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.     

Evidence that MgATP accelerates primary electron transfer in a Clostridium pasteurianum Fe protein-Azotobacter vinelandii MoFe protein nitrogenase tight complex.

The nitrogenase catalytic cycle involves binding of the iron (Fe) protein to the molybdenum-iron (MoFe) protein, transfer of a single electron from the Fe protein to the MoFe protein concomitant with the hydrolysis of at least two MgATP molecules, followed by dissociation of the two proteins. Earlier studies found that combining the Fe protein isolated from the bacterium Clostridium pasteurianum with the MoFe protein isolated from the bacterium Azotobacter vinelandii resulted in an inactive, nondissociating Fe protein-MoFe protein complex. In the present work, it is demonstrated that primary electron transfer occurs within this nitrogenase tight complex in the absence of MgATP (apparent first-order rate constant k = 0.007 s-1) and that MgATP accelerates this electron transfer reaction by more than 10,000-fold to rates comparable to those observed within homologous nitrogenase complexes (k = 100 s-1). Electron transfer reactions were confirmed by EPR spectroscopy. Finally, the midpoint potentials (Em) for the Fe protein [4Fe-4S]2+/+ cluster and the MoFe protein P2+/N cluster were determined for both the uncomplexed and complexed proteins and with or without MgADP. Calculations from electron transfer theory indicate that the measured changes in Em are not likely to be sufficient to account for the observed nucleotide-dependent rate accelerations for electron transfer.     

Klebsiella pneumoniae nitrogenase: formation and stability of putative beryllium fluoride-ADP transition state complexes.

Incubation of the MoFe protein (Kp1) and Fe protein (Kp2), the component proteins of Klebsiella pneumoniae nitrogenase, with BeF(3)(-) and MgADP resulted in a progressive inhibition of nitrogenase activity. We have shown that at high Kp2 to Kp1 molar ratios this inhibition is due to the formation of an inactive complex with a stoichiometry corresponding to Kp1.{Kp2.(MgADP.BeFx)2}2. At lower Kp2:Kp1 ratios, an equilibrium between this 2:1 complex, the partially active 1:1 Kp1.Kp2.(MgADP. BeFx)2 complex, and active nitrogenase components was demonstrated. The inhibition was reversible since incubation of the 1:1 complex in the absence of MgADP and beryllium resulted in complete restoration of activity over 30 h. Under pseudo-first-order conditions with regard to nitrogenase components and MgADP, the kinetics of the rate of inhibition with increasing concentrations of BeF(3)(-) showed a square dependence on [BeF(3)(-)], consistent with the binding of two Be atoms by Kp2 in the complex. Analytical fplc gel filtration profiles of Kp1.Kp2 incubation mixtures at equilibrium resolved the 2:1 complex and the 1:1 complex from free Kp1. Deconvolution of the equilibrium profiles gave concentrations of the components allowing constants for their formation of 2.1 x 10(6) and 5.6 x 10(5) M(-1) to be calculated for the 1:1 and 2:1 complexes, respectively. When the active site concentration of the different species was taken into account, values for the two constants were the same, indicating the two binding sites for Kp2 are the same for Kp1 with one or both sites unoccupied. The value for K(1) we obtain from this study is comparable with the value derived from pre-steady-state studies of nitrogenase. Analysis of the elution profile obtained on gel filtration of a 1:1 ratio incubation mixture containing 20 microM nitrogenase components showed 97% of the Kp2 present initially to be complexed. These data provide the first unequivocal demonstration that Fe protein preparations which may contain up to 50% of a species of Fe protein defective in electron transfer is nevertheless fully competent in complex formation with MoFe protein.     

A transient-kinetic study of the nitrogenase of Klebsiella pneumoniae by stopped-flow calorimetry. Comparison with the myosin ATPase.

The pre-steady-state kinetics of MgATP hydrolysis by nitrogenase from Klebsiella pneumoniae were studied by stopped-flow calorimetry at 6 degrees C and at pH 7.0. An endothermic reaction (delta Hobs. = +36 kJ.mol of ATP-1; kobs. = 9.4 s-1) in which 0.5 proton.mol of ATP-1 was released, has been assigned to the on-enzyme cleavage of MgATP to yield bound MgADP + Pi. The assignment is based on the similarity of these parameters to those of the corresponding reaction that occurs with rabbit muscle myosin subfragment-1 (delta Hobs. = +32 kJ.mol of ATP-1; kobs. = 7.1 s-1; 0.2 proton released.mol of ATP-1) [Millar, Howarth & Gutfreund (1987) Biochem. J. 248, 683-690]. MgATP-dependent electron transfer from the nitrogenase Fe-protein to the MoFe-protein was monitored by stopped-flow spectrophotometry at 430 nm and occurred with kobs. value of 3.0 s-1 at 6 degrees C. Thus, under these conditions, hydrolysis of MgATP precedes electron transfer within the protein complex. Evidence is presented that suggests that MgATP cleavage and subsequent electron transfer are reversible at 6 degrees C with an overall equilibrium constant close to unity, but that, at 23 degrees C, the reactions are essentially irreversible, with an overall equilibrium constant greater than or equal to 10.     

