AZOTOBACTER VINELANDII NITROGENASE MoFe PROTEIN: PRE-STEADY STATE SPECTROSCOPIC STUDIES OF THE METAL COFACTORS

摘要

Rapid freezing of wild-type A. vinelandii nitrogenase at a 3:1 molar ratio of Fe protein to MoFe protein, 23癈, and pH 7.4, elicited two transient electron paramagnetic resonance (EPR) signals (signals Ib and Ic) from the FeMo-cofactor within the MoFeprotein ca. 500 ms after mixing. The first signal (Ib; g = 4.21, 3.76) formed at the expense of the starting S = 3/2 EPR signal (la; g = 4.32, 3.66), whereas the second signal (Ic; g = 4.7, 3.4) formed more slowly and in lower intensity. Both signals occurred independent of substrate (N_2, CJElz, H~+). By either increasing the molar ratio of the component proteins or pre-reducing the MoFe protein, both signal Ib and Ic formed more quickly, indicating that they arose from more-reduced MoFe-protein states. The loss of signal-la intensity was never fully compensated by those of signals Ib plus Ic, suggesting the presence of other EPR-silent species. Simulations of the kinetics of signal-Ib formation indicated that it arose from a 3-electron reduced state(pound 3) of the MoFe protein and so signal Ic likely arose from an even more reduced state (Fisher et al., 2001). Furthermore, signal Ib closely resembled the EPR signal reported for Klebsiella pnewnoniae MoFe protein at high pH (Smith et al., 1973), which presumably reflects deprotonation and a changed conformation at the FeMo-cofactor.