Differential effects on N(2) binding and reduction, HD formation, and azide reduction with alpha-195(His)- and alpha-191(Gln)-substituted MoFe proteins of Azotobacter vinelandii nitrogenase

In contrast to the wild-type MoFe protein, neither the alpha-195(Asn) nor the alpha-191(Lys) MoFe protein catalyzed N(2) reduction to NH(3), when complemented with wild-type Fe protein. However, N(2) was bound by the alpha-195(Asn) MoFe protein and inhibited the reduction of both protons and C(2)H(2). The alpha-191(Lys) MoFe protein did not interact with N(2). With the alpha-195(Asn) MoFe protein, the N(2)-induced inhibition of substrate reduction was reversed by removing the N(2). Surprisingly, even though added H(2) also relieved N(2) inhibition of substrate reduction, the alpha-195(Asn) MoFe protein did not catalyze HD formation under a N(2)/D(2) atmosphere. This observation is the first indication that these two reactions have different chemical origins, prompting a revision of the current hypothesis that these two reactions are consequences of the same nitrogenase chemistry. A rationale that accounts for the dichotomy of the two reactions is presented. The two altered MoFe proteins also responded quite differently to azide. It was a poor substrate for both but, in addition, azide was an electron-flux inhibitor with the 195(Asn) MoFe protein. The observed reactivity changes are correlated with likely structural changes caused by the amino acid substitutions and provide important details about the interaction(s) of N(2,) H(2), D(2), and azide with Mo-nitrogenase.     

N2O reduction and HD formation by nitrogenase from a nifV mutant of Klebsiella pneumoniae.

Dinitrogenase from a nifV mutant of Klebsiella pneumoniae contains an altered form of iron-molybdenum cofactor (FeMoco) that lacks a biologically active homocitric acid molecule. Change in the composition of FeMoco led to substantial variation in the kinetics of nitrogenase action. The KmS of the mutant enzyme for N2 and N2O were 0.244 and 0.175 atm (24,714 and 17,726 kPa), respectively. The km for N2 was higher and the Km for N2O was lower than that for the wild-type enzyme. The mutant enzyme was ineffective in N2 fixation, in N2O reduction, and in HD formation, as indicated by the low Vmax of these reactions with saturating levels of substrate and under conditions of saturating electron flux. These observations provide further support for the concept that N2, N2O, and D2 interact with the same form of dinitrogenase. H2 evolution by the mutant enzyme is only partially inhibited by CO. Observation that different numbers of electrons are stored in CO-inhibited than in noninhibited dinitrogenase before H2 is released suggests that the mutant enzyme has more sites responsible for H2 evolution than the wild-type enzyme, whose H2 evolution is not inhibited by CO.     

The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of an enzyme-bound intermediate in N2 reduction and of NH3 formation.

The reduction of N2 to 2NH3 by Klebsiella pneumoniae nitrogenase was studied by a rapid-quench technique. The pre-steady-state time course for N2H4, formed on quenching by the acid-induced hydrolysis of an enzyme-bound intermediate in N2 reduction, showed a 230 ms lag followed by a damped oscillatory approach to a constant concentration in the steady state. The pre-steady-state time course for NH3 formation exhibited a lag of 500 ms and a burst phase that was essentially complete at 1.5s, before a steady-state rate was achieved. These time courses have been simulated by using a previously described kinetic model for the mechanism of nitrogenase action [Lowe & Thorneley (1984) Biochem. J. 224, 877-886]. A hydrazido(2-) structure (=N-NH2) is favoured for the intermediate that yields N2H4 on quenching. The NH3-formation data indicate enzyme-bound metallo-nitrido (identical to N) or -imido (=NH) intermediates formed after N-N bond cleavage to produce the first molecule of NH3 and which subsequently give the second molecule of NH3 by hydrolysis on quenching. The simulations require stoichiometric reduction of one N2 molecule at each Mo and the displacement of one H2 when N2 binds to the MoFe protein. Inhibition by H2 of N2-reduction activity occurs before the formation of the proposed hydrazido(2-) species, and is explained by H2 displacement of N2 at the active site.     