Nitrogenase of Klebsiella pneumoniae. Kinetic studies on the Fe protein involving reduction by sodium dithionite, the binding of MgADP and a conformation change that alters the reactivity of the 4Fe-4S centre.

The kinetics of reduction of indigocarmine-dye-oxidized Fe protein of nitrogenase from Klebsiella pneumoniae (Kp2ox) by sodium dithionite in the presence and absence of MgADP were studied by stopped-flow spectrophotometry at 23 degrees C and at pH 7.4. Highly co-operative binding of 2MgADP (composite K greater than 4 X 10(10) M-2) to Kp2ox induced a rapid conformation change which caused the redox-active 4Fe-4S centre to be reduced by SO2-.(formed by the predissociation of dithionite ion) with k = 3 X 10(6) M-1.s-1. This rate constant is at least 30 times lower than that for the reduction of free Kp2ox (k greater than 10(8) M-1.s-1). Two mechanisms have been considered and limits obtained for the rate constants for MgADP binding/dissociation and a protein conformation change. Both mechanisms give rate constants (e.g. MgADP binding 3 X 10(5) less than k less than 3 X 10(6) M-1.s-1 and protein conformation change 6 X 10(2) less than k less than 6 X 10(3) s-1) that are similar to those reported for creatine kinase (EC 2.7.3.2). The kinetics also show that in the catalytic cycle of nitrogenase with sodium dithionite as reductant replacement of 2MgADP by 2MgATP occurs on reduced and not oxidized Kp2. Although the Kp2ox was reduced stoichiometrically by SO2-. and bound two equivalents of MgADP with complete conversion into the less-reactive conformation, it was only 45% active with respect to its ability to effect MgATP-dependent electron transfer to the MoFe protein.     

The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of H2 formation.

A comprehensive model for the mechanism of nitrogenase action is used to simulate pre-steady-state kinetic data for H2 evolution in the presence and in the absence of N2, obtained by using a rapid-quench technique with nitrogenase from Klebsiella pneumoniae. These simulations use independently determined rate constants that define the model in terms of the following partial reactions: component protein association and dissociation, electron transfer from Fe protein to MoFe protein coupled to the hydrolysis of MgATP, reduction of oxidized Fe protein by Na2S2O4, reversible N2 binding by H2 displacement and H2 evolution. Two rate-limiting dissociations of oxidized Fe protein from reduced MoFe protein precede H2 evolution, which occurs from the free MoFe protein. Thus Fe protein suppresses H2 evolution by binding to the MoFe protein. This is a necessary condition for efficient N2 binding to reduced MoFe protein.     

Characterization of the nucleotide tight-binding sites of the isolated mitochondrial F1-ATPase

The properties of the nucleotides tightly bound with mitochondrial F1-ATPase were examined. One of three bound nucleotide molecules is localized at the site with Kd approximately 10(-7) M and released with koff approximately 0.1 s-1. The second nucleotide molecule is bound with the enzyme with Kd approximately 10(-8) M and koff for its dissociation is 3 X 10(-4) s-1. The third is never released even in the presence of 1 mM ATP or ADP. The last two nucleotides are believed to be bound at the noncatalytic sites of F1-ATPase. Pyrophosphate promotes liberation of two releasable nucleotide molecules, decreasing the affinity of the enzyme to AD(T)P. From the results obtained it follows that the only suitable criterion for localization of the nucleotide at the F1-ATPase catalytic site is the high rate (koff greater than or equal to 0.1 s-1) of its spontaneous release.     

