Functional characterization of Chlamydomonas mutants defective in cytochrome f maturation

Citation
F. Baymann et al., Functional characterization of Chlamydomonas mutants defective in cytochrome f maturation, J BIOL CHEM, 274(33), 1999, pp. 22957-22967
Citations number
48
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
274
Issue
33
Year of publication
1999
Pages
22957 - 22967
Database
ISI
SICI code
0021-9258(19990813)274:33<22957:FCOCMD>2.0.ZU;2-U
Abstract
We have altered the N terminus of cytochrome f by site-directed mutagenesis of the chloroplast petA gene in Chlamydomonas reinhardtii, We have replace d the tyrosine residue, Tyr(32), located immediately downstream of the proc essing site Ala(29)-Gln(30)-Ala(31) by a proline. Tyr(32) in the N terminus of the mature protein and serves as the sixth axial ligand to the heme iro n. This mutant, F32P, accumulated different forms of holocytochrome f and a ssembled them into the cytochrome b(6)f complex. The strain was able to gro w phototrophically. Our results therefore contradict a previous report (Zho u, 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 charact erization 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 Lu-band p eak at 552 nm, Both hemes have significantly lower redox potentials than wi ld type cytochrome f, We attribute the high spin form to uncleaved pre-holo cytochrome f and the low spin form to misprocessed forms of cytochrome f th at 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 func tional cytochrome f; presumably cleaved at Ala(29), with characteristics cl ose to those of wild type cytochrome f, The latter form would account for c ytochrome b(6)f turnover and photosynthetic electron transfer that sustain phototrophic growth of F32P.