Electron-transfer studies involving flavodoxin and a natural redox partner, the iron protein of nitrogenase. Conformational constraints on protein-protein interactions and the kinetics of electron transfer within the protein complex.

The kinetics of electron-transfer reactions involving flavodoxins from Klebsiella pneumoniae (KpFld), Azotobacter chroococcum (AcFld), Anacystis nidulans (AnFld) and Megasphaera elsdenii (MeFld), the free, MgADP-bound and MgATP-bound forms of the Fe protein component of nitrogenase from K. pneumoniae [Kp2, Kp2(MgADP)2 and Kp2(MgATP)2] and Na2S2O4 were studied by stopped-flow spectrophotometry. Kinetic evidence was obtained for the formation of binary protein complexes involving KpFldSQ (semiquinone) with either Kp2(MgADP)2 (KD = 49 microM) or Kp2(MgATP)2 (KD = 13 microM) but not with Kp2 (KD greater than 730 microM). The binding of 2MgATP or 2MgADP to Kp2 therefore not only shifts the midpoint potential (Em) of the [4Fe-4S] centre from -200 mV to -320 mV or -350 mV respectively but also changes the affinity of Kp2 for KpFldSQ. Thermodynamically unfavourable electron from Kp2(MgADP)2 and Kp2(MgATP)2 to KpFldSQ occurs within the protein complexes with k = 1.2 s-1 (delta E = -72 mV) and 0.5 s-1 (delta E = -120 mV) respectively. Although AcFldSQ is reduced by Kp2, Kp2(MgADP)2 and Kp2(MgATP)2 (k = 8 x 10(3), 2.4 x 10(3) and 9 x 10(2) M-1.s-1 respectively), protein-complex formation is weak in each case (KD greater than 700 microM). Electron transfer in the physiologically important and thermodynamically favourable direction from Kp2FldHQ (hydroquinone) and AcFldHQ to Kp2ox.(MgADP)2 (the state of Kp2 that accepts electrons from FldHQ in the catalytic cycle of nitrogenase) is rapid (k greater than 10(6) M-1.s-1). The second-order rate constants for the reduction of KpFldSQ, AcFldSQ, AnFldSQ and MeFldSQ by SO2.- (active reductant formed by the predissociation of S2O4(2-) ion) exhibited the linear free-energy relationship predicted by the Marcus theory of electron transfer.     

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.     

Construction and characterization of a heterodimeric iron protein: defining roles for adenosine triphosphate in nitrogenase catalysis

One molecule of MgATP binds to each subunit of the homodimeric Fe protein component of nitrogenase. Both MgATP molecules are hydrolyzed to MgADP and P(i) in reactions coupled to the transfer of one electron into the MoFe protein component. As an approach to assess the contributions of individual ATP binding sites, a heterodimeric Fe protein was produced that has an Asn substituted for residue 39 in the ATP binding domain in one subunit, while the normal Asp(39) residue within the other subunit remains unchanged. Separation of the heterodimeric Fe protein from a mixed population with homodimeric Fe proteins contained in crude extracts was accomplished by construction of a seven His tag on one subunit and a differential immobilized-metal-affinity chromatography technique. Three forms of the Fe protein (wild-type homodimeric Fe protein [Asp(39)/Asp(39)], altered homodimeric Fe protein [Asn(39)/Asn(39)], and heterodimeric Fe protein [Asp(39)/Asn(39)]) were compared on the basis of the biochemical and biophysical changes elicited by nucleotide binding. Among those features examined were the MgATP- and MgADP-induced protein conformational changes that are manifested by the susceptibility of the [4Fe-4S] cluster to chelation and by alterations in the electron paramagnetic resonance, circular dichroism, and midpoint potential of the [4Fe-4S] cluster. The results indicate that changes in the [4Fe-4S] cluster caused by nucleotide binding are the result of additive conformational changes contributed by the individual subunits. The [Asp(39)/Asn(39)] Fe protein did not support substrate reduction activity but did hydrolyze MgATP and showed MgATP-dependent primary electron transfer to the MoFe protein. These results support a model where each MgATP site contributes to the rate acceleration of primary electron transfer, but both MgATP sites must be functioning properly for substrate reduction. Like the altered homodimeric [Asn(39)/Asn(39)] Fe protein, the heterodimeric [Asp(39)/Asn(39)] Fe protein was found to form a high affinity complex with the MoFe protein, revealing that alteration on one subunit is sufficient to create a tight complex.     