Evidence for the selective population of FeMo cofactor sites in MoFe protein and its molecular recognition by the Fe protein in transition state complex analogues of nitrogenase

We have collected synchrotron x-ray solution scattering data for the MoFe protein of Klebsiella pneumoniae nitrogenase and show that the molecular conformation of the protein that contains only one molybdenum per alpha(2)beta(2) tetramer is different from that of the protein that has full occupancy i.e. two molybdenums per molecule. This structural finding is consistent with the existence of MoFe protein molecules that contain only one FeMo cofactor site occupied and provides a rationale for the 50% loss of the specific activity of such preparations. A stable inactive transition state complex has been shown to form in the presence of MgADP and AlF(4)(-). Gel filtration chromatography data show that the MoFe protein lacking a full complement of the cofactor forms initially a 1:1 complex before forming a low affinity 1:2 complex. A similar behavior is found for the MoFe protein with both cofactors occupied, but the high affinity 1:2 complex is formed at a lower ratio of Fe protein/MoFe protein. The 1:1 complex, MoFe protein-Fe protein x (ADP x AlF(4)(-))(2), formed with MoFe protein that lacks one of the cofactors, is stable. X-ray scattering studies of this complex have enabled us to obtain its low resolution structure at approximately 20-A resolution, which confirms the gel filtration finding that only one molecule of the Fe protein binds the MoFe protein. By comparison with the low resolution structure of purified MoFe protein that contains only one molybdenum per tetramer, we deduce that the Fe protein interacts with the FeMo cofactor-binding alpha-subunit of the MoFe protein. This observation demonstrates that the conformation of the alpha-subunit or the alpha beta subunit pair that lacks the FeMo cofactor is altered and that the change is recognized by the Fe protein. The structure of the 1:1 complex reveals a similar change in the conformation of the Fe protein as has been observed in the low resolution scattering mask and the high resolution crystallographic study of the 1:2 complex where both cofactors are occupied and with the Fe protein bound to both subunits. This extensive conformational change observed for the Fe protein in the complexes is, however, not observed when MgATP or MgADP binds to the isolated Fe protein. Thus, the large scale conformational change of the Fe protein is associated with the complex formation of the two proteins.     

Nitrogenase of Klebsiella pneumoniae. Kinetics of the dissociation of oxidized iron protein from molybdenum-iron protein: identification of the rate-limiting step for substrate reduction.

Stopped-flow spectrophotometry and e.p.r. spectroscopy were used to study the kinetics of reduction by dithionite of the oxidized Fe protein of nitrogenase from Klebsiella pneumoniae (Kp2ox.) in the presence of MgADP at 23 degrees C at pH 7.4. The active reductant, SO2.-, produced by the predissociation of S2O4(2-) in equilibrium 2SO2.-, reacts with Kp2ox. (MgADP)2, with k4 = 3.0 X 10(6) +/- 0.4 X 10(6) M-1 X s-1. The inhibition of this reaction by the Mo-Fe protein (Kp1) has enabled the rate of dissociation of Kp2ox. (MgADP)2 from Kp1+ (the Kp2-binding site on Kp1) to be measured (k-3 = 6.4 +/- 0.8 s-1). Comparison with the steady-state rate of substrate reduction shows that the dissociation (k-3) of the complex Kp2ox. (MgADP)2-Kp1+, which is formed after MgATP-induced electron transfer from Kp2 to Kp1+, is the rate-limiting step in the catalytic cycle for substrate reduction.     

Hydrolysis of nucleoside triphosphates other than ATP by nitrogenase

The hydrolysis of ATP to ADP and P(i) is an integral part of all substrate reduction reactions catalyzed by nitrogenase. In this work, evidence is presented that nitrogenases isolated from Azotobacter vinelandii and Clostridium pasteurianum can hydrolyze MgGTP, MgITP, and MgUTP to their respective nucleoside diphosphates at rates comparable to those measured for MgATP hydrolysis. The reactions were dependent on the presence of both the iron (Fe) protein and the molybdenum-iron (MoFe) protein. The oxidation state of nitrogenase was found to greatly influence the nucleotide hydrolysis rates. MgATP hydrolysis rates were 20 times higher under dithionite reducing conditions (approximately 4,000 nmol of MgADP formed per min/mg of Fe protein) as compared with indigo disulfonate oxidizing conditions (200 nmol of MgADP formed per min/mg of Fe protein). In contrast, MgGTP, MgITP, and MgUTP hydrolysis rates were significantly higher under oxidizing conditions (1,400-2,000 nmol of MgNDP formed per min/mg of Fe protein) as compared with reducing conditions (80-230 nmol of MgNDP formed per min/mg of Fe protein). The K(m) values for MgATP, MgGTP, MgUTP, and MgITP hydrolysis were found to be similar (330-540 microM) for both the reduced and oxidized states of nitrogenase. Incubation of Fe and MoFe proteins with each of the MgNTP molecules and AlF(4)(-) resulted in the formation of non-dissociating protein-protein complexes, presumably with trapped AlF(4)(-) x MgNDP. The implications of these results in understanding how nucleotide hydrolysis is coupled to substrate reduction in nitrogenase are discussed.     

