Oxidized Fe Protein Research Articles

Negative cooperativity in the nitrogenase Fe protein electron delivery cycle

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen (N2) to two ammonia (NH3) molecules through the participation of its two protein components, the MoFe and Fe proteins. Electron transfer (ET) from the Fe protein to the catalytic MoFe protein involves a series of synchronized events requiring the transient association of one Fe protein with each αβ half of the α2β2 MoFe protein. This process is referred to as the Fe protein cycle and includes binding of two ATP to an Fe protein, association of an Fe protein with the MoFe protein, ET from the Fe protein to the MoFe protein, hydrolysis of the two ATP to two ADP and two Pi for each ET, Pi release, and dissociation of oxidized Fe protein-(ADP)2 from the MoFe protein. Because the MoFe protein tetramer has two separate αβ active units, it participates in two distinct Fe protein cycles. Quantitative kinetic measurements of ET, ATP hydrolysis, and Pi release during the presteady-state phase of electron delivery demonstrate that the two halves of the ternary complex between the MoFe protein and two reduced Fe protein-(ATP)2 do not undergo the Fe protein cycle independently. Instead, the data are globally fit with a two-branch negative-cooperativity kinetic model in which ET in one-half of the complex partially suppresses this process in the other. A possible mechanism for communication between the two halves of the nitrogenase complex is suggested by normal-mode calculations showing correlated and anticorrelated motions between the two halves.

Evidence That the Pi Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle.

Nitrogenase reduction of dinitrogen (N2) to ammonia (NH3) involves a sequence of events that occur upon the transient association of the reduced Fe protein containing two ATP molecules with the MoFe protein that includes electron transfer, ATP hydrolysis, Pi release, and dissociation of the oxidized, ADP-containing Fe protein from the reduced MoFe protein. Numerous kinetic studies using the nonphysiological electron donor dithionite have suggested that the rate-limiting step in this reaction cycle is the dissociation of the Fe protein from the MoFe protein. Here, we have established the rate constants for each of the key steps in the catalytic cycle using the physiological reductant flavodoxin protein in its hydroquinone state. The findings indicate that with this reductant, the rate-limiting step in the reaction cycle is not protein-protein dissociation or reduction of the oxidized Fe protein, but rather events associated with the Pi release step. Further, it is demonstrated that (i) Fe protein transfers only one electron to MoFe protein in each Fe protein cycle coupled with hydrolysis of two ATP molecules, (ii) the oxidized Fe protein is not reduced when bound to MoFe protein, and (iii) the Fe protein interacts with flavodoxin using the same binding interface that is used with the MoFe protein. These findings allow a revision of the rate-limiting step in the nitrogenase Fe protein cycle.

A confirmation of the quench-cryoannealing relaxation protocol for identifying reduction states of freeze-trapped nitrogenase intermediates.

We have advanced a mechanism for nitrogenase catalysis that rests on the identification of a low-spin EPR signal (S = 1/2) trapped during turnover of a MoFe protein as the E4 state, which has accumulated four reducing equivalents as two [Fe–H–Fe] bridging hydrides. Because electrons are delivered to the MoFe protein one at a time, with the rate-limiting step being the off-rate of oxidized Fe protein, it is difficult to directly control, or know, the degree of reduction, n, of a trapped intermediate, denoted En, n = 1–8. To overcome this previously intractable problem, we introduced a quench-cryoannealing relaxation protocol for determining n of an EPR-active trapped En turnover state. The trapped “hydride” state was allowed to relax to the resting E0 state in frozen medium, which prevents additional accumulation of reducing equivalents; binding of reduced Fe protein and release of oxidized protein from the MoFe protein both are abolished in a frozen solid. Relaxation of En was monitored by periodic EPR analysis at cryogenic temperature. The protocol rests on the hypothesis that an intermediate trapped in the frozen solid can relax toward the resting state only by the release of a stable reduction product from FeMo-co. In turnover under Ar, the only product that can be released is H2, which carries two reducing equivalents. This hypothesis implicitly predicts that states that have accumulated an odd number of electrons/protons (n = 1, 3) during turnover under Ar cannot relax to E0: E3 can relax to E1, but E1 cannot relax to E0 in the frozen state. The present experiments confirm this prediction and, thus, the quench-cryoannealing protocol and our assignment of E4, the foundation of the proposed mechanism for nitrogenase catalysis. This study further gives insights into the identity of the En intermediates with high-spin EPR signals, 1b and 1c, trapped under high electron flux.