Kinetics of nitrogenase of Klebsiella pneumoniae. Heterotropic interactions between magnesium-adenosine 5''-diphosphate and magnesium-adenosine 5''-triphosphate.

The effects of MgADP and MgATP on the kinetics of a pre-steady-state electron-transfer reaction and on the steady-state kinetics of H2 evulution for nitrogenase proteins of K. pneumoniae were studied. MgADP was a competitive inhibitor of MgATP in the MgATP-induced electron transfer from the Fe-protein to the Mo-Fe-protein. A dissociation constant K'i = 20 micron was determined for MgADP. The release of MgADP or a coupled conformation change in the Fe-protein of K.pneumoniae occurred with a rate comparable with that of electron transfer, k approximately 2 X 10(2)S-1. Neither homotropic nor heterotropic interactions involving MgATP and MgADP were observed for this reaction. Steady-state kinetic data for H2 evolution exhibited heterotropic effects between MgADP and MgATP. The data have been fitted to symmetry and sequential-type models involving conformation changes in two identical subunits. The data suggest that the enzyme can bind up to molecules of either MgATP or MgADP, but is unable to bind both nucleotides simultaneously. The control of H2 evolution by the MgATP/MgADP ratio is not at the level of electron transfer between the Fe- and Mo-Fe-proteins.     

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.     

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.     

Vanadium nitrogenase of Azotobacter chroococcum. MgATP-dependent electron transfer within the protein complex.

The kinetics of MgATP-induced electron transfer from the Fe protein (Ac2V) to the VFe protein (AclV) of the vanadium-containing nitrogenase from Azotobacter chroococcum were studied by stopped-flow spectrophotometry at 23 degrees C at pH 7.2. They are very similar to those of the molybdenum nitrogenase of Klebsiella pneumoniae [Thorneley (1975) Biochem. J. 145, 391-396]. Extrapolation of the dependence of kobs. on [MgATP] to infinite MgATP concentration gave k = 46 s-1 for the first-order electron-transfer reaction that occurs with the Ac2V MgATPAclV complex. MgATP binds with an apparent KD = 230 +/- 10 microM and MgADP acts as a competitive inhibitor with Ki = 30 +/- 5 microM. The Fe protein and VFe protein associate with k greater than or equal to 3 x 10(7) M-1.s-1. A comparison of the dependences of kobs. for electron transfer on protein concentrations for the vanadium nitrogenase from A. chroococcum with those for the molybdenum nitrogenase from K. pneumoniae [Lowe & Thorneley (1984) Biochem. J. 224, 895-901] indicates that the proteins of the vanadium nitrogenase system form a weaker electron-transfer complex.     

Entropies of Redox Reactions between Proteins and Mediators: The Temperature Dependence of Reversible Electrode Potentials in Aqueous Buffers

The temperature dependencies of the reversible electrode potentials for a number of charge transfer reactions of redox mediators were used to evaluate the corresponding charge transfer entropies in Tris–HCl (pH 8) buffer. The redox mediator thermodynamic data, along with reaction enthalpy data for mediator redox protein electron transfer, were used to evaluate the charge transfer entropy for the cytochrome c redox couple [(cytc)_ox/(cytc)_red] in Tris–HCl (pH 8) buffer and were found to be equal to −16 cal/°K mol. Reversible electrode potentials at 298°K for the redox mediator half-reactions were observed to vary from −528 to +657 mV (vs NHE). Charge transfer entropies were observed to depend upon the structure of the redox mediators and to vary from −13.8 to −29.7 cal/°K mol for a closely related series of organic dications (viologens) and a value of −43.6 cal/°K mol was observed for the [Fe(CN)₆]³⁻/[Fe(CN)₆]⁴⁻couple under the same conditions. A procedure for determining charge transfer entropies of protein redox couples which cannot be studied by direct electrochemical methods is outlined. The factors contributing to the magnitude of the charge transfer entropies are discussed.     

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.     

