IRON-SULFUR CLUSTERS IN THE MOLYBDENUM-IRON PROTEIN-COMPONENT OF NITROGENASE - ELECTRON-PARAMAGNETIC RESONANCE OF THE CARBON-MONOXIDE INHIBITED STATE

CO inhibits reduction of N2 by purified nitrogenase from Azotobacter vinelandii and Clostridium pasteurianum in a noncompetitive manner (Kii and Kis = 1.4 .times. 10-4 and 4.5 .times. 10-4 and 7 .times. 10-4 atm and 14 .times. 10-4 atm for the 2 enzymes, respectively). The onset of inhibition is within the turnover time of the enzyme, and CO does not affect the electron flux to the H2-evolving site. The kinetics of CO inhibition of N2 reduction are simple, but CO inhibition of acetylene reduction is complicated by substrate inhibition effects. When low-temperature (.apprx. 13.degree. K) electron paramagnetic resonance (EPR) spectra of CO-inhibited nitrogenase are examined, low concentrations of CO ([CO] = [enzyme]) induce the appearance of a signal with g values near 2.1, 1.98 and 1.92 with t1/2 [half-life] 4 s, while higher concentrations of CO lead to the appearance of a signal with g values near 2.17, 2.1 and 2.05 with a similar time course. The MoFe proteins from Rhizobium japonicum and Rhodospirillum rubrum, reduced with Azotobacter Fe protein in the presence of CO, give similar results. Under conditions which promote the accumulation of H2 in the absence of CO, an additional EPR signal with g values near 2.1, 2.0 and 1.98 is observed. The use of Azotobacter nitrogenase components enriched selectively with 57Fe or 95Mo, and the use of 13CO, permitted the assignment of the center(s) responsible for the induced signals. Only 57Fe, when present in the MoFe protein, yielded broadened EPR signals. The MoFe protein of nitrogenase contains one or more iron-sulfur clusters of the type found in the simple ferredoxins. It is further proposed that the CO-induced signals arise from states of the MoFe protein in which CO inhibits electron flow to the N2-reducing site so that Fe-S cluster achieves steady-state net charges of -1 (high CO complex) and -3 (low CO complex) in analogy to the normal paramagnetic statesof high-potential iron-sulfur proteins and ferredoxins, respectively. The no-CO signal may be either an additional center or the N2-reducing site with H2 bound competitively.