Reduction of nitrogenase Fe protein from Azotobacter vinelandii by dithionite: quantitative and qualitative effects of nucleotides, temperature, pH and reaction buffer

Oxidized Fe protein from Azotobacter vinelandii (Av2 0) was reduced by dithionite (DT) in the absence and presence of nucleotides, over the temperature range 10–40 °C, over the pH range 7–8, and in various buffers—inorganic phosphate, TES, HEPES, and Tris. The reduction of each species of Fe protein—Av2 0, Av2 0(MgATP) 2, and Av2 0(MgADP) 2—was resolved into at least three exponential phases, with relative amplitudes of each phase varying over the range of experimental conditions, suggesting a dynamic population shift of kinetically distinct species. The rapid phase of Av2 0 reduction predominated at low temperature and pH, and in Tris buffer; rapid Av2 0(MgATP) 2 reduction was favored at high temperature and pH, and in phosphate buffer; and Av2 0(MgADP) 2 reduction was favored under more physiologically relevant conditions of 20 °C, pH 7.5, and in phosphate buffer. The rates of reduction of Fe protein species did not change with buffer, but temperature and pH do have an effect on the rates. With the appropriate constants, an empirically derived equation estimates the rate of Fe protein reduction at any temperature and pH within the limits 10–40 °C and pH 7–8, for a given species of Fe protein, and a given phase of the reaction. At 23.0 °C and pH 7.4, the rate of the dominant phase of Av2 0 reduction is 1.9×10 8 M −1 s −1. Under the same conditions, the rates of the two dominant phases of Av2 0(MgATP) 2 reduction are 1.2×10 6 and 1.5×10 5 M −1 s −1; and the rate of the dominant phase of Av2 0(MgADP) 2 reduction is 3.5×10 6 in M −1 s −1. Thermodynamic activation parameters for each phase of reduction were calculated. No breaks in the Arrhenius plots for any Fe protein species were observed.

Multiple inequivalent metal-nucleotide coordination environments in the presence of the VO2+-inhibited nitrogenase iron protein: pH-dependent structural rearrangements at the nucleotide binding site.

Nitrogenase naturally requires adenosine nucleoside triphosphates and divalent metal cations for catalytic activity. Their energy of hydrolysis controls several mechanistic functions, most probably via separate structural conformers of the nitrogenase Fe protein. To characterize the ligand environment of the divalent metal in the ternary complex, with ADP or ATP and the Fe protein from Klebsiella pneumoniae, the hyperfine structures have been investigated by electron paramagnetic resonance (EPR) spectroscopy by substituting naturally occurring diamagnetic Mg(2+) by paramagnetic oxovanadium. This metal replacement leads to inhibition of nitrogenase activity. Moreover, depending on pH, two distinctly different VO(2+) EPR spectra are detected. At pH 7.4 each of the vanadyl EPR hyperfine lines is further split into two. This indicates that several spectroscopically distinguishable metal coordination environments coexist for VO(2+)-nucleotide chelate complexes in the presence of the reduced Fe protein. Overall, a total of at least three distinct local metal coordination environments have been identified. We report the EPR parameters for each of the disparate metal coordinations measured at different pH values with ADP and ATP bound. EPR spectra have also been recorded for the oxidized Fe protein showing essentially similar spectra to that of the reduced protein. The EPR parameters of VO-nucleotides in the presence of the Fe protein are consistent, for all metal coordination environments, with direct metal ligation by nucleotide phosphate groups and the formation of mononucleotide complexes. The nucleotide binding environment with the highest ligand field strength is compatible with a metal coordination structure that is also found in various G-proteins with GTP bound. No significant EPR line width change is detected after exchange into D(2)O buffer solution for any of the pH forms although differences exist between the pH forms. The missing difference between the EPR parameters in the presence of ADP or ATP suggests that there is little or no conformational rearrangement between these two forms; this contrasts with behavior of G-proteins that undergo substantial conformational changes upon hydrolysis. This could be related to the inhibition of nitrogenase by VO(2+).

