COMPARATIVE RESONANCE RAMAN-STUDY OF CYTOCHROME-C-OXIDASE FROM BEEF-HEART AND PARACOCCUS-DENITRIFICANS

Well-resolved, Soret band excited resonance Raman spectra were measure d from the fully oxidized and fully reduced cytochrome c oxidase from beef heart and Paracoccus denitrificans. The vibrational patterns in t he marker band region (1450-1700 cm-1) were analyzed, and a complete a ssignment of heme a and heme a3 vibrational modes is presented, permit ting a detailed structural comparison of the mammalian and bacterial e nzymes. Similar frequencies of the porphyrin modes for the reduced hem e a and the reduced and oxidized heme a3 are found, indicating a close relationship of the ground-state conformations in all oxidase species studied. In oxidized heme a, however, significant frequency differenc es are observed and interpreted in terms of a ruffled porphyrin struct ure in the three- and two-subunit forms of the Paracoccus enzyme compa red to the planar heme a of beef heart oxidase. The structural distort ions, which also perturb the conformation of the formyl substituent an d its electronic coupling with the porphyrin, reflect the specific hem e-protein interactions at heme a. Since in the fully reduced state hem e a appears to be largely planar in all oxidase species, the redox-lin ked conformational transition requires a more drastic rearrangement of the heme a-protein interactions in the bacterial than in the mammalia n oxidase. For both heme a and heme a3 in the reduced state and for he me a3 in the oxidized state, frequency, intensity, and bandwidth diffe rences of the formyl stretching vibration and intensity differences of some porphyrin modes are noted between the three oxidase forms. The s ame modes are also affected by quaternary structure changes in the bov ine oxidase caused by different detergents and isolation procedures. T hese effects are attributed to differences of the dielectric propertie s of the heme environment, due to subtle structural changes in the hem e pockets, induced by protein-protein interactions of subunit III with subunits I and/or II.