Effect of the interdomain basic region of cytochrome f on its redox reactions in vivo

The prominent interdomain basic surface region seen in the high-resolution structure of the active lumen-side C-terminal fragment of turnip cytochrome f, containing the conserved Lys58,65,66 (large domain) and Lys187 (small domain), has been inferred from in vitro studies to bt responsible for docking of its physiological oxidant, plastocyanin. The effect of the putative docking region of cyt f on its reactivity in vivo was tested by site-directed mutagenesis in Chlamydomonas reinhardtii. Three charge-neutralizing mutants were constructed involving: (i) the two lysines (Lys188Asn-Lys189Gln) in the small domain, (ii) the three lysines (Lys58Gln-Lys65Ser-Lys66Glu) in the large domain, and (iii) all five of these lysines spanning both domains. All mutants grew phototrophically. The mutants displayed a 20-30% increase in average generation time, and comparable decreases in rates of steady-state oxygen evolution and the slow (millisecond) electrochromic 515 nm band shift, The magnitude of the changes was greatests in the 5-fold Lys-minus mutant (Lys58Gln-Lys65Ser-Lys66Glu-Lys188Asn-Lys189Gln). The mutants showed a small increase (similar to 25%) in the t(1/2), from 0.2 to 0.25 ms, of cyt f photooxidation, far less than anticipated (ca. 100-fold) from in vitro studies of the effect of high ionic strength on the cyt f-PC interaction. The t(1/2) of cyt f dark reduction via the Rieske protein increased from 5-6 ms in the wild type to 11-12 ms in the 5-fold Lys-minus mutant. Cells grown phototrophically in the absence of Cu, where cyt c(6) is the electron acceptor of cyt f; displayed net rates of cytochrome photooxidation that were slightly faster than those in the presence of Cu, which also decreased by a factor of less than or equal to 25% in the Lys-minus mutants. II was concluded that (a) the net effect of electrostatic interaction between cytochrome Sand its electron acceptor ii? vivo is much smaller than measured in vitro and is not rate-limiting, This may be a consequence of a relatively high ionic strength environment and the small diffusional space available for collision and docking in the internal thylakoid lumen of log phase C, reinhardtii. (b) The efficiency of electron transfer to cytochrome f from the Rieske protein is slightly impaired by the neutralization of the lysine-rich domain.