Effect of the Interdomain Basic Region of Cytochromefon Its Redox Reactionsin Vivo

Abstract

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 be 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 greatest in the 5-fold Lys-minus mutant (Lys58Gln-Lys65Ser-Lys66Glu-Lys188Asn-Lys189G ln). The mutants showed a small increase (approximately 25%) in the t1/2, 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 t1/2 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 c6 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 < or = 25% in the Lys-minus mutants. It was concluded that (a) the net effect of electrostatic interaction between cytochrome f and its electron acceptor in 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.

Dynamic Interaction of Plastocyanin with the Cytochrome bf Complex

“…Therefore, at ionic strengths below 50 mM, the rate constant of plastocyanin with a neutral patch of residues 42-45 is more than an order of magnitude smaller than that of wildtype plastocyanin. At ionic strengths higher than 150 mM, the relative effect of these mutations is masked by screening of the charged residues consistent with previous results found with mutant cyt f (28).…”

The Interactions of Cyanobacterial Cytochromec6 and Cytochrome f, Characterized by NMR

“…Although Ana-cytc 6 has a net charge close to zero, the presence of positive residues in the vicinity of the heme group promotes favorable electrostatic docking to cytf. The role of electrostatics in this complex is therefore analogous to the complex formed between plant cytf and Pc in vitro (14,27,(37)(38)(39). As illustrated in Fig.…”

Nanodomains of Cytochromeb 6 fand Photosystem II Complexes in Spinach Grana Thylakoid Membranes

“…However, mutagenesis of cytochrome f in C. reinhardtii found that electrostatic interactions exerted a much weaker influence on cytochrome f oxidation kinetics in vivo (Soriano et al, 1996(Soriano et al, , 1998, and other factors undoubtedly come into play in chloroplasts such as restricted diffusional space and the higher osmotic strength found in the thylakoid lumen. An NMR study of the complex between cytochrome f and plastocyanin from the cyanobacterium Nostoc showed that the binding interface involves hydrophobic residues with charged residues at the edge (Díaz-Moreno et al, 2005).…”