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

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