Functional characterization of Chlamydomonas mutants defective in cytochrome f maturation

Abstract

We have altered the N terminus of cytochrome f by site-directed mutagenesis of the chloroplast petA gene in Chlamydomonas reinhardtii. We have replaced the tyrosine residue, Tyr32, located immediately downstream of the processing site Ala29-Gln30-Ala31 by a proline. Tyr32 is the N terminus of the mature protein and serves as the sixth axial ligand to the heine iron. This mutant, F32P, accumulated different forms of holocytochrome f and assembled them into the cytochrome b6f complex. The strain was able to grow phototrophically. Our results therefore contradict a previous report (Zhou, Jo., Fernandez-Velasco, J.G., and Malkin, R. (1996) J. Biol. Chem. 271, 1-8) that a mutation, considered to be identical to the mutation described here, prevented cytochrome b6f assembly. A comparative functional characterization of F32P with F29L-31L, a site-directed processing mutant in which we had replaced the processing site by a Leu29-Gln30-Leu31 sequence (2), revealed that both mutants accumulate high spin cytochrome f, with an unusual orientation of the heme and low spin cytochrome f with an α- band peak at 552 nm. Both hemes have significantly lower redox potentials than wild type cytochrome f. We attribute the high spin form to uncleaved pre-holocytochrome f and the low spin form to misprocessed forms of cytochrome f that were cleaved at a position different from the regular Ala29-Gln-Ala31 motif. In contrast to F29L-31L, F32P displayed a small population of functional cytochrome f, presumably cleaved at Ala29, with characteristics close to those of wild type cytochrome f. The latter form would account for cytochrome b6f turnover and photosynthetic electron transfer that sustain phototrophic growth of F32P.