Influence of pN2 and pD2 on HD formation by various nitrogenases

Formation of HD from D2 has been demonstrated with nitrogenase preparations from Azotobacter vinelandii, Clostridium pasteurianum, Klebsiella pneumoniae, and Azospirillum sp. We conclude that the formation of HD from D2 is a general property of nitrogenases. However, the nitrogenases differ in their Ki values for D2 (N2 fixation) and in their rates of catalyzing HD formation; among the nitrogenases tested, C. pasteurianum nitrogenase had the lowest activity for formation of HD. When contaminating N2 was removed from the atmospheres above reaction mixtures, less than 1% of the total electron flux in the system was directed to HD formation; hence, we doubt that N2-independent HD formation is significant. A working hypothesis is suggested that operates without invoking an N2-independent reaction for forming HD.     

Diazene (HN=NH) is a substrate for nitrogenase: insights into the pathway of N2 reduction

Nitrogenase catalyzes the sequential addition of six electrons and six protons to a N2 that is bound to the active site metal cluster FeMo-cofactor, yielding two ammonia molecules. The nature of the intermediates bound to FeMo-cofactor along this reduction pathway remains unknown, although it has been suggested that there are intermediates at the level of reduction of diazene (HN=NH, also called diimide) and hydrazine (H2N-NH2). Through in situ generation of diazene during nitrogenase turnover, we show that diazene is a substrate for the wild-type nitrogenase and is reduced to NH3. Diazene reduction, like N2 reduction, is inhibited by H2. This contrasts with the absence of H2 inhibition when nitrogenase reduces hydrazine. These results support the existence of an intermediate early in the N2 reduction pathway at the level of reduction of diazene. Freeze-quenching a MoFe protein variant with alpha-195His substituted by Gln and alpha-70Val substituted by Ala during steady-state turnover with diazene resulted in conversion of the S = 3/2 resting state FeMo-cofactor to a novel S = 1/2 state with g1 = 2.09, g2 = 2.01, and g3 approximately 1.98. 15N- and 1H-ENDOR establish that this state consists of a diazene-derived [-NHx] moiety bound to FeMo-cofactor. This moiety is indistinguishable from the hydrazine-derived [-NHx] moiety bound to FeMo-cofactor when the same MoFe protein is trapped during turnover with hydrazine. These observations suggest that diazene joins the normal N2-reduction pathway, and that the diazene- and hydrazine-trapped turnover states represent the same intermediate in the normal reduction of N2 by nitrogenase. Implications of these findings for the mechanism of N2 reduction by nitrogenase are discussed.     

Nitrogenase of Klebsiella pneumoniae. Hydrazine is a product of azide reduction.

Klebsiella pneumoniae nitrogenase reduced azide, at 30 degrees C and pH 6.8-8.2, to yield ammonia (NH3), dinitrogen (N2) and hydrazine (N2H4). Reduction of (15N = 14N = 14N)-followed by mass-spectrometric analysis showed that no new nitrogen-nitrogen bonds were formed. During azide reduction, added 15N2H4 did not contribute 15N to NH3, indicating lack of equilibration between enzyme-bound intermediates giving rise to N2H4 and N2H4 in solution. When azide reduction to N2H4 was partially inhibited by 15N2, label appeared in NH3 but not in N2H4. Product balances combined with the labelling data indicate that azide is reduced according to the following equations: (formula: see text); N2 was a competitive inhibitor and CO a non-competitive inhibitor of azide reduction to N2H4. The percentage of total electron flux used for H2 evolution concomitant with azide reduction fell from 26% at pH 6.8 to 0% at pH 8.2. Pre-steady-state kinetic data suggest that N2H4 is formed by the cleavage of the alpha-beta nitrogen-nitrogen bond to bound azide to leave a nitride (= N) intermediate that subsequently yields NH3.     

Nitrogenase. VI. Acetylene reduction assay: Dependence of nitrogen fixation estimates on component ratio and acetylene concentration.

Acetylene reduction, an assay for nitrogenase activity (nitrogen:(acceptor) oxidoreductase, EC 1.7.99.2), Is dependent on the ratio of the two protein components of nitrogenase as well as on C2H2 concentration. As the component I : component II ratio (based on activity) is increased, the C2H2 reduction : N2 fixation ratio decreases to a minimum of 3.4 and then increases. The minimum is found at a ratio near 1 : 1. At a component I : component II ratio of 20 : 1, the C2H2 reduction : N2 fixation ratio is 5.3. Acetylene exhibits substrate inhibition in assays for nitrogenase activity. Both the apparent Km and Ki for acetylene vary as a function of the relative concentrations of components I and II present in the assay. When the more labile component II is limiting in the assay and "saturating" levels of C2H2 (above 0.1 atm) are used, N2-fixation capacity may be greatly under-estimated.     

