Electron paramagnetic resonance analysis of different Azotobacter vinelandii nitrogenase MoFe-protein conformations generated during enzyme turnover:Evidence for S = (3)/(2) spin states from reduced MoFe-protein intermediates

Rapid-freezing experiments elicited two transient EPR signals, designated 1 b and Ic, during Azotobacter vinelandii nitrogenase turnover at 23 degreesC and pH 7.4. The first of the signals to form, signal Ib, exhibited g value s of 4.21 and 3.76. Its formation was at the expense of the starting EPR si gnal (signal la with g values of 4.32, 3.66, and 2.01). The second signal t o arise, signal Ic, with a characteristic g value of 4.69, formed very slow ly and was always of low intensity. Both signals occurred independently of the substrate being reduced. Increased electron flux through the MoFe prote in caused these signals to form more rapidly. Moreover, after a MoFe-protei n solution had been pretreated (using conditions of extremely low electron flux) to set up an equimolar mixture of its resting state and one-electron reduced state, these signals appeared even more rapidly when this mixture w as exposed to an excess of the Fe protein. We have simulated the kinetics o f formation of these EPR features using the published kinetic model for nit rogenase catalysis [Lowe, D. J., and Thorneley, R. N. F. (1984) Biochem. J. 224, 887-909] and propose that they arise from reduced states of the MoFe protein and reflect different conformations of the FeMo cofactor with diffe rent protonation states.