The temperature dependence of the pre-steady-state MgATP-dependent electron transfer from the MoFe protein to the Fe protein of the nitrogenase from Azotohacter vinelandii has been investigated between 6°C and 31 "C by stopped-flow spectrophotometry. Below 1 4 T , the data are consistent with a model in which interaction of MgATP with nitrogenase is fast and irreversible, and is followed by reversible electron transfer. From the extent and from the rate of the absorbance change, the rate constants for electron transfer from Fe protein to MoFe protein and of the reverse reaction were calculated. The direct rate constant increases with temperature (6-14°C) from about 1 s-l to about 26 s-'. The rate constant for the reverse reaction was found to be approximately 4 s -l and invariant with the reaction temperature. Analysis of the data obtained in the temperature range between 6°C and 12°C within the framework of the transition-state theory show that electron transfer from the Fe protein to the MoFe protein occurs via a highly disordered transition state with activation parameters AH": = 289 kJ . mol-' and A S ' ; = 792 J . K -l . mol-'. The Eyring plot of the stoppedflow data displays an inflection point around 14°C. From the stopped-flow data obtained between 18°C and 27°C the activation parameters AH": and AS" for the reduction of the MoFe protein by Fe protein are calculaled to be 90 kJ . mol-' and 99 J . K -' . mol-' respectively. A second inflection point in the Eyring plot could exist around 28°C.

The pre-steady-state ATPase activity of nitrogenase has been reinvestigated. The exceptionally high burst in the hydrolysis of MgATP by the nitrogenase from Amtobarter vinelandii communicated by Cordewener et al. (1987) 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"C, a pre-steady-state phase of H t production was observed, with a first-order rate constant of 2.2 s-l, whereas electron transfer occurred with a first-order rate constant of 4.9 s-'. At 20.0"C, MgATP-dependent H + production and electron transfer in the pre-steady-state phase were characterized by observed rate constants of 9.4 s-l and 104 s -', rcspcctively. 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. Eady et al. [8] have published a combined stopped-llow spectrophotometry and rapid quench study in which pre-steady-state electron transfer and MgATP hydrolysis were investigated. A1 l O T , both reactions were characterized by a burst phase which could both be fitted to a single exponential function. Since the rate constants were found to be identical within experimental error, these results were interpreted as evidence that electron transfer from the Fe Enzj~me. Nitrogcnase (EC 1.1.1.37).

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