Resonance raman studies of ‘Blue’ copper proteins

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‘Blue’ copper proteins possess intense visible absorption bands at ≅600 nm due to (Cys)S − → Cu(II) charge transfer. The type 1 copper in these proteins also exhibits anomalously low hyperfine splitting in the EPR spectra and high redox potentials. Crystallographic and EXAFS investigations on two simple blue copper proteins, plastocyanin and azurin, have provided fairly detailed pictures on their active site structures. The resonance Raman (RR) spectra of blue copper proteins are remarkably similar to one another; all have three or more strong bands in the 350–450 cm −1 range; all exhibit intensity enhancement by resonance with the ≅600 nm LMCT band; all spectra have two nearly constant frequency components at ≅750 and ≅250 cm −1 which appear to be vibrational modes of the coordinated cysteinate ligand; and, finally, many weaker spectral features are resolvable in high quality spectra (Fig. 1). The complexity of the RR spectra of the type 1 copper sites suggests the vibrational modes of the chromophore in addition to CuS(Cys) stretching must also contribute. Interpretation is complicated by the fact that the principal peaks in the RR spectra are at unprecedently high frequencies for copper complexes involving sulfur and nitrogen coordination. We recently undertook a normal coordinate analysis of the CuN 2S(S′) chromophore structure of Pseudomonas aeruginosa azurin. We found that the anomalously high frequencies can be explained by the shortness of the CuS (cysteinate) bond and the ▪ t001 Raman Frequencies (cm −1) for CuLADH at 77 K. Protein ν a ν b ν c ν d CuLADH 415 350 254 199 + NADH 420 359 263 202 + pyrazole 413 368 246 196 strongly coupled nature of the vibrations. Good agreement with observed azurin frequencies was obtained for a trigonal pyramidal model with Cu(II) coordinated to two histidines and one cysteinate [1]. The structure of the zinc enzyme, liver alcohol dehydrogenase (LADH), is well documented. Substitution of the catalytic zinc by Cu(II) yields a protein that has type 1 copper properties. We have investigated the RR spectrum of this system and find it to be an excellent model for the spectra of blue copper proteins [2]. A spectrum of CuLADH is shown in Fig. 1. The main spectral features are also at high frequencies and correlate well with those observed for azurin. The catalytic Zn(II) ion in LADH is coordinated by two cysteinates, one histidine, and one water molecule in a tetrahedral environment. We attribute the sharp Raman peak at 415 cm −1 in CuLADH to a predominantly Cuhistidine vibration and the broader band at ≅ 350 cm −1 to contributions from the two Cu-cysteinates. The high frequencies of the chromophore vibrations lead to the prediction that the CuS bonds distances in CuLADH are also close to 21.1 Å, as in azurin and plastocyanin. Addition of the coenzyme, NADH, or pyrazole, a competitive inhibitor of alcohol oxidation in the native enzyme, to the copper proteins causes only minor changes in the RR spectrum of the Cu(II) chromophore (Table I). Thus, despite the large shift of the absorbance maximum from 580 to 495 nm upon pyrazole binding, the copper site appears to maintain its near tetrahedral geometry. Since normal coordinate analyses indicate that vibrational frequencies are sensitive to geometric variations in this type of system [1], any structural changes responsible for the altered λ max and hyperfine splitting in the Cu-LADH/pyrazole complex must be fairly subtle ones.