Distinct Structures and Environments for the Three Hemes of the Cytochrome bc₁Complex fromRhodospirillum rubrum. A Resonance Raman Study Using B-Band Excitations

Abstract

The B-band excited resonance Raman (RR) spectra (100-1700 cm-1) of the bacterial cytochrome bc1 complex purified from Rhodospirillum rubrum are reported. Four redox states, i.e., the persulfate-oxidized, "as prepared", and ascorbate- and dithionite-reduced states of the complex, were investigated with the laser excitations at 406.7, 413.1, and 441.6 nm. Following the different absorption properties of the b- and c-type hemes and the different resonance enhancements of the vibrational modes of oxidized and reduced hemes, RR contributions from the b- and c-type hemes were characterized. For the nu2, nu10, and nu8 porphyrin vibrational modes, individual contributions of hemes c1, bH, and bL were determined. The data show that the macrocycle conformation of the three hemes of the cytochrome bc1 complex is different. In particular, the frequencies assigned to ferrous heme bL (1580, 1610, and 352 cm-1, respectively) reveal that its porphyrin is more strongly distorted than that of ferrous heme bH (1584, 1614, and 344 cm-1, respectively). The frequencies of the nu11 modes (1543, 1536, and 1526 cm-1 for ferrous heme c1, heme bH, and heme bL, respectively) confirm that the axial histidylimidazole ligands of heme bL have a marked anionic character. Strong differences in the peripheral interactions of the three hemes with the proteins were also detected through the frequency differences of the nu5, nu13, nu14, and nu42 modes. Considering that hemes bH and bL are inserted into a four-helice bundle, the RR data are interpreted in the frame of a strong protein constraint on heme bL.

Photo-induced dynamics of the heme centers in cytochrome bc1

“…17 The efficient photo-oxidation of the b-heme is presumably linked to the stronger physical constraints exerced by the protein backbone onto the b-heme. 40 We suggest that these constraints hinder the flexibility of the adjacent His residues, and along with the induced ionic character of these same His ligand, 40 favor the electron transfer process. The subsequent charge recombination takes place in 6.8 ps, in agreement with our estimate of the electron residing on the axial His residue.…”

“…(1) Stronger physical constraints are exerted by the protein backbone onto the b-heme, resulting in the loss of planarity of the b L heme. 40 The higher protein constraint that is exerted on the hemes certainly stiffens the His-heme-His ligation and impedes the possible dissociation of these axial ligands. Since photo-dissociation is hindered, other processes are expected to prevail.…”

Relative Orientation of the Hemes of the Cytochromebc 1 Complexes from Rhodobacter sphaeroides, Rhodospirillum rubrum, and Beef Heart Mitochondria

“…In agreement with this proposal, our recent resonance Raman study of the Cyt bc 1 complex from Rs. rubrum suggests that the b L macrocycle was much more distorted compared with the b H macrocycle (36). Examination of the crystal structure reveals that it is the opposite case in the beef heart mitochondria.…”

Spectro-Electrochemical Investigation of the bc1 Complex from the Yeast Saccharomyces cerevisiae using Surface Enhanced B-Band Resonance Raman Spectroscopy

“…(Kitagawa, Kyogoku et al 1975;Spiro 1975;Kitagawa, Ozaki et al 1978;Spiro 1978) RR spectra were obtained mostly from B-band (Soret) excitations, (Spiro 1988) whereas Q-band excited spectra are less intensive and informative at low protein concentrations. (Le Moigne, Schoepp et al 1999) Soret excitation and Q-band resonance was applied for investigations of the bacterial (Hobbs, Kriauciunas et al 1990;Le Moigne, Schoepp et al 1999) and mitochondrial bc 1 complex (Gao, Qin et al 1998), respectively. In these cases the redox state of the hemes was altered by adding soluble redox compounds such as ascorbate and sodium hydrosulphite to the protein, which was present in the detergent-solubilized form.…”

“…Spectra are analyzed on the basis of previous investigations of the bc 1 complex in different oxidation states obtained by adding reducing compounds in solution. (Hobbs, Kriauciunas et al 1990;Le Moigne, Schoepp et al 1999) These data are used to find out whether or not electrochemical reduction/oxidation of the bc 1 complex can be accomplished under the experimental conditions described above.…”