Spectroscopic evidence for changes in the redox state of the nitrogenase P-cluster during turnover

Biological nitrogen fixation catalyzed by nitrogenase requires the particip ation of two component proteins called the Fe protein and the MoFe protein. Each alpha beta catalytic unit of the MoFe protein contains an [8Fe-7S] cl uster and a [7Fe-9S-Mo-homocitrate] cluster, respectively designated the P- cluster and FeMo-cofactor. FeMo-cofactor is known to provide the site of su bstrate reduction whereas the P-cluster has been suggested to function in n itrogenase catalysis by providing an intermediate electron-transfer site. I n the present work, evidence is presented for redox changes of the P-cluste r during the nitrogenase catalytic cycle from examination of an altered MoF e protein that has the beta-subunit serine-188 residue substituted by cyste ine, This residue was targeted for substitution because it provides a rever sible redox-dependent ligand to one of the P-cluster Fe atoms. The altered beta-188(Cys) MoFe protein was found to reduce protons, acetylene, and nitr ogen at rates approximately 30% of that supported by the wild-type MoFe pro tein. In the dithionite-reduced state, the beta-188(Cys) MoFe protein exhib ited unusual electron paramagnetic resonance (EPR) signals arising from a m ixed spin state system (S = 5/2, 1/2) that integrated to 0.6 spin/alpha bet a-unit. These EPR signals were assigned to the P-cluster because they were also present in an ape-form of the beta-188(Cys) MoFe protein that does not contain FeMo-cofactor. Mediated voltammetry was used to show that the inte nsity of the EPR signals was maximal near -475 mV at pH 8.0 and that the P- cluster could be reversibly oxidized or reduced with concomitant loss in in tensity of the EPR signals. A midpoint potential (E-m) of -390 mV was appro ximated for the oxidized/resting state couple at pH 8.0, which was observed to be pH dependent. Finally, the EPR signals exhibited by the beta-188(Cys ) MoFe protein greatly diminished in intensity under nitrogenase turnover c onditions and reappeared to the original intensity when the MoFe protein re turned to the resting state.