Evidence for a synergistic salt-protein interaction -- complex patterns of activation vs. inhibition of nitrogenase by salt

The molybdenum nitrogenase enzyme system, comprised of the MoFe protein and the Fe protein, catalyzes the reduction of atmospheric N(2) to NH(3). Interactions between these two proteins and between Fe protein and nucleotides (MgADP and MgATP) are crucial to catalysis. It is well established that salts are inhibitors of nitrogenase catalysis that target these interactions. However, the implications of salt effects are often overlooked. We have reexamined salt effects in light of a comprehensive framework for nitrogenase interactions to offer an in-depth analysis of the sources of salt inhibition and underlying apparent cooperativity. More importantly, we have identified patterns of salt activation of nitrogenase that correspond to at least two mechanisms. One of these mechanisms is that charge screening of MoFe protein-Fe protein interactions in the nitrogenase complex accelerates the rate of nitrogenase complex dissociation, which is the rate-limiting step of catalysis. This kind of salt activation operates under conditions of high catalytic activity and low salt concentrations that may resemble those found in vivo. While simple kinetic arguments are strong evidence for this kind of salt activation, further confirmation was sought by demonstrating that tight complexes that have previously displayed little or no activity due to the inability of Fe protein to dissociate from the complex are activated by the presence of salt. This occurs for the combination Azotobacter vinelandii MoFe protein with: (a) the L127Delta Fe protein; and (b) Clostridium pasteurianum Fe protein. The curvature of activation vs. salt implies a synergistic salt-protein interaction.     

Cross-linking of nitrogenase components. Structure and activity of the covalent complex

The nitrogenase complex from Azotobacter vinelandii is composed of the MoFe protein (Av1), an alpha 2 beta 2 tetramer, and the Fe protein (Av2), a gamma 2 dimer. During turnover of the enzyme, electrons are transferred from Av2 to Av1 in parallel with the hydrolysis of MgATP. Using the cross-linking reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, we have identified some of the properties of the complex between the two components. The cross-linking reaction was highly specific yielding a single apparent Mr = 97,000 protein. The amount of cross-linked product was essentially independent of whether MgATP or MgADP were in the reaction. Also, the amount was maximum at high ratios of Av2 to Av1. The Mr = 97,000 protein was characterized by amino acid analysis and Edman degradation and was found to be consistent with a 1:1 complex of an Av2 gamma subunit and an Av1 beta subunit (the amino terminal serine subunit). The complex was no longer active in the nitrogenase reaction which supports, but does not prove, the requirement for dissociation of the complex after each electron transferred. Nitrogenase activity and cross-linking were inhibited in an identical way by NaCl, which suggests that electrostatic forces are critical to the formation of the electron transfer complex.     

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.     

Activation of the dynein adenosinetriphosphatase by microtubules

Previous work has indicated that following the rapid adenosine 5'-triphosphate (ATP) induced dissociation of the microtubule-dynein complex, the rate-limiting step in the ATPase cycle is product release [Johnson, K. A. (1983) J. Biol. Chem. 258, 13825-13832], which occurs at a rate of approximately 2-6 s-1. In this report we complete the analysis of the ATPase cycle by examining the effect of microtubules on the rate of product release. For these studies we used repolymerized Tetrahymena axonemal microtubules and microtubule-associated protein (MAP) free bovine brain microtubules which were shown to be free of any measureable ATPase activity. Tetrahymena 22S dynein bound to these microtubules predominantly by the ATP-sensitive site and at a rate giving an apparent second-order rate constant of (0.2-1) X 10(6) M-1 s-1, which is 50-fold greater than the rate observed with brain microtubules containing MAPs. ATP induced the rapid dissociation of the microtubule-dynein complex with an apparent second-order rate constant vs. ATP concentration equal to 1.6 X 10(6) M-1 s-1; this value is only slightly lower than that observed in the presence of MAPs. After the ATP-induced dissociation, the dynein reassociated with the microtubules following a lag period due to the time required to hydrolyze the ATP. The duration of the lag time for reassociation decreased with increasing microtubule concentration, suggesting that microtubules increased the rate of ATP turnover. Direct measurements at steady state showed that the specific activity of the dynein increased with increasing microtubule concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

The vanadium- and molybdenum-containing nitrogenases of Azotobacter chroococcum. Comparison of mid-point potentials and kinetics of reduction by sodium dithionite of the iron proteins with bound magnesium adenosine 5''-diphosphate.

