A Spectroscopic Investigation of a Tridentate Cu-Complex Mimicking the Tyrosine-Histidine Cross-Link of Cytochrome c Oxidase

Heme-copper oxidases have a crucial role in the energy transduction mechanism, catalyzing the reduction of dioxygen to water. The reduction of dioxygen takes place at the binuclear center, which contains heme a(3) and Cu-B. The X-ray crystal structures have revealed that the C6' of tyrosine 244 (bovine heart numbering) is cross-linked to a nitrogen of histidine 240, a ligand to Cu-B. The role of the cross-linked tyrosine at the active site still remains unclear. In order to provide insight into the function of the cross-linked tyrosine, we have investigated the spectroscopic and electrochemical properties of chemical analogues of the Cu-B-His-Tyr site. The analogues, a tridentate histidine-phenol cross-linked ether ligand and the corresponding Cu-containing complex, were previously synthesized in our laboratory (White, K.; et al. Chem. Commun. 2007, 3252-3254). Spectrophotometric titrations of the ligand and the Cu-complex indicate a pK(a) of the phenolic proton of 8.8 and 7.7, respectively. These results are consistent with the cross-linked tyrosine playing a proton delivery role at the cytochrome c oxidase active site. The presence of the phenoxyl radical was investigated at low temperature using electron paramagnetic resonance (EPR) and Fourier transform infrared (FT-IR) difference spectroscopy. UV photolysis of the ligand, without bound copper, generated a narrow g = 2.0047 signal, attributed to the phenoxyl radial. EPR spectra recorded before and after UV photolysis of the Cu-complex showed a g = 2 signal characteristic of oxidized copper, suggesting that the copper is not spin-coupled to the phenoxyl radical. An EPR signal from the phenoxyl radical was not observed in the Cu-complex, either due to spin relaxation of the two unpaired electrons or to masking of the narrow phenoxyl radical signal by the strong copper contribution. Stable isotope (C-13) labeling of the phenol ring (C1') Cu-complex, combined with photoinduced difference FT-IR spectroscopy, revealed bands at 1485 and 1483 cm(-1) in the C-12-minus C-13-isotope-edited spectra of the ligand and Cu-complex, respectively. These bands are attributed to the radical v(7a) stretching frequency and are shifted to 1468 and 1472 cm(-1), respectively, with (13)C1' labeling. These results show that a radical is generated in both the ligand and the Cu-complex and support the unambiguous assignment of a vibrational band to the phenoxyl radical V-7a stretching mode. These data are discussed with respect to a possible role of the cross-linked tyrosine radical in cytochrome c oxidase.