The mechanism of Klebsiella pneumoniae nitrogenase action. The determination of rate constants required for the simulation of the kinetics of N2 reduction and H2 evolution.

Kinetic data for Klebsiella pneumoniae nitrogenase were used to determine the values of nine of the 17 rate constants that define the scheme for nitrogenase action described by Lowe & Thorneley [(1984) Biochem. J. 224, 877-886]. Stopped-flow spectrophotometric monitoring of the MgATP-induced oxidation of the Fe protein (Kp2) by the MoFe protein (Kp1) was used to determine the rates of association (k+1) and dissociation (k-1) of reduced Kp2(MgATP)2 with Kp1. The dependences of the apparent KNm2 on Fe protein/MoFe protein ratio and H2 partial pressure were used to determine the mutual displacement rates of N2 and H2 (k+10, k-10, k+11 and k-11). These data also allowed the rate constants for H2 evolution from progressively more reduced forms of Kp1 to be determined (k+7, k+8 and k+9). A mechanism for N2-dependent catalysis of 1H2H formation from 2H2 that requires H2 to be a competitive inhibitor of N2 reduction is also presented.     

N2O as a substrate and as a competitive inhibitor of nitrogenase

We have investigated the inhibitory effect of N2O on NH3 formation by purified component proteins from Klebsiella pneumoniae and have confirmed that the inhibition is competitive with respect to N2 and that N2O is reduced to N2, which in turn is further reduced to NH3. In addition, we have shown that N2O is unable to support HD formation from D2 and H2O. N2-supported HD formation from D2 and H2O was found to be inhibited by N2O. In contrast to N2, N2O was found to suppress nitrogenase-mediated H2 evolution completely at infinitely high pN2O. H2 was found to inhibit N2O-supported NH3 production but not N2O-supported N2 production. The steady-state kinetics of N2O reduction showed a good fit to Michaelis-Menten kinetics with a Km for N2O of 5 mM at 30 degrees C, corresponding to 24 kPa of N2O. A model is proposed that fits the observed results.     

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.     

Molybdenum nitrogenase catalyzes the reduction and coupling of CO to form hydrocarbons

The molybdenum-dependent nitrogenase catalyzes the multi-electron reduction of protons and N(2) to yield H(2) and 2NH(3). It also catalyzes the reduction of a number of non-physiological doubly and triply bonded small molecules (e.g. C(2)H(2), N(2)O). Carbon monoxide (CO) is not reduced by the wild-type molybdenum nitrogenase but instead inhibits the reduction of all substrates catalyzed by nitrogenase except protons. Here, we report that when the nitrogenase MoFe protein α-Val(70) residue is substituted by alanine or glycine, the resulting variant proteins will catalyze the reduction and coupling of CO to form methane (CH(4)), ethane (C(2)H(6)), ethylene (C(2)H(4)), propene (C(3)H(6)), and propane (C(3)H(8)). The rates and ratios of hydrocarbon production from CO can be adjusted by changing the flux of electrons through nitrogenase, by substitution of other amino acids located near the FeMo-cofactor, or by changing the partial pressure of CO. Increasing the partial pressure of CO shifted the product ratio in favor of the longer chain alkanes and alkenes. The implications of these findings in understanding the nitrogenase mechanism and the relationship to Fischer-Tropsch production of hydrocarbons from CO are discussed.     

Continuous monitoring, by mass spectrometry, of H2 production and recycling in Rhodopseudomonas capsulata.

Hydrogen evolution and consumption by cell and chromatophore suspensions of the photosynthetic bacterium Rhodopseudomonas capsulata was measured with a sensitive and specific mass spectrometric technique which directly monitors dissolved gases. H2 production by nitrogenase was inhibited by acetylene and restored by carbon monoxide. An H2 evolution activity coupled with HD formation and D2 uptake (H-D exchange) was unaffected by C2H2 and CO. Cultures lacking nitrogenase activity also exhibited H-D exchange activity, which was catalyzed by a membrane-bound hydrogenase present in the chromatophores of R. capsulata. A net hydrogen uptake, mediated by hydrogenase, was observed when electron acceptors such as CO2, O2, or ferricyanide were present in the medium.     

