We have studied the pre-steady-state kinetics of wild type and altered nitrogenases during turnover in order to characterize the different MoFe-protein catalytic intermediates using rapid-freeze EPR and UV/visible stopped-flow spectroscopy. The major drawback with such experiments, in addition to the large amounts of protein required, is that the reduced states of the MoFe-protein which bind substrates, are only generated under turnover conditions and therefore the intermediates of interest are only a small proportion of the total protein present. EPR and stopped-flow spectroscopy are particularly useful techniques because they allow us to detect conformational and redox changes that are likely to control the delivery of electrons and protons to the FeMo-cofactor during substrate reduction.

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