Direct assessment of the reduction potential of the [4Fe-4S](1+/0) couple of the Fe protein from Azotobacter vinelandii.

Recently, it has been demonstrated that the [4Fe-4S] cluster of the Fe protein of nitrogenase from Azotobacter vinelandii can be reduced to an unprecedented all-ferrous state. In this work, the reduction potential for the formation of the all-ferrous state is measured by the reactions of the reduced and oxidized Fe protein with a variety of chemical redox active agents, and by mediated spectroelectrochemical titration. Redox titrations obtain a potential ca. -790 mV/NHE for the formation of the all-ferrous state, a value consistent with the chemical reactivity experiments and with recent theoretical calculations. At present, no known redox protein in A. vinelandii is capable of generating the all-ferrous Fe protein.

Formation of a tight 1:1 complex of Clostridium pasteurianum Fe protein-Azotobacter vinelandii MoFe protein: evidence for long-range interactions between the Fe protein binding sites during catalytic hydrogen evolution.

It has been well documented that the combination of the MoFe protein of Azotobacter vinelandii nitrogenase (Av1) with the Fe protein (Cp2) from Clostridium pasteurianum nitrogenase produces an inactive, stable complex. However, we report that this heterologous nitrogenase has a low level of activity for H(2) evolution, with a specific activity of 12 nmol min(-)(1) mg(-)(1) of Av1. This activity does not arise from contaminating hydrogenase since it required the presence of both Cp2 and Av1 and showed saturation kinetics when increasing amounts of Cp2 were added to the assay. Incubation of the two proteins at a 4:1 Cp2:Av1 ratio in the absence of MgATP followed by analytical gel filtration showed, surprisingly, that the stoichiometry of the isolated complex was Av1.Cp2 instead of Av1.(Cp2)(2) as determined previously. The presence of MgATP in the elution buffer did not change the elution profile of the complex. The hydrodynamic radius of the isolated complex determined by dynamic light scattering was 5.93 +/- 0.14 nm, intermediate between Av1 and a stable 2:1 nitrogenase complex, consistent with a 1:1 assignment for the Av1.Cp2 complex. When assayed with Av2, the isolated Av1.Cp2 complex showed full half-site reactivity with a specific activity of 750 nmol of C(2)H(2) reduced min(-)(1) mg(-)(1) of Av1. The EPR spectrum of the isolated complex showed the Cp2 to be oxidized and the Av1 to retain the S = (3)/(2) signal characteristic of FeMoco. In the presence of MgATP, under turnover conditions at a 2:1 ratio of Cp2:Av1, the [4Fe-4S] center of Cp2 was protected from the chelator 2,2'-bipyridyl. This is consistent with the formation of a tight 2:1 complex of Av1.(Cp2)(2) which is more stable than the homologous Cp nitrogenase. Assuming that the Lowe-Thorneley model for nitrogenase applies and that a rate-limiting dissociation of the complex is required for H(2) evolution, then with a rate of 0.032 s(-)(1) the 1:1 complex is too stable to be involved in catalysis. The differences in the stability of the 2:1 and 1:1 complexes indicate cooperativity between the Fe protein binding sites of Av1, which structural data show to be separated by 105 A. On the basis of these observations, we propose a model for nitrogenase catalysis in which the stable 1:1 complex formed between oxidized Fe protein and the one-electron-reduced MoFe protein plays an essential role. In this scheme, the two Fe protein binding sites of the MoFe protein alternately bind and release Fe protein in a shuttle mechanism associated with long-range conformational changes in the MoFe protein.

