Analysis of steady state Fe and MoFe protein interactions during nitrogenase catalysis

Steady state kinetic measurements are reported for nitrogenase from Azotobacter vinelandii (Av) and Clostridium pasteurianum (Cp) under a variety of conditions, using dithionite as reductant. The specific activities of Av1 and Cp1 are determined as functions of Av2:Av1 and Cp2:Cp1, respectively, at component protein ratios from 0.4 to 50, and conform to a simple hyperbolic rate law for the interaction of Av2 with Av1 and Cp2 with Cp1. The specific activities of Av2 and Cp2 are also measured as a function of increasing Av1 and Cp1 concentrations, producing 'MoFe inhibition' at large MoFe:Fe ratios. When the rate of product formation under MoFe inhibited conditions is re-plotted as increasing Av2:Av1 or Cp2:Cp1 ratios, sigmoidal kinetic behavior is observed, suggesting that the rate constants in the Thorneley and Lowe (T&L) model are more dependent upon the oxidation level of MoFe protein than previously suspected [R.N.F. Thorneley, D.J. Lowe, Biochem. J. 224 (1984) 887-894], at least when applied to Av and Cp. Calculation of Hill coefficients gave values of 1.7-1.9, suggesting a highly cooperative Fe-MoFe protein interaction in both Av and Cp nitrogenase catalysis. The T&L model lacks analytical solutions [R.N.F. Thorneley, D.J. Lowe, Biochem. J. 215 (1983) 393-404], so the ease of its application to experimental data is limited. To facilitate the study of steady state kinetic data for H(2) evolution, analytical equations are derived from a different mechanism for nitrogenase activity, similar to that of Bergersen and Turner [Biochem. J. 131 (1973) 61-75]. This alternative cooperative model assumes that two Fe proteins interact with one MoFe protein active site. The derived rate laws for this mechanism were fitted to the observed sigmoidal behavior for low Fe:MoFe ratios (<0.4), as well as to the commonly observed hyperbolic behavior for high values of Fe:MoFe for both Av and Cp.     

Electron paramagnetic resonance analysis of different Azotobacter vinelandii nitrogenase MoFe-protein conformations generated during enzyme turnover: evidence for S = 3/2 spin states from reduced MoFe-protein intermediates

Rapid-freezing experiments elicited two transient EPR signals, designated 1b and 1c, during Azotobacter vinelandii nitrogenase turnover at 23 degrees C and pH 7.4. The first of the signals to form, signal 1b, exhibited g values of 4.21 and 3.76. Its formation was at the expense of the starting EPR signal (signal 1a with g values of 4.32, 3.66, and 2.01). The second signal to arise, signal 1c, with a characteristic g value of 4.69, formed very slowly and was always of low intensity. Both signals occurred independently of the substrate being reduced. Increased electron flux through the MoFe protein caused these signals to form more rapidly. Moreover, after a MoFe-protein solution had been pretreated (using conditions of extremely low electron flux) to set up an equimolar mixture of its resting state and one-electron reduced state, these signals appeared even more rapidly when this mixture was exposed to an excess of the Fe protein. We have simulated the kinetics of formation of these EPR features using the published kinetic model for nitrogenase catalysis [Lowe, D. J., and Thorneley, R. N. F. (1984) Biochem. J. 224, 887-909] and propose that they arise from reduced states of the MoFe protein and reflect different conformations of the FeMo cofactor with different protonation states.     

The Chatt cycle and the mechanism of enzymic reduction of molecular nitrogen

 The Chatt Cycle is a hypothetical model for the mechanism of nitrogenase action at the atomic level and is based upon reactions of certain well-defined molybdenum compounds. The proton and electron-transfer chemistry of the cycle is reviewed, revised and extended. An explanation for general and obligatory hydrogen evolution by the nitrogenase system is advanced; this involves carboxylate acting as a tethered leaving group at the Mo centre and the formation of mono-, di- and tri-hydric intermediates. The chemical model is integrated with the biochemical model proposed by Lowe and Thorneley. Received and accepted: 21 August 1996     

The mechanism of Klebsiella pneumoniae nitrogenase action. Simulation of the dependences of H2-evolution rate on component-protein concentration and ratio and sodium dithionite concentration.

