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

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, (1)H and (13)C NMR, mass spectrometry, and single-crystal X-ray analysis. Spectrophotometric titrations indicated that the pK(a) of the phenolic proton on compound 1 (8.34) was lower than the pK(a) of tyrosine (10.1) or of p-cresol (10.2). This decrease in pK(a) 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 approximately 330 and approximately 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 beta-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.

The Met-Tyr-Trp Cross-link in Mycobacterium tuberculosis Catalase-peroxidase (KatG)

“…A peptide fragment in the former but not latter digest was identified that exhibited an unusual UV-visible spectrum for a polypeptide, with large bathochromic shifts of the major absorption bands. This alone suggested the presence of the Met-Tyr-Trp cross-link, as shifts for comparable aromatic-ring substitutions are predicted from theory (58) and observed in similar small molecule analogs, most notably for the cross-linked histidine-phenol mimic of cytochrome c oxidase (71), which exhibits a red-shift of ϳ20 nm of the phenol B (tyrosine) band (from 280 to ϳ300 nm).…”

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

“…Tyr I-288 is at the end of the K channel, hydrogen-bonded to the water on heme a 3 ( Figure 2). Oxygen reduction chemistry is likely to involve a coupled electron (92)(93)(94) and proton (75,95,96) transfer from this Tyr to O 2 . The Tyr could also serve as a proton acceptor when the BNC is reduced, if it is deprotonated in the fully oxidized state.…”

Identification of a histidine-tyrosine cross-link in the active site of the cbb 3 -type cytochrome c oxidase from Rhodobacter sphaeroides

“…Several studies of histidine-tyrosine model compounds have been reported (25)(26)(27)(28)(29)(30)(31)(32) supporting stabilization of a neutral tyrosine radical. However, the ligated copper, not included in these models, also is likely to affect the properties of the histidine-tyrosine dimer, which is the actual ligand of Cu B (33), and might increase its acidity and stabilize the neutral radical beyond that shown for the model compounds.…”