An atomic-level mechanism for molybdenum nitrogenase. Part 1. Reduction of dinitrogen

The properties of the Fe and Mo sites of the iron-molybdenum cofactor of nitrogenase with respect to binding and activation of N(2) have been studied by molecular mechanics calculations on the local protein environment and by density functional theory (DFT) calculations on subsections of the cofactor. The DFT calculations indicate that the homocitrate ligand of the cofactor can become monodentate on reduction, allowing N(2) to bind at Mo. In addition, the neighboring Fe atom plays a crucial role in N(2) reduction by stabilizing the initial reduced N(2) species and by facilitating cleavage of the N-N bond. The various possible isomers for partially reduced N(2) intermediates have been compared by DFT, and a detailed model for the reduction of N(2) is developed based on these results, together with chemical precedents and the available biochemical data for nitrogenase.     

Kinetics and mechanism of the reaction of cyanide with molybdenum nitrogenase from Azotobacter vinelandii

The steady-state kinetic behavior of the six-electron reduction of N2 by nitrogenase is known to differ markedly from the six-electron reduction of cyanide in two ways. First, on extrapolation to infinite concentration of cyanide, the H2 evolution reaction is almost completely suppressed whereas at extrapolated infinite concentration of N2, H2 evolution continues. Second, as the ratio of the Fe protein to the MoFe protein increases, the reduction of N2 is favored over H2 evolution, whereas the reduction of cyanide becomes less favored relative to H2 evolution. We have extended these steady-state experiments with Azotobacter vinelandii nitrogenase to include a third observation, that the six-electron reduction of N2 is favored over H2 evolution at high total protein concentrations whereas cyanide reduction is less favored over H2 evolution at high total protein concentrations. All three steady-state observations can be explained by a model whereby cyanide is proposed to bind to a redox state of the MoFe protein more oxidized than that reactive toward H2 evolution and N2 reduction. To test this model, we have examined the pre-steady-state kinetic behavior of both cyanide reduction by A. vinelandii nitrogenase and cyanide inhibition of total electron flow through nitrogenase. The data show that in the presence or absence of cyanide there is a short lag of 100 ms before H2 is detected, followed by a linear phase of H2 evolution lasting for about 3 s, during which time no effects of cyanide are observable. After 3 s electron flow is finally inhibited by cyanide, and the cyanide reduction product CH4 is finally formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Model of the Regulation of Nitrogenase Electron Allocation in Legume Nodules (II. Comparison of Empirical and Theoretical Studies in Soybean)

In N2-fixing legumes, the proportion of total electron flow through nitrogenase (total nitrogenase activity, TNA) that is used for N2 fixation is called the electron allocation coefficient (EAC). Previous studies have proposed that EAC is regulated by the competitive inhibition of H2 on N2 fixation and that the degree of H2 inhibition can be affected by a nodule's permeability to gas diffusion. To test this hypothesis, EAC was measured in soybean (Glycine max L. Merr.) nodules exposed to various partial pressures of H2 and N2, with or without changes in TNA or nodule permeability to gas diffusion, and the results were compared with the predictions of a mathematical model that combined equations for gas diffusion and competitive inhibition of N2 fixation (A. Moloney and D.B. Layzell [1993] Plant Physiol 103: 421-428). The empirical data clearly showed that decreases in EAC were associated with increases in external pH2, decreases in external pN2, and decreases in nodule permeability to O2 diffusion. The model predicted similar trends in EAC, and the small deviations that occurred between measured and predicted values could be readily accounted for by altering one or more of the following model assumptions: K1(H2) of nitrogenase (range from 2-4% H2), Km(N2) of nitrogenase (range from 4-5% N2), the allocation of less than 100% of whole-nodule respiration to tissues within the diffusion barrier, and the presence of a diffusion pathway that is open pore versus closed pore. The differences in the open-pore and closed-pore versions of the model suggest that it may be possible to use EAC measurements as a tool for the study of legume nodule diffusion barrier structure and function. The ability of the model to predict EAC provided strong support for the hypothesis that H2 inhibition of N2 fixation plays a major role in the in vivo control of EAC and that the presence of a variable barrier to gas diffusion affects the H2 and N2 concentration in the infected cell and, therefore, the degree of H2 inhibition.     