The rate constants from Table 1 and Scheme 2 of Lowe & Thorneley [(1984) Biochem. J. 224, 877-886] were used to simulate the rate of H2 evolution, under various conditions, from nitrogenase isolated from Klebsiella pneumoniae. These rates depend on both the ratio and concentrations of the MoFe protein and Fe protein that comprise nitrogenase. The simulations explain the shapes of 'protein titration' and 'dilution effect' curves. The concept of an apparent Km for the reductant Na2S2O4 is shown to be invalid, since the dependence of H2-evolution rate on the square root of S2O4(2-) concentration is not hyperbolic and depends on the ratio and absolute concentrations of the MoFe protein and Fe protein.     

Mechanistic interpretation of the dilution effect for Azotobacter vinelandii and Clostridium pasteurianum nitrogenase catalysis

Nitrogenase activity for Clostridium pasteurianum (Cp) at a Cp2:Cp1 ratio of 1.0 and Azotobacter vinelandii (Av) at Av2:Av1 protein ratios (R) of 1, 4 and 10 is determined as a function of increasing MoFe protein concentration from 0.01 to 5 microM. The rates of ethylene and hydrogen evolution for these ratios and concentrations were measured to determine the effect of extreme dilution on nitrogenase activity. The experimental results show three distinct types of kinetic behavior: (1) a finite intercept along the concentration axis (approximately 0.05 microM MoFe); (2) a non-linear increase in the rate of product formation with increasing protein concentration (approximately 0.2 microM MoFe) and (3) a limiting linear rate of product formation at high protein concentrations (>0.4 microM MoFe). The data are fitted using the following rate equation derived from a mechanism for which two Fe proteins interact cooperatively with a single half of the MoFe protein. (see equation) The equation predicts that the cubic dependence in MoFe protein gives rise to the non-linear rate of product formation (the dilution effect) at very low MoFe protein concentrations. The equation also predicts that the rate will vary linearly at high MoFe protein concentrations with increasing MoFe protein concentration. That these limiting predictions are in accord with the experimental results suggests that either two Fe proteins interact cooperatively with a single half of the MoFe protein, or that the rate constants in the Thorneley and Lowe model are more dependent upon the redox state of MoFe protein than previously suspected [R.N. Thornley and D. J. Lowe, Biochem. J. 224 (1984) 887-894]. Previous Klebsiella pneumoniae and Azotobacter chroococcum dilution results were reanalyzed using the above equation. Results from all of these nitrogenases are consistent and suggest that cooperativity is a fundamental kinetic aspect of nitrogenase catalysis.     

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.     

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.     

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.     

Simulating large nuclear quantum mechanical corrections in hydrogen atom transfer reactions in metalloenzymes

The role of nuclear quantum mechanical effects in enzyme catalysis has recently attracted significant interest both from theoretical and experimental points of view. From a theoretical point of view, it is undoubtedly a challenge to try to account for the observed tunneling in the protein by microscopic simulations without adjustable parameters. One of the most spectacular examples of nuclear quantum mechanical effects is the reaction of lipoxygenase, which is characterized by a very large kinetic isotope effect and, thus, provides an excellent benchmark for simulation approaches. In the present study, we report a microscopic simulation of the large kinetic isotope effect in soybean lipoxygenase and its temperature dependence. This is, to the best of our knowledge, the first time that a very large nuclear quantum mechanical contribution to the activation free energy of a hydrogen atom transfer reaction and its temperature dependence have been evaluated by microscopic simulation. The simulation reproduces quite well the experimental kinetic information and the corresponding difference between the classical and quantum mechanical activation free energies for the H and D transfer reactions.     

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

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

Substrate interactions with nitrogenase: Fe versus Mo

Biological nitrogen reduction is catalyzed by a complex two-component metalloenzyme called nitrogenase. For the Mo-dependent enzyme, the site of substrate reduction is provided by a [7Fe-9S-Mo-X-homocitrate] metallocluster, where X is proposed to be an N atom. Recent progress with organometallic model compounds, theoretical calculations, and biochemical, kinetic, and biophysical studies on nitrogenase has led to the formulation of two opposing models of where N(2) or alternative substrates might bind during catalysis. One model involves substrate binding to the Mo atom, whereas the other model involves the participation of one or more Fe atoms located in the central region of the metallocluster. Recently gathered evidence that has provided the basis for both models is summarized, and a perspective on future research in resolving this fundamental mechanistic question is presented.     

