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J Biol Chem, Vol. 274, Issue 33, 22957-22967, August 13, 1999

Functional Characterization of Chlamydomonas Mutants Defective in Cytochrome f Maturation

Franke Baymann, Francesca Zito, Richard Kuras, Limor Minai^¶, Wolfgang Nitschke, and Francis-André Wollman

From the  Institut de Biologic Physico-Chimique, 13 rue Pierre et Marie Curie, Paris 75005, France, the ^¶ Hebrew University of Jerusalem, Jerusalem 91904, Israel, and the  BIP/CNRS, UPR 9036, Institut de Biologic Structurale at Microbiologique, Marseille 13402, France

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 heme 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, 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 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 -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.

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