Distinct structures and environments for the three hemes of the cytochromebc(1) complex from Rhodospirillum rubrum. A resonance Raman study using B-band excitations

The B-band excited resonance Raman (RR) spectra (100-1700 cm(-1)) of the ba cterial cytochrome bc(1) complex purified from Rhodospirillum rubrum are re ported. Four redox states, i.e., the persulfate-oxidized, "as prepared", an d ascorbate- and dithionite-reduced states of the complex, were investigate d with the laser excitations at 406.7, 413.1, and 441.6 nm, Following the d ifferent absorption properties of the b- and c-type hemes and the different resonance enhancements of the vibrational modes of oxidized and reduced he mes, RR contributions from the b- and c-type hemes were characterized. For the nu(2), nu(10), and nu(8) porphyrin vibrational modes, individual contri butions of hemes c(1), b(H), and b(L) were determined. The data show that t he macrocycle conformation of the three hemes of the cytochrome bc(1) compl ex is different. In particular, the frequencies assigned to ferrous heme b( L) (1580, 1610, and 352 cm(-1), respectively) reveal that its porphyrin is more strongly distorted than that of ferrous heme b(H) (1584, 1614, and 344 cm(-1), respectively). The frequencies of the nu(11) modes (1543, 1536, an d 1526 cm(-1) for ferrous heme c(1), heme b(H), and heme b(L), respectively ) confirm that the axial histidylimidazole ligands of heme b(L) have a mark ed anionic character. Strong differences in the peripheral interactions of the three hemes with the proteins were also detected through the frequency differences of the nu(5), nu(13), nu(14), and nu(42) modes. Considering tha t hemes b(H) and b(L) are inserted into a four-helice bundle, the RR data a re interpreted in the frame of a strong protein constraint on heme b(L).