Spectroscopic and electrochemical studies, incorporating electronic spectra, electron paramagnetic resonance (EPR) spectra, resonance Raman (RR) spectra, and measurements of the redox potential, have been carried out on the blue copper protein azurin, from Alcaligenes denitrificans. These data are correlated with the refined crystal structure of this azurin and with corresponding data for other blue copper proteins. The electronic spectrum, characterized by an intense (epsilon = 5100 M-1 cm-1) charge-transfer band at 619 nm, the EPR spectral parameters (g perpendicular = 2.059, g parallel of = 2.255, A parallel of = 60 X 10(-4) cm-1), and the resonance Raman spectrum are similar to those obtained from other azurins and from plastocyanins. Both the electronic spectrum and the EPR spectrum are unchanged over the pH range 4-10.5, but major changes occur above pH 12 and below pH 3.5. A small reversible change occurs at pH approximately 11.4. In the RR spectrum the Cu-S stretching mode is shown to contribute to all of the five principal RR peaks. Deuterium substitution produces shifts in at least seven of the peaks; these shifts may be attributable, at least in part, to the NH...S hydrogen bond to the copper-ligated Cys-112. Measurements of the redox potential, using spectroelectrochemical methods, over the temperature range 4.8-40.0 degrees C, give values for delta H0' and delta S0' of -55.6 kJ mol-1 and -97.0 J K-1 mol-1, respectively. The redox potential of A. denitrificans azurin at pH 7.0, Eo', is 276 mV. These data are interpreted in terms of a copper site, in azurin, comprising three strong bonds, in an approximately trigonal plane, from Cys-112, His-46, and His-117 and much longer axial approaches from Met-121 and the peptide carbonyl oxygen of Gly-45. Spectral differences within the azurin family and between azurin and plastocyanin are attributed to differences in the strengths of these axial interactions. Likewise, the distinctly lower Eo values for azurins, as compared with plastocyanins, are related to the more copper(II)-like site in azurin [with a weaker Cu-S(Met) interaction and a Cu-O interaction not found in plastocyanin]. On the other hand, the relative constancy of the EPR parameters between azurin and plastocyanin suggests they are not strongly influenced by weakly interacting axial groups.

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The preparation of the complexes [ (CuLX),] (H L = 2-formylpyridine thiosemicarbazone) from the interaction of H L with the appropriate copper salt (X = CH,COO, CI, NO,, or CIO,) or by reaction of an HL-KX mixture w i t h copper(ti) acetate monohydrate (X = Br or I) is described. Dissolution of [{Cu L( CH,COO)},] in H,SO, or HCI yields the protonated ligand complexes [{Cu ( H L) (SO,)},] and [{Cu (H L) CI,},].2H2O respectively. The complexes have been characterized by a variety of spectroscopic techniques and the crystal and molecular structures of [{CuL(CH,COO)},] and [{Cu( H L) (SO,,)},] determined by single-crystal X-ray diffraction techniques from diffractometer data. Crystals of [{CuL(CH,COO)},] are triclinic, space group P r , w i t h a = 8.827(3), b = 8.81 3(3), c = 8.997(3) A, a = 117.49(2), p = 11 0.96(3), y = 91.65(3)", and Z = 1. Crystals of [{Cu(HL) (SO,)},] are monoclinic, space group C2/c, w i t h a = 14.751 (3), b = 9.1 38(2), c = 17.468(4) A, j3 = 104.91 ( 2)", and Z = 4. After full-matrix leastsquares refinement the final R value was 0.027 for both complexes ( 2 21 6 and 2 206 observed reflections were used respectively). Both complexes consist of discrete centrosymmetric dimers, the monomeric units being bridged by two acetato or sulphato ligands. The copper atoms have a distorted square-pyramidal co-ordination geometry w i t h three donor atoms (NNS) coming from L or H L t o form a tricyclic ligating system. The fourth donor atom (oxygen) comes from the bridging CH,COO-or SO,'ion. The fifth co-ordination position is occupied by a less strongly bound oxygen from the second bridging anion.