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New resonance Raman (RR) spectra at 15 K are reported for poplar (Populus nigra) and oleander (Oleander nerium) plastocyanins and for Alcaligenes faecalis pseudoazurin. The spectra are compared with those of other blue copper proteins (cupredoxins). In all cases, nine or more vibrational modes between 330 and 460 cm-1 can be assigned to a coupling of the Cu-S(Cys) stretch with Cys ligand deformations. The fact that these vibrations occur at a relatively constant set of frequencies is testimony to the highly conserved ground-state structure of the Cu-Cys moiety. Shifts of the vibrational modes by 1-3 cm-1 upon deuterium exchange can be correlated with N-H...S hydrogen bonds from the protein backbone to the sulfur of the Cys ligand. There is marked variability in the intensities of these Cys-related vibrations, such that each class of cupredoxin has its own pattern of RR intensities. For example, plastocyanins from poplar, oleander, French bean, and spinach have their most intense feature at approximately 425 cm-1; azurins show greatest intensity at approximately 410 cm-1, stellacyanin and ascorbate oxidase at approximately 385 cm-1, and nitrite reductase at approximately 360 cm-1. These variable intensity patterns are related to differences in the electronic excited-state structures. We propose that they have a basis in the protein environment of the copper-cysteinate chromophore. A further insight into the vibrational spectra is provided by the structures of the six cupredoxins for which crystallographic refinements at high resolution are available (plastocyanins from P. nigra, O. nerium, and Enteromorpha prolifera, pseudoazurin from A. faecalis, azurin from Alcaligenes denitrificans, and cucumber basic blue protein). The average of the Cu-S(Cys) bond lengths is 2.12 +/- 0.05 A. Since the observed range of bond lengths falls within the precision of the determinations, this variation is considered insignificant. The Cys ligand dihedral angles are also highly conserved. Cu-S gamma-C beta-C alpha is always near -170 degrees and S gamma-C beta-C alpha-N near 170 degrees. As a result, the Cu-S gamma bond is coplanar with the Cys side-chain atoms and part of the polypeptide backbone. The coplanarity accounts for the extensive coupling of Cu-S stretching and Cys deformation modes as seen in the RR spectrum. The conservation of this copper-cysteinate conformation in cupredoxins may indicate a favored pathway for electron transfer.

The copper center of the Pseudomonas aeruginosa His117Gly azurin mutant is accessible to exogenous ligands through an aperture in its surface created by the removal of the endogenous imidazole ligand. Depending on the exogenous ligand, a surprising variety of type 1 and type 2 copper sites can be obtained that are readily distinguished by electronic, EPR, and resonance Raman (RR) spectroscopy. The RR spectrum of type 1 H117G with exogenous imidazole is nearly identical to that of wild-type azurin, indicating that the trigonal geometry and short Cu-S(Cys) bond of approximately 2.15 A have been maintained. With anionic ligands (e.g., Cl-, Br-, N3-), the RR spectra show increased intensity at 370 and 400 cm-1 and a corresponding decrease in intensity at 410 cm-1, suggesting a lengthening of the Cu-S(Cys) bond as the site achieves a more tetrahedral character. An extreme example is the hydroxide adduct of H117G which is green in color and has optical and RR spectra reminiscent of the tetrahedral type 1 site in Achromobacter cycloclastes nitrite reductase. The fact that the basic RR pattern is little changed in most of the type 1 adducts indicates that the RR spectrum is due primarily to vibrations of the Cu-cysteinate moiety and that its coplanar conformation is conserved. Type 2 H117G proteins are formed by the addition of bidentate exogenous ligands such as histidine and histamine. They have their absorption maxima blue-shifted to 400 nm and their EPR A parallel values increased to approximately 160 x 10(-4) cm-1, both of which are characteristic of tetragonal Cu sites with Cu-S(thiolate) bonds of > 2.25 A. The RR spectra of the type 2 H117G proteins are still dominated by multiple cysteinate-related vibrational modes. However, the vibrational modes with the greatest intensity and Cu-S(Cys) stretching character have shifted approximately 100 cm-1 to lower energy compared to the type 1 sites, consistent with a longer (Cys)S-Cu bond. It is proposed that the tetragonal type 2 character of the bidentate ligand complexes is due to the addition of a fourth strong ligand in the equatorial ligand plane.

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In the redox center of azurin, the Cu(II) is strongly coordinated to one thiolate S from Cys 112 and two imidazole Ns from His 46 and 117. This site yields a complex resonance Raman (RR) spectrum with `20 vibrational modes between 200 and 1500 cm -1 . We have investigated the effects of ligand-selective isotope replacements on the RR spectrum of Pseudomonas aeruginosa azurin to determine the relative spectral contribution from each of the copper ligands. Growth on 34 S-sulfate labels the cysteine ligand and allows the identification of a cluster of bands with Cu-S(Cys) stretching character between 370 and 430 cm -1 whose frequencies are consistent with the trigonal or distorted tetrahedral coordination in type 1 sites. In type 2 copper-cysteinate sites, the lower n (Cu-S) frequencies between 260 and 320 cm -1 are consistent with square-planar coordination. Addition of exogenous 15 N-labeled imidazole or histidine to the His117Gly mutant generates type 1 or type 2 sites, respectively. Because neither the above nor the His46Gly mutant reconstituted with 15 N-imidazole exhibits significant isotope dependence, the histidine ligands can be ruled out as important contributors to the RR spectrum. Instead, a variety of evidence, including extensive isotope shifts upon global substitution with 15 N, suggests that the multiple RR modes of azurin are due principally to vibrations of the cysteine ligand. These are resonance-enhanced through kinematic coupling with the Cu-S stretch in the ground state or through an excited-state A-term mechanism involving a Cu-cysteinate chromophore that extends into the peptide backbone.

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Cyanide has been investigated as a potential ligand-directed probe of the coordination chemistry of Cu(II) and Cu(I) active sites via vibrational spectroscopic studies of CN" coordinated to the metal centers in non-blue copper proteins. Native superoxide dismutase (SOD) was found to bind one CN" that had IR and Raman frequencies at 2137 cm"1 2.The assignment of this band was confirmed by using 13CN", which gave the theoretically expected isotope shift, and by comparison with an inorganic model complex, [Cu(PMAS)]2+, which was found to form a monocyano complex with vcn = 2125 cm"1. Ultrafiltration experiments definitively identified the 2137-cnr1 band as vCn of a prote inbound species. These results are consistent with a linear end-on-bonded CN" on Cu(II)-SOD as proposed from earlier EPR and EXAFS experiments. Replacing H20 in the medium by D20 gave no isotope shift of the 2137-cm"1 band, indicating that CN" is most likely not involved in hydrogen-bonding interactions in the active-site cavity. With increasing concentrations of cyanide, the 2137-cm"1 band became weaker and was accompanied by the appearance of strong vibrational modes characteristic of di-, tri-, and tetracyano Cu(I) complexes arising from Cu removal from the protein. These studies demonstrate the potential importance of cyanides (and isocyanides) as ligand-directed vibrational spectroscopic probes of non-blue copper proteins.

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