Perturbation of the internal water chain in cytochrome f of oxygenic photosynthesis: Loss of the concerted reduction of cytochromes f and b(6)

The 1.96 Angstrom structure of turnip cytochrome f revealed a linear intern al chain of H2O molecules with the oxygen atoms of the chain having occupan cies and "B" factors comparable to those of neighboring atoms [Martinez et al, (1996) Protein Sci. 5, 1081-1092.]. Four waters extend 11 Angstrom from the heme toward Lys66 on the cytochrome surface. All residues that contrib ute an atom to the 15 H-bonds of five internal H2O molecules are essentiall y conserved in 23 cytochrome sequences. With only Gin and Asn side chains i nvolved in H-bonding, the water chain resembles a "proton wire". The functi on of the conserved H2O chain was tested through site-directed mutagenesis of these Asn and Gin residues. Four of the five conserved Asn/Gln residues were changed in six mutants generated in the green alga, Chlamydomonas rein hardtii. Except for the N168F mutant, all grew photosynthetically. Although the rates of oxidation of cyt f oxidation and of reduction of cyt b6 (5-6 ms in the wild type) were not significantly affected, the rates of cyt f re duction and generation of the slow electrochromic band shift (Delta psi(s)) were markedly decreased, the half-times increasing to as much as 38 and 18 ms, respectively. Thus, in these mutants, reduction of cyt bg reduction cl early precedes that of cyt f, Retardation of Delta psi(s) in the absence of an observable change in the rate of cyt bg reduction implied that the rate of Hi translocation decreased in the mutants, and electron transfer was co ncomitantly retarded, most likely between the ISP and cyt f. The following was concluded: (i) proton and electron transfer are coupled in reduction of cyt f, and the cyt f water chain functions in H+ transfer; (ii) reduction of the high- and low-potential chains in the b(6)f complex is not concerted in the water chain mutants; and (iii) quinol deprotonation and electron tr ansfer from reduced quinone are initiated by an early event, probably the m ovement of the ISP triggered by oxidation of cyt f.