The structural changes in oxidized yeast iso-1-cytochrome c and fully oxidized bovine cytochrome c oxidase that are induced upon complex formation have been analyzed by resonance Raman spectroscopy. The main spectral changes could be ascribed to cytochrome c, which in the case of the wild-type protein are essentially the same as previously observed in the complex of horse heart cytochrome c and bovine cytochrome c oxidase [Hildebrandt et al. (1990) Biochemistry 29, 1661-1668]. These spectral changes are attributed to the formation of the conformational state II (approximately 45%) which exhibits an open heme pocket structure. The structural changes are assumed to be induced by the electrostatic interactions between the negatively charged binding domain on cytochrome c oxidase and the positively charged lysine residues on the front surface of cytochrome c. Substituting one of these lysine residues (i.e., Lys-72) by an alanine significantly lowers the state II content (< 15%), implying that this lysine is essential for controlling the conformational equilibrium of the bound protein. On the other hand, the replacement of lysine-79 by alanine only slightly lowers the state II content (approximately 35%). However, the analysis of the spectra suggests that lysine-79 may be involved in controlling conformational details within the heme pocket of the bound cytochrome c. Due to the underlying structural changes and the lowered redox potential, formation of state II may be of functional importance for the physiological electron-transfer process by lowering the reorganization energy and increasing the driving force. The spectral changes caused by complex formation that are attributable to cytochrome c oxidase indicate structural changes of the vinyl and formyl substituents while the ground-state conformations of the porphyrin macrocycles are preserved. This finding implies that the conformational changes in the heme pockets of cytochrome c oxidase are much smaller than those in cytochrome c. These changes refer not only to heme a but also to heme a3, located remote from the cytochrome c binding site, pointing to a long-range structural communication between the binding domain and the oxygen reduction site. The possible functional implications of these structural changes are discussed.

S u r f a~l u d Raman (SER) spectra of the xantbine dyes rbodamine 66 (R6G) and o x h 1 (0x1) were! measured with near-infrared excitation employing Fourier transform Raman spectroscopy. Both dyes exhibit strong SER signals on Ag and Au surfaces. The enhancement factors were evaluated based on the determination of the number of adsorbed molecules. In the absence of additional electrolyte, the enhancement factors for R6G on Ag and Au sols are 13 x 10' and 1.6 x lo', respectively. This enhancement is operative for all adsorption sites and is ascribed to the classical electromagnetic mechanism. On activation of the Ag sol by chloride ions, the enhancement factors for R6G a d OX1 are 4 . 3 x lo' and 7.6 x lo5, respectively. This increrrsed enhancement which occurs at a small number of specific adsorption sites is ascribed to the charge-transfer mechanism. For R6G this enhancement mechanism is stronger by a factor of 47 than for visible excitation. The electrolyte-induced activation is not possible for Au sols or precipitates of Ag or Au on filter-papers. These metal-coated filter-papers may provide appropriate SER-active surfaces for analytical applications of the SER effect in combination with Fourier tramform Raman spectroscopy.

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The resonance Raman spectra of yeast ferri- and ferro-iso-1-cytochrome c dissolved in H2O and D2O are reported. Hydrogen exchange in the protein leads to distinct spectral changes of heme vibrational bands, particularly in the region between 670 and 710 cm-1 and at approximately 443 and approximately 450 cm-1. The latter two bands, which have previously been assigned to porphyrin modes including bending vibrations of the propionate side chains [Hildebrandt, P. (1991) J. Mol. Struct. 242, 379-395], reveal frequency shifts by up to 4 cm-1. These shifts are attributed to structural changes of the propionate groups caused by the energetic differences of the hydrogen and deuterium bonds between these substituents and the adjacent amino acid residues. The frequency shifts of the bands between 670 and 710 cm-1 most likely reflect structural differences of the tetrapyrrole macrocycle itself. Time-dependent experiments revealed that the hydrogen exchange processes associated with the changes in the resonance Raman spectra are complete in less than 15 min. The protons which are involved are those in the interior of the heme pocket as concluded by comparison with the exchange rate constants previously determined by NMR spectroscopy [Mayne, L., Paterson, Y., Cerasoli, D., & Englander, S. W. (1992) Biochemistry 31, 10678-10685]. These protons are part of a hydrogen bonding network including the amide protons of Asn-52, Met-80, and Lys-79, the side chain protons of Asn-52, Tyr-67, Thr-78, Trp-59, and Thr-49, and the water molecules 121 and 166.(ABSTRACT TRUNCATED AT 250 WORDS)

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