modulator protein from brain substituted for the natural activator protein and stimulated Ca2+ uptake in reconstituted vesicles.