Regulation of Dinitrogenase Reductase ADP-ribosyltransferase and Dinitrogenase Reductase-activating Glycohydrolase by a Redox-dependent Conformational Change of Nitrogenase Fe Protein

The nitrogenase-regulating enzymes dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase-activating glycohydrolase (DRAG), from Rhodospirillum rubrum, were shown to be sensitive to the redox status of the [Fe(4)S(4)](1+/2+) cluster of nitrogenase Fe protein from R. rubrum or Azotobacter vinelandii. DRAG had <2% activity with oxidized R. rubrum Fe protein relative to activity with reduced Fe protein. The activity of DRAG with oxygen-denatured Fe protein or a low molecular weight substrate, N(alpha)-dansyl-N(omega)-(1,N(6)-etheno-ADP-ribosyl)-arginine methyl ester, was independent of redox potential. The redox midpoint potential of DRAG activation of Fe protein was -430 mV versus standard hydrogen electrode, coinciding with the midpoint potential of the [Fe(4)S(4)] cluster from R. rubrum Fe protein. DRAT was found to have a specificity opposite that of DRAG, exhibiting low (<20%) activity with 87% reduced R. rubrum Fe protein relative to activity with fully oxidized Fe protein. A mutant of R. rubrum in which the rate of oxidation of Fe protein was substantially decreased had a markedly slower rate of ADP-ribosylation in vivo in response to 10 mM NH(4)Cl or darkness stimulus. It is concluded that the redox state of Fe protein plays a significant role in regulation of the activities of DRAT and DRAG in vivo.

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.

Nucleotide binding by the nitrogenase Fe protein: a 31P NMR study of ADP and ATP interactions with the Fe protein of Klebsiella pneumoniae.

Investigation of the interaction of MgADP- and MgATP2- with the Fe protein of Klebsiella pneumoniae nitrogenase by 31P NMR showed that the adenine nucleotides are reversibly bound in slow exchange with free nucleotides. Dissociation of the MgADP--Fe protein complex was slow enough to enable its isolation by gel filtration, thus permitting the assignment of resonances to bound nucleotides. Spectra of ADP bound to Kp2 were similar to spectra of ADP bound to the myosin motor domain. Oxidative inactivation of a Kp2-MgADP- complex with excess ferricyanide ion eliminated exchange between bound and free ADP, indicating that the intact iron sulphur cluster, located 20 A from the binding sites, is required for the reversible binding of MgADP-. A change in conformation on controlled oxidation of Kp2 with indigocarmine increased the chemical shift of the beta phosphate resonance of bound MgADP-. Both oxidized and reduced conformers were observed transiently in the absence of dithionite. The 31P resonances of both the beta and gamma phosphates of bound MgATP2- indicated major changes in environment and labilization of both groups on binding to the Fe protein. Highly purified Kp2 slowly hydrolysed ATP, resulting in mixtures of bound nucleotides. Partial occupation of Kp2 MgATP2--binding sites (N=1.9+/-0.2, Kd=145 microM) in concentrated protein solutions was demonstrated by flow dialysis. Scatchard plots of data for bound and free ligand obtained after equilibration with Kp2 were linear and no co-operative interactions were detected. We conclude that MgADP- stabilizes the oxidized Fe protein conformer and this conformation in turn triggers the dissociation of the Fe protein from the MoFe protein in the rate-limiting step of the overall process of dinitrogen reduction.

