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

Abstract

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.

Direct observation of protonation reactions during the catalytic cycle of cytochrome c oxidase

“…However, three positive bands in the P m -O difference spectrum indicate the presence of a tyrosine radical in P m . The 1,587-cm Ϫ1 band concurs with the CAC stretching vibration observed at 1,587 cm Ϫ1 in the deprotonated neutral radical form of orthoimidazole-bound para-cresol, a model compound for the crosslinked histidine-tyrosine (33). Either of the two positive bands at 1,528 cm Ϫ1 and 1,517 cm Ϫ1 in the P m -O difference spectrum can be assigned to the C-C stretch of the radical form of cross-linked histidine-phenol observed at 1,522 cm Ϫ1 (34).…”

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.…”

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

“…Nyquist and coworkers attributed bands at 1528 and 1517 cm −1 to the C-C ring stretching frequencies of the tyrosine-histidine radical in R. sphaeroides cytochrome c oxidase 16. However, previous Raman spectroscopic studies of ortho -imidazole-bound para -cresol-based model compounds, with and without copper, have attributed bands around 1530 – 1533 cm −1 to the CO stretching vibration 35, 38, 47. The good agreement between the vibrational assignments presented here for the ligand and the Cu-complex and those observed in the spectra of the P M intermediate of cytochrome c oxidase from different species provides further support for the presence of a tyrosyl radical in the P M intermediate of cytochrome oxidase.…”