Ferrous-carbonyl complexes of the soluble Pseudomonas putida cytochrome P-450,,, and a denatured form (P-420) of this enzyme, as well as cytochromes P-450 and P-448 in liver microsomes from rats pretreated with phenobarbital and 3-methylcholanthrene, have been studied by infrared spectroscopy. The FeCO bonding was examined in order to probe the spatial relationship between the dioxygen-and substrate-binding sites and to determine whether the presence of a unique axial ligand bound trans to carbon monoxide could be responsible for the red-shifted Soret absorbance band maximum at 450 nm. The d-camphor(K+)-bound cytochrome P-450,,, yielded a single infrared absorbance band for the heme-bound carbonyl (uc-, 1940 cm-') having a bandwidth a t half-height ( A v l p ) of 13 ern-', while the camphor-free enzyme gave rise to two stretching frequencies of equal area a t 1963 and 1942 cm-' ( A u l / 2 11-12 and 19-21 cm-', respectively). Addition of d-camphor and a monovalent metal ion (K+) to the camphor-free ferrous carbonyl-enzyme converted the infrared spectrum back to that of the original camphor-bound enzyme. The area of the 1940-cm-I band was found to equal that of the combined areas of the 1963-and 1942-cm-l bands. Conversion of the native enzyme to a denatured form (P-420) yielded a w-, 1966 cm-' with A u l p 23 1 nfrared spectroscopy is a powerful technique for probing the oxygen binding site of heme proteins. Carbon monoxide has been the ligand of choice for such studies because the C-0 stretching frequency is much more easily observed in aqueous media than is that of the 0-0 stretching frequency (Maxwell and Caughey, 1978) and because its substitution for 0 2 is assumed to leave the structure of the protein unaltered. The infrared stretching frequency is related to the C-0 bond energy which is very sensitive to differences in both bond type and environment. The present study was initiated to directly probe, by infrared spectroscopy, the dioxygen-binding site of cyto-