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.

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.

Steady-state kinetic studies of dithionite utilization, component protein interaction, and the formation of an oxidized iron protein intermediate during Azotobacter vinelandii nitrogenase catalysis.

Steady-state kinetic analysis of the two-component protein system of Azotobacter vinelandii (Av) nitrogenase is reported. A precisely obeyed half-order reaction in dithionite was observed at concentrations up to 21 mM with no indication of saturation by this substrate. This behavior was monitored by optical, amperometric, and manometric kinetic techniques, and the results were mathematically fit to establish the half-order reaction in dithionite. Under conditions where the MgATP and dithionite concentrations remain unchanged, Av2 (the Fe protein component) interacts with Av1 (the MoFe protein component according to the rate law, suggesting a rapid 1:1 Av2-Av1 interaction: [formula: see text]. with [Av2] the free Fe protein concentration, K = 5.9 microM, and Vmax = 2314 nmol of H2 min-1 (mg of Av1)-1. Under dithionite-depleted conditions, Av2 undergoes an Av1-mediated, one-electron oxidation, consistent with its proposed role as a specific, single-electron reductant for Av1. During steady-state turnover as a function of Av2/Av1 ratio, optical spectroscopy demonstrated the presence of 25-30% oxidized Av2 as an enzyme intermediate. Computer-averaged EPR spectra showed that Av1 was > 95% EPR-silent and Av2 was up to 30% oxidized (Av2ox), consistent with the optical measurements. These optical and EPR results show that up to six Av2ox per Av1 can accumulate in the presence of dithionite during catalysis, suggesting that the conversion of Av2ox back into Av2red is a relatively slow process.