The heme redox center of chloroplast cytochrome f is linked to a buried five‐water chain

Abstract

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.

Characterization and Crystallization of the Lumen Side Domain of the Chloroplast Rieske Iron-Sulfur Protein

“…If the slope of the temperature dependence of the pK was in the range of Ϫ0.02 to Ϫ0.03°C Ϫ1 , a value found in common buffers (42), the pK at 0°C, just before the frozen state is reached, would be increased from 6.5 to 7.0 -7.2. Some of the difference in pK ox may arise from protein-protein interactions in the intact complex that would shift the pK ox observed in the soluble Rieske protein in the present work, as previously discussed for the pK ox of soluble cytochrome f (5).…”

“…5 in a buffer medium of constant composition and ionic strength, as a function of pH, by titrating the amplitude of the short wavelength peak minus trough difference (394 and 408 nm, respectively) in the reduced minus oxidized difference spectrum. The possible spectral interference caused by the presence of ferricyanide (absorbance maximum, 420 nm, ⑀ mM ϭ 1) was eliminated by using a reference cuvette that contained the same concentration of all reagents except the protein and to which equal additions of reductant (sodium ascorbate) or oxidant were made.…”

“…The first crystal structure for an active domain of the cyt b 6 f or bc 1 complexes was the 252-residue soluble lumen side fragment of the 285 residue cyt f, whose structure has been determined to a resolution of 1.96 Å (4,5). The cytochrome was found to consist of two domains made mostly of ␤ sheet in an elongate (approximately 75 ϫ 35 ϫ 25 Å) geometry.…”

“…The elongate structure of cytochrome f raises questions that are defined in a structural context about the pathway of electron transfer from the membrane-bound hydrogen donor, plastoquinol, to the Rieske protein, and from the Rieske protein to cytochrome f, whose heme appears to be far from the membrane surface. In addition, the existence of a buried ordered linear H 2 O chain, a seeming proton wire with virtually all H-bond donors and acceptors conserved in evolution, which extends 11 Å from the N␦1 nitrogen of His 25 toward Lys 66 of the prominent basic surface region of cytochrome f, suggests that cytochrome f may be the exit port for protons translocated by the b 6 f complex (5). Structures of the other protein subunits of the complex are needed to resolve these questions about the pathways of electron transfer and proton translocation.…”

Electron Transfer Between Spinach Plastocyanin Mutants and Photosystem 1

“…surface (Martinez et al, 1994(Martinez et al, , 1996. The distance between the Tyr83 hydroxyl group and the copper ion is approximately 1.2 nm in poplar plastocyanin.…”