SPIN-STATE CHANGES IN CYTOCHROME P-450camON BINDING OF SPECIFIC SUBSTRATES

The electron paramagnetic resonance signals of the soluble P-450 cytochrome from Pseudomonas putidawere observed at temperatures from 4.2 to 80°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, 1.915). 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-1; D= 3.8 cm-1; E/D= 0.087).We conclude that P-450camas 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.