UV Resonance Raman Characterization of Model Compounds of Tyr²⁴⁴ of Bovine Cytochrome c Oxidase in Its Neutral, Deprotonated Anionic, and Deprotonated Neutral Radical Forms:  Effects of Covalent Binding between Tyrosine and Histidine

A model compound of Tyr 244 -His 240 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 p K a of phenolic OH and imidazolic N δ H 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 18 O 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 C 2 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 C 2 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.