Modeling the Active Site of Cytochrome Oxidase: Synthesis and Characterization of a Cross-Linked Histidine−Phenol

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Abstract
A cross-linked histidine−phenol compound was synthesized as a chemical analogue of the active site of cytochrome c oxidase. The structure of the cross-linked compound (compound 1) was verified by IR, 1H and 13C NMR, mass spectrometry, and single-crystal X-ray analysis. Spectrophotometric titrations indicated that the pKa of the phenolic proton on compound 1 (8.34) was lower than the pKa of tyrosine (10.1) or of p-cresol (10.2). This decrease in pKa is consistent with the hypothesis that a cross-linked histidine−tyrosine may facilitate proton delivery to the binuclear site in cytochrome c oxidase. Time-resolved optical absorption spectra of compound 1 at room temperature, generated by excitation at 266 nm in the presence and absence of dioxygen, indicated a species with absorption maxima at ∼330 and ∼500 nm, which we assign to the phenoxyl radical of compound 1. The electron paramagnetic resonance (EPR) spectra of compound 1, obtained after UV photolysis, confirmed the generation of a paramagnetic species at low temperature. Because the cross-linked compound lacks β-methylene protons, the EPR line shape was dramatically altered when compared to that of the tyrosyl radical. However, simulation of the EPR line shape and measurement of the isotropic g value was consistent with a small coupling to the imidazole nitrogen and with little spin density perturbation in the phenoxyl ring. The ground-state Fourier transform infrared (FT-IR) spectrum of compound 1 showed that addition of the imidazole ring perturbs the frequency of the tyrosine ring stretching vibrations. The difference FT-IR spectrum, associated with the oxidation of the cross-linked compound, detected significant perturbations of the phenoxyl radical vibrational bands. We postulate that phenol oxidation produces a small delocalization of spin density onto the imidazole nitrogen of compound 1, which may explain its unique optical spectral properties.

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