Inhibitory Copper Binding Site on the Spinach Cytochromeb6fComplex: Implications for QoSite Catalysis

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Abstract
The isolated cytochrome (cyt) b6f complex from spinach is inhibited by Cu2+ with a KD of about 1 μM at pH 7.6 in the presence of 1.6 μM decyl−plastoquinol (C10−PQH2) as a substrate. Inhibition was competitive with respect to C10−PQH2 but noncompetitive with respect to horse heart cyt c or plastocyanin (PC). Inhibition was also pH-sensitive, with an apparent pK at about 7, above which inhibition was stronger, suggesting that binding occurred at or near a protonatable amino acid residue. Equilibrium binding titrations revealed ca. 1.4 tight Cu2+ binding sites with a KD of about 0.5 μM and multiple (>8) weak (KD > 50 μM) binding sites per complex. Pulsed electron paramagnetic resonance (EPR) techniques were used to identify probable binding sites for inhibitory Cu2+. A distinct enhancement of the relaxation time constant for the EPR signal from bound Cu2+ was observed when the cyt f was paramagnetic. The magnitude and temperature-dependence of this relaxation enhancement were consistent with a dipole interaction between Cu2+ and the cyt f (Fe3+) heme at a distance of between 30 and 54 Å, depending upon the relative orientations of Cu2+ and cyt f heme g-tensors. Two-pulse electron spin−echo envelope modulation (ESEEM) and 4-pulse 2-dimensional hyperfine sublevel correlation (2D HYSCORE) measurements of Cu2+ bound to isolated cyt b6f complex indicated the presence of a weakly coupled nitrogen nucleus. The nuclear quadrupole interaction (NQI) and the hyperfine interaction (HFI) parameters identified one Cu2+ ligand as an imidazole nitrogen of a His residue, and electron−nuclear double resonance (ENDOR) confirmed the presence of a directly coordinated nitrogen. A model of the 3-dimensional structure of the cytochrome b6f complex was constructed on the basis of sequences and structural similarities with the mitochondrial cyt bc1 complex, for which X-ray structures have been solved. This model indicated three possible His residues as ligands to inhibitory Cu2+. Two of these are located on the “Rieske” iron−sulfur protein protein (ISP) while the third is found on the cyt f protein. None of these potential ligands appear to interact directly with the quinol oxidase (Qo) binding pocket. A model is thus proposed wherein Cu2+ interferes with the interaction of the ISP protein with the Qo site, preventing the binding and subsequent oxidation of plastoquinonol. Implications for the involvement of ISP “domain movement” in Qo site catalysis are discussed.