Azotobacter vinelandii nitrogenases containing altered MoFe proteins with substitutions in the FeMo-cofactor environment: effects on the catalyzed reduction of acetylene and ethylene

Altered MoFe proteins of Azotobacter vinelandii Mo-nitrogenase, with amino acid substitutions in the FeMo-cofactor environment, were used to probe interactions among C(2)H(2), C(2)H(4), CO, and H(2). The altered MoFe proteins used were the alpha-195(Asn) or alpha-195(Gln) MoFe proteins, which have either asparagine or glutamine substituting for alpha-histidine-195, and the alpha-191(Lys) MoFe protein, which has lysine substituting for alpha-glutamine-191. On the basis of K(m) determinations, C(2)H(2) was a particularly poor substrate for the nitrogenase containing the alpha-191(Lys) MoFe protein. Using C(2)D(2), a correlation was shown between the stereospecificity of proton addition to give the products, cis- and trans-C(2)D(2)H(2), and the propensity of nitrogenase to produce ethane. The most extensive loss of stereospecificity occurred with nitrogenases containing either the alpha-195(Asn) or the alpha-191(Lys) MoFe proteins, which also exhibited the highest rate of ethane production from C(2)H(2). These data are consistent with the presence of a common ethylenic intermediate on the enzyme, which is responsible for both ethane production and loss of proton-addition stereochemistry. C(2)H(4) was not a substrate of the nitrogenase with the alpha-191(Lys) MoFe protein and was a poor substrate of the nitrogenases incorporating either the wild-type or the alpha-195(Gln) MoFe protein, both of which had a low V(max) and high K(m) (120 kPa). Ethylene was a somewhat better substrate for the nitrogenase with the alpha-195(Asn) MoFe protein, which exhibited a K(m) of 48 kPa and a specific activity for C(2)H(6) formation from C(2)H(4) 10-fold higher than the others. Neither the wild-type nitrogenase nor the nitrogenase containing the alpha-195(Asn) MoFe protein produced cis-C(2)D(2)H(2) when turned over under trans-C(2)D(2)H(2). These results suggest that the C(2)H(4)-reduction site is affected by substitution at residue alpha-195, although whether the effect is related to the substrate-reduction site directly or is mediated through disturbance of the delivery of electrons/protons is unclear. Ethylene inhibited total electron flux, without uncoupling MgATP hydrolysis from electron transfer, to a similar extent for all four A. vinelandii nitrogenases. This observation indicates that this C(2)H(4) flux-inhibition site is remote from the C(2)H(4)-reduction site. Added CO eliminated C(2)H(4) reduction but did not fully relieve its electron-flux inhibition with all four A. vinelandii nitrogenases, supporting the suggestion that electron-flux inhibition by C(2)H(4) is not directly connected to C(2)H(4) reduction. Thus, C(2)H(4) has two binding sites, and the presence of CO affects only the site at which it binds as a substrate. When C(2)H(2) was added, it also eliminated C(2)H(6) production from C(2)H(4) and also did not relieve electron-flux inhibition fully. Thus, C(2)H(2) and C(2)H(4) are likely reduced at the same site on the MoFe protein. Two schemes are presented to integrate the results of the interactions of C(2)H(2) and C(2)H(4) with the MoFe proteins.     

Dinitrogenase with altered substrate specificity results from the use of homocitrate analogues for in vitro synthesis of the iron-molybdenum cofactor

