Spin-state changes in cytochrome P-450cam on binding of specific substrates.

The electron paramagnetic resonance signals of the soluble P-450 cytochrome from Pseudomonas putida were observed at temperatures from 4.2 to 80 degrees K. As isolated, P-450 has a signal typical of a low spin ferric-heme compound with sulfur as one of the axial ligands (g = 2.45, 2.26, 1.91(5)). 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 degrees 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(-1); D = 3.8 cm(-1); E/D = 0.087). We conclude that P-450(cam) 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.