The first atomic structure of a subunit of either the cytochrome b6f complex or of the related cytochrome bc1 complex has been obtained. The structure of cytochrome f allows prediction of the approximate docking site of plastocyanin and should allow systematic studies of the mechanism of intra- and inter-protein electron transfer between the cytochrome heme and plastocyanin copper, which are approximately isopotential. The unprecedented axial heme iron ligand also provides information on the sequence of events (i.e. cleavage of signal peptide and ligation of heme) associated with translocation of the cytochrome across the membrane and its subsequent folding.

The crystal structure of the 252-residue lumen-side domain of reduced cytochrome f, a subunit of the protonpumping integral cytochrome b6 f complex of oxygenic photosynthetic membranes, was determined to a resolution of 1.96 A from crystals cooled to -35". The model was refined to an R-factor of 15.8% with a 0.013-A RMS deviation of bond lengths from ideality. Compared to the structure of cytochrome f at 20°, the structure at -35" has a small change in relative orientation of the two folding domains and significantly lower isotropic temperature factors for protein atoms. The structure revealed a n L-shaped array of five buried water molecules that extend in two directions from the N61 of the heme ligand His 25. The longer branch extends 11 A within the large domain, toward Lys 66 in the prominent basic patch at the top of the large domain, which has been implicated in the interaction with the electron acceptor, plastocyanin. The water sites are highly occupied, and their temperature factors are comparable to those of protein atoms. Virtually all residues that form hydrogen bonds with the water chain are invariant among 13 known cytochrome f sequences. The water chain has many features that optimize it as a proton wire, including insulation from the protein medium. It is suggested that this chain may function as the lumen-side exit port for proton translocation by the cytochrome b6 f complex.

The cytochrome b6f complex functions in oxygenic photosynthetic membranes as the redox link between the photosynthetic reaction center complexes II and I and also functions in proton translocation. It is an ideal integral membrane protein complex in which to study structure and function because of the existence of a large amount of primary sequence data, purified complex, the emergence of structures, and the ability of flash kinetic spectroscopy to assay function in a readily accessible ms-100 mus time domain. The redox active polypeptides are cytochromes f and b6 (organelle encoded) and the Rieske iron-sulfur protein (nuclear encoded) in a mol wt = 210,000 dimeric complex that is believed to contain 22-24 transmembrane helices. The high resolution structure of the lumen-side domain of cytochrome f shows it to be an elongate (75 A long) mostly beta-strand, two-domain protein, with the N-terminal alpha-amino group as orthogonal heme ligand and an internal linear 11-A bound water chain. An unusual electron transfer event, the oxidant-induced reduction of a significant fraction of the p (lumen)-side cytochrome b heme by plastosemiquinone indicates that the electron transfer pathway in the b6f complex can be described by a version of the Q-cycle mechanism, originally proposed to describe similar processes in the mitochondrial and bacterial bc1 complexes.

Size analysis of the cytochrome bdcomplex by FPLC Superose-12 chromatography and Blue Native PAGE indicated a predominantly dimeric component with M, = (1.9-2.5) X lo5. The true dimer molecular weight including bound lipid, but not detergent, was estimated to be 2.3 X lo5. Size and shape analysis by negative-stain single-particle electron microscopy indicated that the preparation of dimeric complexes contains a major population that has a protein cross section 40% larger than the monomer, binds more negative stain, and has a geometry with a distinct 2-fold axis of symmetry compared to the monomeric complex. The dimeric species is more stable at higher ionic strength with respect to conversion to the monomeric species. SDS-PAGE of monomer and dimer preparations indicated that both contain the four major polypeptides in approximately equal stoichiometry and also contain thepetG M, 4000 subunit. One bound chlorophyll a per monomer, part of the bound lipid, is present in monomer and dimer. The in vitro electron-transport activity (decyl-PQHz -PC-ferricyanide) of the separated dimer was comparable to that of the isolated bdcomplex and was 4-5-fold greater than that of the monomer preparation, whose activity could be attributed to residual dimer. No difference in the properties of the dimer and monomer was detected by SDS-PAGE or redox difference spectrophotometry that could account for the difference in activities. However, the concentration of the Rieske [2Fe-2S] center was found by EPR analysis of the g , = 1.90 signal to be lower in the monomer fraction by a factor of 3.5 relative to the dimer. The presence of active dimer at high levels in the detergent-extracted bdcomplex, the absence of activity in the monomer, and the absence of a monomer preparation that is not degraded in its spectral properties and activity suggest that the simplest inference is that the dimer is the active complex in the membrane. The possibility that cytochrome b d and bcl are primitive trans-membrane-signaling complexes is noted.The cytochrome bsf complex is one of three integral membrane protein complexes involved in electron transport in membranes that carry out oxygenic photosynthesis. The bsfcomplex occupies an electrochemically central position in the noncyclic electron-transport chain, accepting electrons from the photosystem I1 reaction center that is associated with 0 2 evolution, and donating them to the photosystem I reaction center that reduces ferredoxin and nicotinamide adenine dinucleotide phosphate (NADP+). The bsfcomplex bears many similarities to the cytochrome bcl complex of the

The cytochrome b 6 f complex of oxygenic photosynthesis carries out "dark reactions" of electron transfer that link the light-driven reactions of the reaction centers, and coupled proton transfer that generates part of the electrochemical potential utilized for ATP synthesis. In contrast to the bc 1 complex of the respiratory chain, with which there are many structural and functional homologies, the b 6 f complex contains bound pigment molecules. Along with the specifically bound chlorophyll a previously found to be bound stoichiometrically in the dimeric b 6 f complex, it was found in the present study that ␤-carotene is also present in the b 6 f complex at stoichiometric levels or nearly so. Chlorophyll and carotenoid pigments were quantitatively extracted from b 6 f complex purified from (i) the thermophilic cyanobacterium, Mastigocladus laminosus, (ii) spinach chloroplasts, and (iii) the green alga, Chlamydomonas reinhardtii. Visible and mass spectra showed the carotenoid to be a ␤-carotene of molecular weight ‫؍‬ 536, with a stoichiometry of 1.0:1 relative to cytochrome f in the highly active M. laminosus complex but somewhat lower stoichiometries, 0.77 and 0.55, in the b 6 f complex obtained from spinach chloroplasts and C. reinhardtii. A photoprotective function for the ␤-carotene was inferred from the findings that the rate of photobleaching of the chlorophyll a bound in the complex was found to vary inversely with ␤-carotene content and to decrease markedly in the presence of ambient N 2 instead of air. The presence of ␤-carotene in the b 6 f complex, and not in the related bc 1 complexes of the mitochondrial respiratory chain and photosynthetic bacteria, suggests that an additional function is to protect the protein complexes in oxygenic photosynthetic membranes against toxic effects of intramembrane singlet O 2 .