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Resonance Raman (RR) and Fourier transform infrared (FTIR) spectra at 12K have been obtained for Pseudomonas aeruginosa azurin, spinach plastocyanin, stellacyanin, and tree laccase. The temperature dependence of the azurin, plastocyanin, and stellacyanin spectra have been recorded as have the RR excitation profiles at 12 K. Room temperature RR spectra have been obtained for azurins from Alcaligenes fecalis, Alcaligenes sp., Bortadella pretussis and Bortadella bronchiseptica; bean plastocyanin; fungal laccase, human ceruloplasmin; and zucchini squash ascorbate squash ascorbate oxidase. Isotope studies employing 63 Cu/ 65 Cu and H/D substitution have been performed on the azurins from Ps. aeruginosa, Alc. fecalis , and Alc. sp . Principal conclusion include the following; The intense RR modes near 400 cm −1 include internal ligand deformations and the Cu-S(cys) stretch, rather than the Cu-S(cys) stretch and Cu-N(his-Im) stretches as previously supposed. The Cu-N(his-Im) stretches are assignable to the ubiquitous feature near 265 cm −1 ,consistent with the frequencies of similar motions in other proteins and in model complexes. Spinach plastocyanin exhibits a frequency shift of 14 cm −1 in its cysteine CS stretching frequency ( ca. 750 cm −1 ) upon freezing of the protein solution, suggesting that extra-protein forces (e.g.,solvent structure, crystallization, or substrate binding.) can influence the conformation of the active site. Above the freezing point of the solvent the plastocyanin RR modes are unusually broad, suggesting either extremely facile due to thermally accessible conformations in the high-temperature form of the active site. No selective enhancement of either the strong or weak RR modes is observed in the S(cys)→ Cu charge transfer excitation profiles of azurin, plastocyanin, or stellacyanin at 12 K. The azurin species other than Ps. aeruginosa exhibit an ‘extra» strong RR peak near 400 cm −1 which is, however, seen to be related to an unresolved shoulder in the 12 K Ps. aeruginosa spectrum. It is therefore unnecessary to invoke higher coordination numbers than four for copper to explain the RR spectra of the azurins other than Ps. aeruginosa . RR peaks appear in the azurin spectrum below 200 K which may be due to methionine C-S stretching modes and Cu-S(met) stretch or methionine C-S-C angle bend. If these low-temperature features are indeed due to such motions,methionine must be closely coordinated to copper in azurin at low temperature. The RR spectra are consistent with a monotonic relationship between the force constant of the Cu-S(cys)bond and the energy of the ligand-field transitions of the various proteins. The isotope studies lead to tentative identification of the RR modes which contain significant contributions from M-L stretches and internal histidine motions. The results provide an initial basis for reliable structural interpretation of the RR spectra of the blue copper proteins.

The authors report the results of a pressure-dependent study of the electronic absorption and Raman spectroscopy of Re/sub 2/Cl/sub 8//sup 2 -/ in CH/sub 2/Cl/sub 2/ solution. The behavior observed is in marked contrast to that reported for the Br/sup -/ and I/sup -/ complexes. Their results are best interpreted as arising from a gradual change in the torsional coordinate, accompanied by decreasing Re-Re distance, with increasing pressure due to pressure dependence of the shape of the ground-state potential surface. The present studies were carried out on approx. 5 mM solutions of ((C/sub 4/H/sub 9/)/sub 4/N)/sub 2/Re/sub 2/Cl/sub 8/ in CH/sub 2/Cl/sub 2/ utilizing diamond anvil cells to attain the static high pressures. Methylene chloride freezes at approx. 2.2 GPa (1 GPa = 10/sup 4/ atm) at room temperatures, and no discontinuities in the spectroscopic data for Re/sub 2/Cl/sub 8//sup 2 -/ were detected on passing through this phase transition. Absorption energy maxima and resonance Raman frequencies for the metal-metal stretching mode are reversible on cycling of the pressure, indicating negligible sample decomposition at high pressures. These data are quantitatively reproducible independent of sample and regardless of the direction of pressure change. However, while all samples behave qualitatively the samemore » with regard to absorption intensity, for experimental reasons related to diamond cell techniques, the intensity data are from a single sample with points obtained only for increasing pressure.« less

Cytochrome oxidase contains four redox-active centers per functional unit. These reducing equivalents are transferred to the binuclear cyt a{sub 3}{hor ellipsis}Cu{sub B} center, which binds O{sub 2} and reduces it to H{sub 2}O. Although the reaction between O{sub 2} and cytochrome oxidase occurs too quickly to be studied by conventional stopped-flow techniques, Gibson and Greenwood showed that photolysis of the cytochrome a{sub 3}{sup 2+}-CO complex of the enzyme in the presence of O{sub 2} could be used to circumvent this limitation. The authors have developed techniques that allow them to carry out low-frequency Raman detection under flow/flash conditions; here they report v(Fe{sup 2+}-O) for an early intermediate in the cytochrome oxidase/dioxygen reaction.

Localized sample heating by the exciting radiation is a constant problem in Raman spectroscopy of absorbing solutions. Static temperature control devices such as the Miller-Harney cell are ineffective when the sample has significant absorbance at the wavelength of excitation. Previous workers have circumvented this difficulty by employing either rotating or stirred sample cells. These methods allow the use of high exciting energy while maintaining sample temperature near ambient. However, these methods do not allow control of the sample temperature, without resorting to rather complicated accessories and scattering geometries. Furthermore, we find that the rotating cell technique sometimes leads to baseline instability in resonance Raman spectra of samples having appreciable background fluorescence.

Magnetochemical and resonance-Raman spectroscopic measurements have been made on oxygenated solutions of {2,6-bis[1-(2-imidazol-4-ylethylimino)ethyl]pyridine}copper(I) cation and related complexes to characterize more fully the nature of the copper–dioxygen interaction. The role, if any, of the imidazole–nitrogen proton in the reversible oxygenation process has been examined. A possible correlation between the redox potential of the CuII⇌ CuI couple and the reaction of the copper(I) centre towards O2 has been studied, and for completeness, copper(II) and zinc(II) analogues of all the copper(I) species have also been prepared and fully characterized. Although inconclusive in an absolute sense, the present studies are consistent with the oxygenation products of these copper(I) species being relatively stable Cu–O2 or Cu–O2–Cu adducts, at least in the initial stages of oxygenation.