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The effects of protein ~ protein interactions and substrate binding on the structure of the active site of rabbit liver microsomal cytochrome P-450 LM2 have been analyzed by resonance Kaman spectroscopy of the monomeric and oligomeric protein in solution. Also Hz02-dependent catalytic activities of the two states have been compared.The two vinyl substituents of the heme exhibit different orientations, as indicated by the frequencies and intensities of their stretching vibrations. One group lies in the plane of the heme and remains unchanged in the two states of cytochrome P-450 LM2, the other is tilted out of the plane. The tilting angle in oligomers was smaller than in monomers. These vinyl stretching modes together with some porphyrin modes, were found to be sensitive indicators of the quaternary structure and of substrate binding. In both the oligomer and the monomer, substrate binding causes changes of the relative intensities of some porphyrin modes and the vinyl stretching vibrations which may reflect modifications of the electronic transitions due to hydrophobic interactions between the bound substrate and the heme. In contrast to the monomeric cytochrome P-450 LM2, benzphetamine binding to the oligomcrs of this isozyme additionally produces a shift of the spin-state equilibrium. This indicates that in the oligomer the substrate-binding pocket is converted by protein -protein interaction to a structure that forces substrates to interfere with the sixth ligands, inducing an increase of the five-coordinated high-spin configuration. In the monomcr the substrate-binding pocket can accommodate benzphetamine without affecting the spin state. Binding of imidazole to the monomeric and oligomeric cytochrome P-450 LM2 produces essentially the same resonance Kaman spectra. Apparently the replacement of the native sixth ligand by imidazole disturbs the structure of the active site in such a way that it becomes insensitive to protein-protein interactions.Hz02-dependent IV-demethylation of bcnzphetamine and aniline p-hydroxylation by cytochrome P-450 LM2 did not depend on its state of aggregation.In mammalian liver miocrosomes a large variety of xenobiotics are oxygenated by an enzyme complex whose central protein is cytochrome P-450 [I]. This heme protein runs through a cyclic process including substrate binding. electron transfer. and oxygen activation. So far no details are known about the molecular mechanism of individual reaction steps. Recently, howcver, good progress has been made in the elucidation of the structure/function relationship of bacterial cytochrome P-450 ( P-450,,,n) whose crystal structure has been determined to 0.16-nm resohition [2]. Thus important information could be obtained about the conformation of the active site and the substrate-binding pocket and the modes of interaction of the substrate with the heme. Based on sequence alignments of eukaryotic cytochrome P-450 and P-450,,,, it was concluded that essential structural features should be largely conserved [2 -41. A fundamental difference o...