Circular Dichroism and X-ray Spectroscopies of Azotobacter vinelandii Nitrogenase Iron Protein: MgATP AND MgADP INDUCED PROTEIN CONFORMATIONAL CHANGES AFFECTING THE [4Fe-4S] CLUSTER AND CHARACTERIZATION OF A [2Fe-2S] FORM

Nucleotide interactions with nitrogenase are a central part of the mechanism of nitrogen reduction. Previous studies have suggested that MgATP or MgADP binding to the nitrogenase iron protein (Fe protein) induce protein conformational changes that control component protein docking, interprotein electron transfer, and substrate reduction. In the present study, we have investigated the effects of MgATP or MgADP binding to the Azotobacter vinelandii nitrogenase Fe protein on the properties of the [4Fe-4S] cluster using circular dichroism (CD) and x-ray absorption spectroscopies. Previous CD and magnetic CD studies on nitrogenase Fe protein suggested that binding of either MgATP or MgADP to the Fe protein resulted in identical changes in the CD spectrum arising from transitions of the [4Fe-4S]2+ cluster. We present evidence that MgADP or MgATP binding to the oxidized nitrogenase Fe protein results in distinctly different CD spectra, suggesting distinct changes in the environment of the [4Fc-4S] cluster. The present results are consistent with previous studies such as chelation assays, electron paramagnetic resonance, and NMR, which suggested that MgADP or MgATP binding to the nitrogenase Fe protein induced different conformational changes. The CD spectrum of a [2Fe-2S]2+ form of the nitrogenase Fe protein was also investigated to address the possibility that the MgATP- or MgADP-induced changes in the CD spectrum of the native enzyme were the result of a partial conversion from a [4Fe-4S] cluster to a [2Fe-2S] cluster. No evidence was found for a contribution of a [2Fe-2S]2+ cluster to the CD spectrum of oxidized Fe protein in the absence or presence of nucleotides. A novel two-electron reduction of the [2Fe-2S]2+ cluster in Fe protein was apparent from absorption, CD, and electron paramagnetic resonance data. Fe K-edge x-ray absorption spectra of the oxidized Fe protein revealed no changes in the structure of the [4Fe-4S] cluster upon MgATP binding to the Fe protein. The present results reveal that MgATP or MgADP binding to the oxidized state of the Fe protein result in different conformational changes in the environment around the [4Fe-4S] cluster.

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)

Nitrogenase of Klebsiella pneumoniae. Reversibility of the reductant-independent MgATP-cleavage reaction is shown by MgADP-catalysed phosphate/water oxygen exchange

The steady-state kinetics of reductant-independent ATP hydrolysis by Klebsiella pneumoniae nitrogenase at 23 degrees C at pH 7.4 were determined as a function of component protein ratio (optimal at an oxidized Fe protein/MoFe protein ratio of 3:1) and MgATP concentration (Km 400 microM). Competitive inhibition was observed for MgADP (Ki 145 microM), [beta gamma-methylene]ATP (Mgp[CH2]ppA) (Ki 115 microM), [beta gamma-monofluoromethylene]ATP (Mgp[CHF]ppA) (Ki 53 microM) and [beta gamma-difluoromethylene]ATP (Mgp[CF2]ppA) (Ki 160 microM). The tighter binding of MgADP to free oxidized Fe protein (KD less than 10 microM) than to the oxidized Fe protein-MoFe protein complex (Ki 145 microM) is proposed as the driving force that induces rate-limiting protein dissociation in the catalytic cycle of nitrogenase. The reversible nature of the reductant-independent MgATP-cleavage reaction was demonstrated by an MgADP-induced enhancement of the rate of the phosphate/water oxygen exchange reaction with 18O-labelled phosphate ion. This enhancement, like the reductant-independent ATPase reaction, only occurred with the complex formed by oxidized Fe protein and MoFe protein and not with the individual proteins. The results are discussed in terms of the mechanism of ATP hydrolysis by nitrogenase and other systems involving protein-protein interactions.