Use of stopped-flow spectrophotometry to establish midpoint potentials for redox proteins

Stopped-flow spectrophotometry was examined as a tool to assign midpoint potentials to protein redox half-reactions. The method involves the rapid mixing of protein and electron transfer mediator solutions and the determination of the absorbance of at least one of the reacting species or products at equilibrium. The utility of the method was demonstrated with two different redox proteins (nitrogenase iron protein and cytochrome c) with very different midpoint potentials. The overall errors ranged from about +/-0.5 to 3 mV. Advantages of the method include short times required for the experiments, the precision and accuracy of the method in comparison to other methods, the conservative use of valuable protein in the experiments and the ease of obtaining midpoint potentials for redox protein half-reactions, and the potential range covered by a single electron transfer mediator when the method involves mediated electron transfer. It is concluded that the stopped-flow spectrophotometry should be considered the method of choice for determining protein midpoint potentials.     

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.     

Nitrogenase IX. Effect of the MgATP generator on the catalytic and EPR properties of the enzyme in vitro.

Nitrogenase(nitrogen:(acceptor) oxidoreduction, EC 1.7.99.2) of Clostridium pasteuranium is very sensitive to the ratio of MgADP/MgATP in dithionite oxidation assays. Variation of concentration of creatine kinase, an ATP-regenerating enzyme, can be used to control the ratio of ADP/ATP and thereby the dithionite oxidation activity of nitrogenase. The in vitro properties of nitrogenase support the suggestion of Haaker (Haaker, H., deKok, A. and Veeger, C. (1974) Biochim. Biophys. Acta 357, 344-357) that in vivo the nucleotide ratio and not the electron supply normally regulates nitrogenase activity. In EPR experiments it has been shown that the "steady state" varies as a function of the concentration of creatine kinase. The spectral differences are interpreted as being a function of the ratio of MgADP/MgATP obtained in the pseudo steady-state condition, which occurs as a result of variation in relative rates of ATP-utilizing and ATP-generating reactions, that is, the relative nitrogenase and creatine kinase activities. Implications of these finding for interpretation of previously reported kinetic and EPR studies are discussed.     

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.     

Oxidation of nitrogenase iron protein by dioxygen without inactivation could contribute to high respiration rates of Azotobacter species and facilitate nitrogen fixation in other aerobic environments.

The kinetics of oxidation of the Fe proteins of nitrogenases from Klebsiella pneumoniae (Kp2) and Azotobacter chroococcum (Ac2) by O2 and H2O2 have been studied by stopped-flow spectrophotometry at 23 degrees C, pH 7.4. With excess O2, one-electron oxidation of Kp2 and Ac2 and their 2 MgATP or 2 MgADP bound forms occurs with rate constants (k) in the range 5.3 x 10(3) M-1.S-1 to 1.6 x 10(5) M-1.S-1. A linear correlation between log k and the mid-point potentials (Em) of these protein species indicates that the higher rates of electron transfer from the Ac2 species are due to the differences in Em of the 4Fe-4S cluster. The reaction of Ac2(MgADP)2 with O2 is sufficiently rapid for it to contribute significantly to the high respiration rate of Azotobacter under N2-fixing conditions and may represent a new respiratory pathway. Excess O2 rapidly inactivates Ac2(MgADP)2 and Kp2(MgADP)2; however, when these protein species are in greater than 4-fold molar excess over the concentration of O2, 4 equivalents of protein are oxidized with no loss of activity. The kinetics of this reaction suggest that H2O2 is an intermediate in the reduction of O2 to 2 H2O by nitrogenase Fe proteins and imply a role for catalase or peroxidase in the mechanism of protection of nitrogenase from O2-induced inactivation.     

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.     

Increasing nitrogenase catalytic efficiency for MgATP by changing serine 16 of its Fe protein to threonine: use of Mn2+ to show interaction of serine 16 with Mg2+.

