Biochemical and spectroscopic characterization of the covalent binding of heme to cytochrome b6.

The three-dimensional structure of the cytochrome b(6)f complex disclosed the unexpected presence of a new heme c(i) [Stroebel, D., Choquet, Y., Popot, J.-L., and Picot, D. (2003) Nature 426, 413-418; Kurisu, G., Zhang, H., Smith, J. L., and Cramer, W. A. (2003) Science 302, 1009-1014]. Here we present a biochemical, spectroscopic, and mutagenesis study of this unusual heme binding in Chlamydomonas reinhardtii. As predicted by the structure data, we identify a Cys(35)-containing proteolytic fragment (Tyr(25)-Lys(111)) from cytochrome b(6) as a peptide that covalently binds a heme. Resonance Raman spectra of cyt b(6)f complexes show particular frequencies in nu(2), nu(3), nu(4), and nu(8) regions that identify this extra heme as a ferrous c'-like heme under a five-coordinated high-spin state. The set of frequencies is consistent with a coordination by either a water molecule or a hydroxide ion. Other changes in resonance Raman bands, observed in the mid- and low-frequency regions, point to a modification in conformation and/or environment of at least one b heme methyl and/or propionate group. Site-directed mutagenesis of apocytochrome b(6), leading to a Cys(35)Val substitution, generates Chlamydomonas strains that are unable to assemble cytochrome b(6)f complexes. On the basis of the mutant phenotype, we discuss the participation, in the covalent binding of heme c(i), of the nuclear CCB factors that we identified previously as controlling the apo to holo conversion of cytochrome b(6) [Kuras, R., de Vitry, C., Choquet, Y., Girard-Bascou, J., Culler, D., Büschlen, S., Merchant, S., and Wollman, F.-A. (1997) J. Biol. Chem. 272, 32427-32435].