Resonance Raman studies of the [4Fe-4S] to [2Fe-2S] cluster conversion in the iron protein of nitrogenase

Resonance Raman spectroscopy has been used to investigate the Fe-S stretching modes of the [4Fe-4S] 2+ cluster in the oxidized iron protein of Clostridium pasteurianum nitrogenase. The results are consistent with a cubane [4Fe-4S] cluster having effective T d symmetry with cysteinyl coordination for each iron. In accord with previous optical and EPR studies [(1984) Biochemistry 23, 2118–2122], treatment with the iron chelator α,α′-dipyridyl in the presence of MgATP is shown to effect cluster conversion to a [2Fe-2S] 2+ cluster. Resonance Raman data also indicate that partial conversion to a [2Fe-2S] 2+ cluster is induced by thionine-oxidation in the presence of MgATP in the absence of an iron chelator. This result suggests new explanations for the dramatic change in the CD spectrum that accompanies MgATP-binding to the oxidized Fe protein and the anomalous resonance Raman spectra of thionine-oxidized Clostridium pasteurianum bidirectional hydrogenase.

Molybdenum nitrogenase of Azotobacter chroococcum. Tight binding of MgADP to the MoFe protein.

The dye-oxidized or dithionite-reduced forms of the MoFe protein of molybdenum nitrogenase of Azotobacter chroococcum were shown to bind 2 mol of MgADP/mol of protein, as determined by column equilibrium techniques. The gel-filtration elution profile of unbound Mg[14C]ADP was not symmetrical, consistent with a low rate of dissociation from the protein. Symmetrical elution profiles were observed for the oxidized Fe protein of nitrogenase, which bound 2 mol of MgADP/mol of protein. The low rate of dissociation of MgADP from MoFe protein was shown by non-equilibrium column techniques, where 1 mol of MgADP/mol of MoFe protein remained tightly bound during chromatography. Very weak binding of MgATP (less than 0.01 mol of MgATP/mol of MoFe protein) to dye-oxidized but not to dithionite-reduced MoFe protein was observed. These results are discussed in terms of their relevance to the catalytic cycle of nitrogenase catalysis.

Nitrogenase of Klebsiella pneumoniae. Rhodanese-catalysed restoration of activity of the inactive 2Fe species of the Fe protein.

The inactive 2Fe species of the Fe protein of the nitrogenase of Klebsiella pneumoniae was generated by treating oxidized Fe protein (Kp2) with MgATP and chelator. Incubation of the 2Fe species of Kp2 with the sulphurtransferase rhodanese in the presence of thiosulphate, ferric citrate and reduced lipoate reproducibly restored activity. The extent of restoration of activity depended on the molar ratio of 2Fe Kp2 to rhodanese and was time-dependent. Re-activation did not occur in the reaction mixture lacking rhodanese.

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.

Reactions with the oxidized iron protein of Azotobacter vinelandii nitrogenase: formation of a 2Fe center.

The Fe-S center of oxidized Fe protein from Azotobacter vinelandii nitrogenase is decomposed by alpha,alpha'-dipyridyl in a biphasic process. In the presence of MgATP, 2 Fe are immediately removed by chelation while the additional irons are removed only after several hours. A slower biphasic Fe release also was observed in the presence of chelator alone. MgADP prevented the Fe release by chelator. An intermediate in the reaction was isolated containing 2 Fe. The visible spectrum of the intermediate was similar to that of 2Fe-2S ferredoxins (epsilon max at 325, 416, and 460 nm of 16.1, 11.3, and 9.0 mM-1 cm-1). The 2Fe form was electron paramagnetic resonance (EPR) silent until partially reduced with sodium dithionite. The EPR spectral properties were similar to 2Fe-2S ferredoxins; namely, the Fe center had resonances at g = 2.00, 1.94, and 1.92 which were detectable, essentially unbroadened at 70 K. The results suggest that in the oxidized (2+) state Fe protein can undergo a 4Fe to 2Fe conversion.

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.

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.