The cytochrome b subunit of the bc1 complexes contains two cytochrome components (bL and bH) and is the locus of both a quinol oxidizing site (Qo or Qz) and a quinone reducing site (Qi or Qc). Sequence alignments of this subunit from over twenty eukaryotic and prokaryotic species has revealed a remarkable degree of conservation, including approximately twenty totally conserved residues. In this paper, site-directed mutagenesis has been used to examine the structural or functional roles of five of these highly conserved residues, Gly48, Gln58, Ser102, Phe104, and Pro202, all predicted to be within transmembrane alpha-helical segments. The mutants were made in the bc1 complex of Rhodobacter sphaeroides, a photosynthetic bacterium. The ability to use spectroscopic, electrochemical, and flash-induced kinetic methods allows the mutants to be analyzed for influences both on cytochrome spectra and thermodynamic properties, and on the kinetics of specific electron transfer reactions.
The results show that none of the five residues is absolutely essential. Substitution of aspartate or valine for Gly48 results in the loss of photosynthetic growth. The G48V mutant assembles a bc1 complex, but with modified cytochromes bH and bL, and a disfunctional quinone reductase (QC-) site; an alanine is tolerated at this position. Possibly, a small residue is important here for heme packing. Gln58 and Ser102 are the only highly conserved polar residues predicted to be within the transmembrane spans, apart from the histidines which are heme axial ligands. Neither Gln58 nor Ser102 is essential for assembly or function of the bc1 complex, although substitution of other amino acids in these positions does cause subtle, but measurable changes. Phe104 lies midway between the axial ligands to cytochromes bL and bH and can be modeled to project in the space separating the two hemes. Replacement of this highly conserved aromatic residue by isoleucine has no measurable influence on the rate of electron transfer through the b-cytochrome chain containing the two hemes. Finally, Pro202 is a totally conserved proline which is in the middle of transmembrane helix D, in between the two histidines which provide ligands to the hemes. No major inhibition of electron transfer resulted from replacing this proline by a leucine, although subtle changes in spectra of the b-cytochromes and their electrochemical properties were noted.