MgATP-binding and hydrolysis are an integral part of the nitrogenase catalytic mechanism. We are exploring the function of MgATP hydrolysis in this reaction by analyzing the properties of the Fe protein (FeP) component of Azotobacter vinelandii nitrogenase altered by site-directed mutagenesis. We have previously (Seefeldt, L.C., Morgan, T.V., Dean, D.R., & Mortenson, L.E., 1992, J. Biol. Chem. 267, 6680-6688) identified a region near the N-terminus of FeP that is involved in interaction with MgATP. This region of FeP is homologous to the well-known nucleotide-binding motif GXXXXGKS/T. In the present work, we examined the function of the four hydroxyl-containing amino acids immediately C-terminal to the conserved lysine 15 that is involved in interaction with the gamma-phosphate of MgATP. We have established, by altering independently Thr 17, Thr 18, and Thr 19 to alanine, that a hydroxyl-containing residue is not needed at these positions for FeP to function. In contrast, an hydroxyl-containing amino acid at position 16 was found to be critical for FeP function. When the strictly conserved Ser 16 was altered to Ala, Cys, Asp, or Gly, the FeP did not support N2 fixation when expressed in place of the wild-type FeP in A. vinelandii. Altering Ser 16 to Thr (S16T), however, resulted in the expression of an FeP that was partially active. This S16T FeP was purified to homogeneity, and its biochemical examination allowed us to assign a catalytic function to this hydroxyl group in the nitrogenase mechanism. Of particular importance was the finding that the S16T FeP had a significantly higher affinity for MgATP than the wild-type FeP, with a measured Km of 20 microM compared to the wild-type FeP Km of 220 microM. This increased kinetic affinity for MgATP was reflected in a significantly stronger binding of the S16T FeP for MgATP. In contrast, the affinity for MgADP, which binds at the same site as MgATP, was unchanged. The catalytic efficiency (kcat/Km) of S16T FeP was found to be 5.3-fold higher than for the wild-type FeP, with the S16T FeP supporting up to 10 times greater nitrogenase activity at low MgATP concentrations. This indicates a role for the hydroxyl group at position 16 in interaction with MgATP but not MgADP. The site of interaction of this residue was further defined by examining the properties of wild-type and S16T FePs in utilizing MnATP compared with MgATP.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Mapping the site(s) of MgATP and MgADP interaction with the nitrogenase of Azotobacter vinelandii. Lysine 15 of the iron protein plays a major role in MgATP interaction.

Nitrogenase binds and hydrolyzes 2MgATP yielding 2MgADP and 2Pi for each electron that is transferred from the iron protein to the MoFe protein. The iron protein alone binds but does not hydrolyze 2MgATP or 2MgADP and the binding of these nucleotides is competitive. Iron protein amino acid sequences all contain a putatitive mononucleotide-binding region similar to a region found in other mononucleotide-binding proteins. To examine the role of this region in MgATP interaction, we have substituted glutamine and proline for conserved lysine 15. The amino acid substitutions, K15Q and K15P, both yielded a non-N2-fixing phenotype when the genes coding for them were substituted into the Azotobacter vinelandii chromosome in place of the wild-type gene. The iron protein from the K15Q mutant was purified to homogeneity, whereas the protein from the K15P mutant could not be purified in its native form. Unlike wild-type iron protein, the purified K15Q iron protein showed no acetylene reduction, H2 evolution, or ATP hydrolysis activities when complemented with wild-type MoFe protein. The K15Q iron protein and the normal iron protein had a similar total iron content and both proteins showed the characteristic rhombic EPR signal resulting from the reduced state of the single 4Fe-4S cluster bridging the two subunits. Unlike the wild-type iron protein, addition of MgATP to the K15Q iron protein did not result in the perturbation necessary to change the EPR signal of its 4Fe-4S center from a rhombic to an axial line shape. Also unlike the wild-type iron protein, addition of MgATP to K15Q iron protein in the presence of the iron chelator, alpha,alpha'-dipyridyl, did not result in a time-dependent transfer of iron to the chelator. Thus, even though the K15Q iron protein contains a normal 4Fe-4S center, it does not respond to MgATP like the wild-type protein. Examination of the ability of the K15Q iron protein to bind MgADP showed no change from the wild-type iron protein, but its ability to bind MgATP decreased to 35% of the wild-type protein. Thus, in A. vinelandii iron protein, lysine 15 is not needed for interaction with MgADP but is involved in the binding of ATP, presumably through charge-charge interaction with the gamma-phosphate. Based on the above data, this lysine appears to be essential for the MgATP induced conformational change of wild-type iron protein that is required for activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.