Earlier studies of electron transfer (ET) from the nitrogenase Fe protein to the MoFe protein concluded that the mechanism for ET changed during cooling from 25 to 5 °C, based on the observation that the rate constant for Fe protein to MoFe protein ET decreases strongly, with a nonlinear Arrhenius plot. They further indicated that the ET was reversible, with complete ET at ambient temperature but with an equilibrium constant near unity at 5 °C. These studies were conducted with buffers having a strong temperature coefficient. We have examined the temperature variation in the kinetics of oxidation of the Fe protein by the MoFe protein at a constant pH of 7.4 fixed by the buffer 3-(N-morpholino)propanesulfonic acid (MOPS), which has a very small temperature coefficient. Using MOPS, we also observe temperature-dependent ET rate constants, with nonlinear Arrhenius plots, but we find that ET is gated across the temperature range by a conformational change that involves the binding of numerous water molecules, consistent with an unchanging ET mechanism. Furthermore, there is no solvent kinetic isotope effect throughout the temperature range studied, again consistent with an unchanging mechanism. In addition, the nonlinear Arrhenius plots are explained by the change in heat capacity caused by the binding of waters in an invariant gating ET mechanism. Together, these observations contradict the idea of a change in ET mechanism with cooling. Finally, the extent of ET at constant pH does not change significantly with temperature, in contrast to the previously proposed change in ET equilibrium.
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