NMR and MD studies on the interaction between ligand peptides and alpha-bungarotoxin

The interaction between alpha-bungarotoxin and linear synthetic peptides, mimotope of the nicotinic acetylcholine receptor binding site, has been characterised extensively by several methods and a wealth of functional, kinetic and structural data are available. Hence, this system represents a suitable model to explore in detail the dynamics of a peptide-protein interaction. Here, the solution structure of a new complex of the protein toxin with a tridecapeptide ligand exhibiting high affinity has been determined by NMR. As observed for three other previously reported mimotope-alpha-bungarotoxin complexes, also in this case correlations between biological activity and kinetic data are not fully consistent with a static discussion of structural data. Molecular dynamics simulations of the four mimotope-toxin complexes indicate that a relevant contribution to the complex stability is given by the extent of the residual flexibility that the protein maintains upon peptide binding. This feature, limiting the entropy loss caused by protein folding and binding, ought to be generally considered in a rational design of specific protein ligands.     

Monte Carlo simulation from proton slip to "coupled" proton flow in ATP synthase based on the bi-site mechanism

ATP synthase couples proton flow to ATP synthesis, but is leaky to protons at very low nucleotide concentration. Based on the bi-site mechanism, we simulated the proton conduction from proton slip to “coupled” proton flow in ATP synthase using the Monte Carlo method. Good agreement is obtained between the simulated and available experimental results. Our model provides deeper insight into the nucleotide dependence of ATP catalysis, and the kinetic cooperativity in three catalysis subunits. The results of simulation support the bi-site mechanism in ATP synthesis.     

Reactions of Azotobacter vinelandii nitrogenase using Ti(III) as reductant

Nitrogenase-catalyzed reactions using Ti(III) were examined under a wide variety of conditions to determine the suitability of Ti(III) to serve as a general nitrogenase reductant. Solutions prepared from H2-reduced TiCl3, aluminum-reduced TiCl3, TiCl2, evaporated TiCl3 from an HCl, solution, and TiF3 were evaluated as reductants. Three general types of reactivity were observed. The first showed that, below Ti(III) concentrations of about 0.50 mM, nitrogenase catalysis utilized Ti(III) in a first-order reaction. The second showed that, above 0.50 mM, the rate of nitrogenase catalysis was zero order in Ti(III), indicating the enzyme was saturated with this reductant. Above 2.0-5.0 mM, nitrogenase catalysis was inhibited by Ti(III) depending on the titanium source used for solution preparation. This inhibition was investigated and found to be independent of the buffer type and pH, while high salt and citrate concentrations caused moderate inhibition. [Ti(IV)] above 2.0-3.0 mM and [Ti(III)] above about 5.0 mM were inhibitory. ATP/2e values were 4-5 for [Ti(III)] at or below 1.0-2.0 mM, 2.0 from 5.0 to 7.0 mM Ti(III) where nitrogenase is not inhibited, and 2.0 above 7.0 mM Ti(III) where severe inhibition occurs. For nitrogenase-catalyzed reactions using Ti(III) as reductant, the potential of the solution changes with time as the Ti(III)/Ti(IV) ratio changes. From the change in the rate of product formation (Ti(III) disappearance) with change in solution potential, the rate of nitrogenase catalysis was determined as a function of solution potential. From such experiments, a midpoint turnover potential of -480 mV was determined for nitrogenase catalysis with an associated n = 2 value.     

Mechanistic origin of the kinetic cooperativity of hexokinase type L1 from wheat germ

A theoretical analysis is presented which shows that initial velocity data for hexokinase L1 catalysis of glucose phosphorylation by MgATP cannot be reconciled with the observed rate of the 'mnemonical' conformational transition which has been proposed to account for the kinetic cooperativity of the enzyme. The basic kinetic properties of hexokinase L1 and other allegedly 'mnemonical' enzymes appear to be fully consistent with an ordered ternary-complex mechanism in which the leading substrate participates in abortive-complex formation. It is concluded that, so far, no enzyme displaying kinetic cooperativity has been convincingly demonstrated to operate by a 'mnemonical' type of reaction mechanism.     

