Interruption of the internal water chain of cytochrome f impairs photosynthetic function

The structure of cytochrome f includes an internal chain of five water mole cules and six hydrogen-bonding side chains, which are conserved throughout the phylogenetic range of photosynthetic organisms from higher plants, alga e, and cyanobacteria. The in vivo electron transfer capability of Chlamydom onas reinhardtii cytochrome f was impaired in site-directed mutants of the conserved Asn and Gln residues that form hydrogen bonds with water molecule s of the internal chain [Ponamarev, M. V., and Cramer, W. A. (1998) Biochem istry 37, 17199-17208]. The 251-residue extrinsic functional domain of C. r einhardtii cytochrome f was expressed in Escherichia coli without the 35 C- terminal residues of the intact cytochrome that contain the membrane anchor . Crystal structures were determined for the wild type and three "water cha in" mutants (N168F, Q158L, and N153Q) having impaired photosynthetic and el ectron transfer function. The mutant cytochromes were produced, folded, and assembled heme at levels identical to that of the wild type in the E. coli expression system. N168F, which had a non-photosynthetic phenotype and was thus most affected by mutational substitution, also had the greatest struc tural perturbation with two water molecules (W4 and W5) displaced from the internal chain. Q158L, the photosynthetic mutant with the largest impairmen t of in vivo electron transfer, had a more weakly hound water at one positi on (W1). N153Q, a less impaired photosynthetic mutant, had an internal wate r chain with positions and hydrogen bonds identical to those of the wild ty pe. The structure data imply that the waters of the internal chain, in addi tion to the surrounding protein, have a significant role in cytochrome f fu nction.