IDENTIFICATION AND DESCRIPTION OF COPPER-THIOLATE VIBRATIONS IN THE DINUCLEAR CU-A SITE OF CYTOCHROME-C-OXIDASE

The Cu-A site of cytochrome c oxidase and the type 1 Cu site of cupred oxins occur in homologous protein fords with a His(2)Cys ligand set, b ut the distinguished by the presence of a second Cys and a second Cu i n Cu-A that result in the formation of a dithiolate-bridged dinuclear Cu cluster. The resonance Raman (RR) spectrum of the soluble Cu-A-cont aining fragment from Paracoccus denitrificans exhibits intense vibrati ons at 260 and 339 cm(-1). Their respective S-34-isotope shifts of -4. 1 and -5.1 cm(-1) allow them to be assigned to two Cu-S stretching mod es, nu(Cu-S), of the Cu(2)S(2)Im(2) moiety. A normal coordinate analys is (NCA) of the RR spectra of Cu-A substituted with isotopes of S, Cu, and N was carried out to determine whether it is possible to distingu ish between dinuclear models with bridging or terminal cysteine ligand s. Whereas the terminal Cys model predicts that both of the nu(Cu-S) m odes lie between 340 and 350 cm(-1). the bridging Cys geometry success fully predicts the S-shifts at 260 and 339 cm(-1). Thus, the Raman dat a and NCA are fully consistent with the bridging cysteine coordination observed by X-ray crystallography. The agreement between predicted an d observed vibrational isotope data is further improved by a trans-til ting of the imidazole nitrogens above and below the Cu2S2 plane, yield ing a distorted tetrahedral geometry fur each of the Cu atoms. Whereas type I protein RR spectra are highly N-dependent due to extended vibr onic coupling with 1 amide vibrations of the cysteine ligand, the vibr ations in CUA are relatively insensitive to N-isotope substitution. Th us, unlike type 1 Cu, the RR spectrum of CUA can be successfully model ed with only the Cu2S2-(Im)(2) core.