A model compound of Tyr244-His240 of bovine cytochrome c oxidase was synthesized and examined with UV resonance Raman (UVRR) as well as UV absorption spectroscopy and pH titration. Owing to the covalent linkage between imidazole and phenol, the pK a of phenolic OH and imidazolic NδH groups were lowered by 1.1 and 2.3, respectively. UVRR measurements of ortho-imidazole-bound para-cresol (Im⧫CrOH), its deprotonated anion (Im⧫CrO-), and deprotonated neutral radical (Im⧫CrO•), and their imidazole perdeuterated, cresol perdeuterated, and 18O derivatives allowed assignments of Raman bands to the imidazole and phenol modes. Unexpectedly, some of imidazole vibrations were resonance enhanced upon ππ* transition of phenol, although they were not observed for the corresponding equimolar mixture of imidazole and p-cresol, indicating delocalization of π electrons between the imidazole and phenol rings via the covalent linkage. Such features were appreciably changed by incorporation of a bulky group at imidazole C2 position, causing staggered conformation. The C−O stretching RR band was observed at 1530 cm-1 only for a radical state. The imidazole substituent at the C2 position seems to increase appreciably a double bond character of the CO bond in the radical state than its absence. The Y8a band is not shifted upon deprotonation, although it was downshifted by 12 cm-1 for the unmodified p-cresol. The UVRR difference spectra of the anion and radical with regard to the neutral states are discussed in relation with the corresponding difference spectra of the enzyme.

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