Limiting Energy Dissipation Induces Glassy Kinetics in Single-Cell High-Precision Responses

Single cells often generate precise responses by involving dissipative out-of-thermodynamic-equilibrium processes in signaling networks. The available free energy to fuel these processes could become limited depending on the metabolic state of an individual cell. How does limiting dissipation affect the kinetics of high-precision responses in single cells? I address this question in the context of a kinetic proofreading scheme used in a simple model of early-time T cell signaling. Using exact analytical calculations and numerical simulations, I show that limiting dissipation qualitatively changes the kinetics in single cells marked by emergence of slow kinetics, large cell-to-cell variations of copy numbers, temporally correlated stochastic events (dynamic facilitation), and ergodicity breaking. Thus, constraints in energy dissipation, in addition to negatively affecting ligand discrimination in T cells, can create a fundamental difficulty in determining single-cell kinetics from cell-population results.     

Only one protomer is active in the dimer of SARS 3C-like proteinase

The severe acute respiratory syndrome coronavirus 3C-like protease has been proposed to be a key target for structurally based drug design against SARS. The enzyme exists as a mixture of dimer and monomer, and only the dimer was considered to be active. In this report, we have investigated, using molecular dynamics simulation and mutational studies, the problems as to why only the dimer is active and whether both of the two protomers in the dimer are active. The molecular dynamics simulations show that the monomers are always inactive, that the two protomers in the dimer are asymmetric, and that only one protomer is active at a time. The enzyme activity of the hybrid severe acute respiratory syndrome coronavirus 3C-like protease of the wild-type protein and the inactive mutant proves that the dimerization is important for enzyme activity and only one active protomer in the dimer is enough for the catalysis. Our simulations also show that the right conformation for catalysis in one protomer can be induced upon dimer formation. These results suggest that the enzyme may follow the association, activation, catalysis, and dissociation mechanism for activity control.     

A kinetic simulation model that describes catalysis and regulation in nitric-oxide synthase

After initiating NO synthesis a majority of neuronal NO synthase (nNOS) quickly partitions into a ferrous heme-NO complex. This down-regulates activity and increases enzyme K(m,O(2)). To understand this process, we developed a 10-step kinetic model in which the ferric heme-NO enzyme forms as the immediate product of catalysis, and then partitions between NO dissociation versus reduction to a ferrous heme-NO complex. Rate constants used for the model were derived from recent literature or were determined here. Computer simulations of the model precisely described both pre-steady and steady-state features of nNOS catalysis, including NADPH consumption and NO production, buildup of a heme-NO complex, changes between pre-steady and steady-state rates, and the change in enzyme K(m,O(2)) in the presence or absence of NO synthesis. The model also correctly simulated the catalytic features of nNOS mutants W409F and W409Y, which are hyperactive and display less heme-NO complex formation in the steady state. Model simulations showed how the rate of heme reduction influences several features of nNOS catalysis, including populations of NO-bound versus NO-free enzyme in the steady state and the rate of NO synthesis. The simulation predicts that there is an optimum rate of heme reduction that is close to the measured rate in nNOS. Ratio between NADPH consumption and NO synthesis is also predicted to increase with faster heme reduction. Our kinetic model is an accurate and versatile tool for understanding catalytic behavior and will provide new perspectives on NOS regulation.     

A MODEL OF HIGH AFFINITY GLUTAMIC ACID TRANSPORT BY RAT CORTICAL SYNAPTOSOMES–A REFINEMENT OF THE ORIGINALLY PROPOSED MODEL

Abstract– In a previous publication (W heeler & H ollingsworth , 1978), a model was presented which accounted for the role of sodium in the high affinity transport of glutamic acid in rat brain synaptosomes. Subsequent studies confirmed a lack of fit of the model to the data at the higher sodium and glutamate concentrations. The model has therefore been reexamined and refined. By removing some of the restrictions placed on the original model, a model emerges which fits the data at all sodium and glutamate concentrations with an average per cent error of only 2.14% per experimental data point. The kinetic constants describing uptake have been redefined and recalculated in accordance with this revised model.     

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.