Carbon monoxide inhibits reduction of dinitrogen (N2) by purified nitrogenase from Azotobacter vinelandii and Clostridium pasteurianum in a noncompetitive manner (Kii and Kis = 1.4 X 10(-4) and 4.5 X 10(-4) and 7 X 10(-4) atm and 14 X 10(-4) atm for the two 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 (approximately 13 K) electron paramagnetic resonance (EPR) spectra of CO-inhibited nitrogenase are examined, it is found that 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 approximately 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 nitogenase components enriched selectively with 57Fe or 95Mo, as well as 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. It is suggested that the MoFe protein of nitrogenase contains one or more iron-sulfur clusters of the type found in the simple ferrodoxins. 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 the iron-sulfur cluster achieves steady-state net charges of -1 (high CO complex) and -3 (low CO complex) in analogy to the normal paramagnetic states of 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.

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Abstract. The electron paramagnetic resonance signals of the soluble P-450 cytochrome from Pseudomonas putida were observed at temperatures from 4.2 to 80'K. As isolated, P450 has a signal typical of a low spin ferric-heme compound with sulfur as one of the axial ligands (g = 2.45, 2.26, 1.915). We also detected a minor signal typical of high spin ferric heme (g = 8, 4, 1.8) equivalent to less than 7% of the heme at temperatures below 20'K. On titration with the substrate, (+)-camphor, the low spin signal decreased and the high spin signal increased, maximally representing about 60% of the heme. For reasons not thus far understood, 40% of the heme is not converted to high spin by either (+) or (-)-camphor. The high spin signal has a rhombic character which is stronger than any previously observed with a heme compound (E = 0.33 cm-'; D = 3.8 cm-'; E/D = 0.087).We conclude that P-450,,.m as isolated is equal to or more than 95% in a low spin form probably having sulfur as one of the axial ligands. The binding of substrate displaces this ligand sufficiently to allow for conversion from a low to a high spin form.Cytochrome P450 (henceforth called P-450) was discovered as a CO-binding pigment in isolated mammalian liver microsomes. 12 Since then similar cytochromes have been found in adrenal mitochondria and in bacteria.A5 Since the proteins from these sources were not readily solubilized and purified, we undertook a study of the pure bacterial protein on the hypothesis that an understanding of the structure and function of this class of heme protein, including the microsomal cytochrome, could be obtained.The visible absorption bands of P-450 from all sources are shifted upon addition of numerous compounds, many of which are known substrates.6 Thus the P450 is thought to be the site of substrate binding for the hydroxylation reactions catalyzed.7-'2 In general, aromatic primary amines and aromatic Nheterocyclic compounds shift the Soret band of the microsomal protein to the red (type II), whereas various drugs, either substrates or inhibitors, shift the Soret peak to the blue (type I). These and the other optical properties of P450 complexes with substrates, etc., prompted the suggestion that the spin state of these heme proteins shift from low spin to high spin on addition of substrates.'3 14 1157