The in vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase requires homocitrate (2-hydroxy-1,2,4-butanetricarboxylic acid). Homocitrate is apparently synthesized by the nifV gene product. In the absence of homocitrate, no FeMo-co is formed in vitro, as determined from coupled C2H2 reduction assays and the lack of 99Mo label incorporation into apodinitrogenase. Several organic acids were tested for their ability to replace homocitrate in the FeMo-co synthesis system. With appropriate homocitrate analogues, aberrant forms of FeMo-co are synthesized that exhibit altered substrate specificity and inhibitor susceptibility. Homoisocitrate (1-hydroxy-1,2,4-butanetricarboxylic acid) and 2-oxoglutarate facilitated the incorporation of 99Mo into apodinitrogenase in the FeMo-co synthesis system, yielding a dinitrogenase that effectively catalyzed the reduction of protons but not C2H2 or N2. Citrate also promoted the incorporation of 99Mo into apodinitrogenase, and the resulting holodinitrogenase reduced protons and C2H2 effectively but not N2. In addition, proton reduction from this enzyme was inhibited by CO. The properties of the homodinitrogenase formed in the presence of citrate were reminiscent of those of the Klebsiella pneumoniae NifV- dinitrogenase. We also observed low rates of HD formation from NifV- dinitrogenase compared to those from the wild-type enzyme. No HD formation was observed with the dinitrogenase activated in vitro in the presence of citrate. We propose that in vivo NifV- mutants utilize citrate for FeMo-co synthesis.     

Reduction of N<Subscript>2</Subscript> by Fe<Superscript>2+</Superscript> via Homogeneous and Heterogeneous Reactions Part 2: The Role of Metal Binding in Activating N<Subscript>2</Subscript> for Reduction; a Requirement for Both Pre-biotic and Biological Mechanisms

Nitrogen reduction by ferrous iron has been suggested as an important mechanism in the formation of ammonia on pre-biotic Earth. This paper examines the effects of adsorption of ferrous iron onto a goethite (alpha-FeOOH) substrate on the thermodynamic driving force and rate of a ferrous iron-mediated reduction of N2 as compared with the homogeneous aqueous reaction. Utilizing density functional theory and Marcus Theory of proton coupled electron transfer reactions, the following two reactions were studied: Fe2+aq + N2aq + H2Oaq --> N2H* + FeOH2+aq and triple bond Fe2+ads + N2aq + 2H2Oaq --> N2H* + alpha-FeOOHs + 2H+aq. Although the rates of both reactions were calculated to be approximately zero at 298 K, the model results suggest that adsorption alters the thermodynamic driving force for the reaction but has no other effect on the direct electron transfer kinetics. Given that simply altering the thermodynamic driving force will not reduce dinitrogen, we can make mechanistic connections between possible prebiotic pathways and biological N2 reduction. The key to reduction in both cases is N2 adsorption to multiple transition metal centers with competitive H2 production.     

Nitrogenase reduction of carbon disulfide: freeze-quench EPR and ENDOR evidence for three sequential intermediates with cluster-bound carbon moieties

Freeze-quenching of nitrogenase during reduction of carbon disulfide (CS(2)) was previously shown to result in the appearance of a novel EPR signal (g = 2.21, 1.99, and 1.97) not previously associated with any of the oxidation states of the nitrogenase metal clusters. In the present work, freeze-quench X- and Q-band EPR and Q-band (13)C electron nuclear double resonance (ENDOR) spectroscopic studies of nitrogenase during CS(2) reduction disclose the sequential formation of three distinct intermediates with a carbon-containing fragment of CS(2) bound to a metal cluster inferred to be the molybdenum-iron cofactor. Modeling of the Q-band (35 GHz) EPR spectrum of freeze-trapped samples of nitrogenase during turnover with CS(2) allowed assignment of three signals designated "a" (g = 2.035, 1.982, 1.973), "b" (g = 2.111, 2.002, and 1.956), and "c" (g = 2.211, 1. 996, and 1.978). Freezing samples at varying times after initiation of the reaction reveals that signals "a", "b", and "c" appear and disappear in sequential order. Signal "a" reaches a maximal intensity at 25 s; signal "b" achieves maximal intensity at 60 s; and signal "c" shows maximal intensity at 100 s. To characterize the intermediates, (13)CS(2) was used as a substrate, and freeze-trapped turnover samples were examined by Q-band (13)C ENDOR spectroscopy. Each EPR signal ("a", "b", and "c") gave rise to a distinct (13)C signal, with hyperfine coupling constants of 4.9 MHz for (13)C(a), 1. 8 MHz for (13)C(b), and 2.7 MHz for (13)C(c). Models for the sequential formation of intermediates during nitrogenase reduction of CS(2) are discussed.