The mid-point potentials of the Fe protein components (Ac2 and Ac2* respectively) of the Mo nitrogenase and V nitrogenase from Azotobacter chroococcum were determined in the presence of MgADP to be -450 mV (NHE) [Ac2(MgADP)2-Ac2*ox.(MgADP)2 couple] and -463 mV (NHE) [Ac2* (MgADP)2-Ac2*ox.(ADP)2 couple] at 23 degrees C at pH 7.2. These values are consistent with a flavodoxin characterized by Deistung & Thorneley [(1986) Biochem. J. 239, 69-75] with Em = -522 mV (NHE) being an effective electron donor to both the Mo nitrogenase and the V nitrogenase in vivo. Ac2*ox.(MgADP)2 and Ac2*ox.(MgADP)2 were reduced by SO2.- (formed by the predissociation of dithionite ion, S2O4(2-)) at similar rates, k = 4.7 X 10(6) +/- 0.5 X 10(6) M-1.s-1 and 3.2 X 10(6) +/- 0.2 X 10(6) M-1.s-1 respectively, indicating structural homology at the electron-transfer site associated with the [4Fe-4S] centre in these proteins.     

The rate of MgADP binding to and dissociation from acto-S1

The rate of binding and dissociation of MgADP from its ternary complex with actin and S1 was measured by following the extent to which fixed concentrations of MgADP slow down MgATP-induced dissociation of acto-S1. The solution of the equations describing this process shows that at any MgADP concentration the apparent rate of acto-S1 dissociation should be proportional to a square root of the equilibrium constant for MgADP dissociation and to MgATP concentration. By measuring the apparent rate of acto-S1 dissociation as a function of MgATP concentration, the rate of MgADP binding and dissociation were determined as 5 X 10(6) M-1 X s-1 and 1400 s-1, respectively. These rates were unchanged by modification of SH1 thiol of S1 by a variety of fluorescence and spin-labels, but dissociation rate was drastically reduced when SH1 was labelled with 5-iodoacetamidofluorescein.     

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.     

Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from Escherichia coli

A kinetic scheme is presented for Escherichia coli dihydrofolate reductase that predicts steady-state kinetic parameters and full time course kinetics under a variety of substrate concentrations and pHs. This scheme was derived from measuring association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance spectroscopy. The binding kinetics suggest that during steady-state turnover product dissociation follows a specific, preferred pathway in which tetrahydrofolate (H4F) dissociation occurs after NADPH replaces NADP+ in the ternary complex. This step, H4F dissociation from the E X NADPH X H4F ternary complex, is proposed to be the rate-limiting step for steady-state turnover at low pH because koff = VM. The rate constant for hydride transfer from NADPH to dihydrofolate (H2F), measured by pre-steady-state transients, has a deuterium isotope effect of 3 and is rapid, khyd = 950 s-1, essentially irreversible, Keq = 1700, and pH dependent, pKa = 6.5, reflecting ionization of a single group in the active site. This scheme accounts for the apparent pKa = 8.4 observed in the steady state as due to a change in the rate-determining step from product release at low pH to hydride transfer above pH 8.4. This kinetic scheme is a necessary background to analyze the effects of single amino acid substitutions on individual rate constants.     

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.     

Nitrogenases from Klebsiella pneumoniae and Clostridium pasteurianum. Kinetic investigations of cross-reactions as a probe of the enzyme mechanism.

In combination with the Mo-Fe protein of nitrogenase from Klebsiella pneumoniae, the Fe protein of nitrogenase from Clostridium pasteurianum forms an active enzyme with novel properties different from those of either of the homologous nitrogenases. The steady-state rates of reduction of acetylene and H+ are 12% of those of the homologous system from C.pasteurianim. Acetylene reductase activity exhibited an approx. 10min lag at 30 degrees C before the rate of reduction became linear, consistent with a once-only activation step being necessary for acetylene reduction to occur. No such lag was observed for H2 evolution. The activity with N2 as a reducible substrate was very low, implying that acetylene reductase activity is not necessarily an accurate indication of nitrogen-fixing ability. This is of particular relevance to studies on mutant and agronomically important organisms. Stopped-flow spectrophotometric studies showed unimolecular electron transfer from the Fe protein to the Mo-Fe protein to occur at the same rate (k2 = 2.5 X 10(2)s-1) and with the same dependence on ATP concentration (apparent KD = 400 muM) as with the homologous Klebsiella nitrogenase. However, an ATP/2e ratio of 50 was obtained for H2 evolution, indicating that ATP hydrolysis had been uncoupled from electron transfer to substrate. These data indicate that ATP has at least two roles in the mechanism of nitrogenase action. The combination of the Mo-Fe protein of nitrogenase of C.pasteurianim and the Fe protein of K.pneumoniae were inactive in all the above reactions, except for a weak adenosine triphosphatase activity, 0.5% of that of the homologous K.pneumoniae system.