Multiple variations of the S=3/2 EPR signal have been previously observed for wild-type MoFe protein. A signal designated as la occurs at neutral pH in the resting MoFe protein, signal lb is observed at higher pH, and signal Ic is only observed during turnover (Smith et al. 1973). We have detected similar S=3/2 signals from the more-reduced states of the wild-type MoFe protein during catalytic turnover and provide evidence that they arise from different conformations of the FeMo-cofactor within the MoFe protein. We attribute signal lb to MoFe protein essentially at the E3 redox level and suggest that the conformational change results as a consequence of protons being incorporated into reduced substrate. Signal Ic is generated much more slowly than lb and, therefore, likely arises from an even more-reduced redox state than lb.
We have extended these studies to include the a-195Gln, a-195Asn, and oc-191Lys MoFe proteins which bind N2 and reduce it very poorly, bind N2 and do not reduce it, and do not bind N2, respectively. The a-191Lys MoFe protein is particularly interesting because it exhibits neither signal lb nor signal Ic under turnover conditions. In addition, stopped-flow spectrophotometric studies with this protein do not show the oxidation after primary electron transfer that is observed with wild-type nitrogenase. This oxidation has been postulated to arise from the P-clusters and to be the step that irreversibly commits N2 to reduction. All evidence suggests that this protein cannot attain the redox levels necessary for N2 binding and reduction. Although the a-195Glnand a-195Asn MoFe proteins show an oxidation, it is short lived and the lb and Ic EPR signals accumulate during catalysis, indicating a problem with the ^-reduction process. Our EPR and stopped-flow spectroscopic data show a correlation between N2 binding and the different intermediate forms of MoFe protein.
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