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

Abstract

The isolated cytochrome (cyt) b(6)f complex from spinach is inhibited by Cu(2+) with a K(D) of about 1 microM at pH 7.6 in the presence of 1.6 microM decyl-plastoquinol (C(10)-PQH(2)) as a substrate. Inhibition was competitive with respect to C(10)-PQH(2) 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 Cu(2+) binding sites with a K(D) of about 0.5 microM and multiple (>8) weak (K(D) > 50 microM) binding sites per complex. Pulsed electron paramagnetic resonance (EPR) techniques were used to identify probable binding sites for inhibitory Cu(2+). A distinct enhancement of the relaxation time constant for the EPR signal from bound Cu(2+) was observed when the cyt f was paramagnetic. The magnitude and temperature-dependence of this relaxation enhancement were consistent with a dipole interaction between Cu(2+) and the cyt f (Fe(3+)) heme at a distance of between 30 and 54 A, depending upon the relative orientations of Cu(2+) 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 Cu(2+) bound to isolated cyt b(6)f complex indicated the presence of a weakly coupled nitrogen nucleus. The nuclear quadrupole interaction (NQI) and the hyperfine interaction (HFI) parameters identified one Cu(2+) 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 b(6)f complex was constructed on the basis of sequences and structural similarities with the mitochondrial cyt bc(1) complex, for which X-ray structures have been solved. This model indicated three possible His residues as ligands to inhibitory Cu(2+). 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 (Q(o)) binding pocket. A model is thus proposed wherein Cu(2+) interferes with the interaction of the ISP protein with the Q(o) site, preventing the binding and subsequent oxidation of plastoquinonol. Implications for the involvement of ISP "domain movement" in Q(o) site catalysis are discussed.

Magnetic Interactions Sense Changes in Distance between Heme bL and the Iron−Sulfur Cluster in Cytochrome bc1

“…Typically, those interactions are examined using EPR spectroscopy with which distances between paramagnetic centers up to about 7 nm can be determined (28−38). Examples of the pairs of the redox centers for which distances were measured include the heme b −3Fe4S cluster of complex II (28), tyrosine radical−nonheme Fe(II) of PSII (31), tyrosine radical−Mn cluster of PSII (39), Cu A −heme a of cytochrome oxidase (30), Cu 2+ −heme of cytochrome f in cytochrome b 6 f (37), and cytochrome c /cytochrome oxidase (in this case estimation of distance distribution in the interprotein complex was reported (38)). In the case of cytochrome bc 1 , early EPR examination of mitochondrial enzyme allowed to approximate a spatial organization of its redox active centers revealing existence of weak spin−spin interactions between the FeS center, heme b 566 ( b L ), and heme c 1 (40).…”

A Caged, Destabilized, Free Radical Intermediate in the Q‐Cycle

“…In particular, the electron transverse relaxation, T M , of SQ in the bacterial protein is strongly enhanced by the heme cofactors. [10a] This paramagnetic relaxation enhancement (PRE) was examined in detail in the mitochondrial cyt bc 1 because PRE is useful for measuring distances between spins in proteins [18] including the Fe 2 S 2 -heme b L distance in cyt bc 1 by Sarewicz et al, [19] copper ion bound to cyt b 6 f , [20] cyt c bound to cyt c oxidase, [21] and spin-labeled metmyoglobin. [22] …”

The Energy Budget of Steady-State Photosynthesis

“…These experiments clearly demonstrated that the cytochrome b 6 f ISP from this complex undergoes large conformational shifts in response to changes in occupancy of the Q o site, providing the first strong support for such conformational changes in cytochrome b 6 f catalysis. The second advance has come from our demonstration that Cu 2+ inhibits the cytochrome b 6 f complex, probably by interfering with ISP domain movements [7,8]. We have since shown that, indeed, metal ions interfere with ISP domain movements [9].…”