F. Baymann et al., Functional characterization of Chlamydomonas mutants defective in cytochrome f maturation, J BIOL CHEM, 274(33), 1999, pp. 22957-22967
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.