Functional characterization of Chlamydomonas mutants defective in cytochrome f maturation.
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, Tyr(32), located immediately downstream of the processing site Ala(29)-Gln(30)-Ala(31) by a proline. Tyr(32) is the N terminus of the mature protein and serves as the sixth axial ligand to the heme iron. This mutant, F32P, accumulated different forms of holocytochrome f and assembled them into the cytochrome b(6)f complex. The strain was able to grow phototrophically. Our results therefore contradict a previous report (Zhou, J., 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 b(6)f 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 Leu(29)-Gln(30)-Leu(31) 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 alpha-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 Ala(29)-Gln-Ala(31) motif. In contrast to F29L-31L, F32P displayed a small population of functional cytochrome f, presumably cleaved at Ala(29), with characteristics close to those of wild type cytochrome f. The latter form would account for cytochrome b(6)f turnover and photosynthetic electron transfer that sustain phototrophic growth of F32P.
Baymann
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F
Institut de Biologic Physico-Chimique, 13 rue Pierre et Marie Curie, Paris 75005, France.
Zito
F
F
Kuras
R
R
Minai
L
L
Nitschke
W
W
Wollman
F A
FA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D003580:Cytochromes / Q000737:chemistry / Q000378:metabolism*; D045348:Cytochromes f; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004792:Enzyme Precursors / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D013050:Spectrometry, Fluorescence
D003580:Cytochromes; D017931:DNA Primers; D004792:Enzyme Precursors; D045348:Cytochromes f