Resonance Raman spectra of the single-copper blue proteins azurin (from the bacteria Pseudomonas putida, Pseudomonas aeruginosa, Iwasaki sp., Bortadella pertussis, Bortadella bronchiseptica, and Alcaligenes faecalis), plastocyanin (from French bean and spinach), and stellacyanin (from the Japanese and Chinese lacquer trees Rhus vernicifera) and the multicopper oxidases lacease (from the fungus Polyporus versicolor and the Japanese and Chinese lacquer trees), ascorbate oxidase (from zucchini squash), and ceruloplasmin (from human blood serum) are reported. Cryoresonance Raman observations (10-77 K) are reported for selected azurins, stellacyanin, the plastocyanins, and the laceases. Isotope studies employing 63Cu/65Cu and H/D substitution are reported for selected azurins and stellacyanin, allowing identification of modes having significant

A comparative study of the magnetic properties of six representative type 1 Cu(II) blue copper centers was carried out by using electron nuclear double resonance (ENDOR). Four centers are EPR single-site proteins: plastocyanins from bean (Phaseolus vulgaris) and poplar leaves (Populus nigra italica), azurin from Pseudomonas aeruginosa, and stellacyanin from Rhus vernicifera. Two are in laceases, fungal (Polyporous versicolor) and tree (R. vernicifera), that have had their type two centers reduced. In each case the low-frequency (v < 35 MHz) region of the spectrum is dominated by the resonances from strongly coupled (AH > 20 MHz) methylene protons of a coordinated cysteinyl mercaptide. In all cases but one, experiments at two microwave frequencies (9.6 and 11.6 GHz) also permitted detection of resonances from two, inequivalent, nitrogenous ligands. The coordination environments in the type 1 Cu(II) sites of the six proteins are broadly similar, but detailed analysis suggests that the stereoelectronic structure of the single-site proteins, as a group, differs in subtle but significant ways from that of the type 1 Cu(II) center of the laceases. The reduction potentials of the single-site type 1 Cu(II) centers correlate well with the bonding within a center, as reflected in the ligand ENDOR parameters: Reduction potentials decrease with decreasing bonding to (or ligand field at) the type 1 Cu(II). This correlation does not hold for the lacease type 1 sites, which appear to have enhanced tr-bonding to mercaptide sulfur. Analysis of these results suggests that fine tuning of the reduction potentials of a type 1 Cu center is primarily achieved by altering the properties of the reduced, Cu(I), state.The mononuclear Cu(II) site of the blue copper (type 1) proteins has unusual optical and magnetic properties.6 The electronic spectrum is characterized by an intense absorption near 600 nm (e ~103-104), in contrast to the weak absorptions (< <102) exhibited by mononuclear copper complexes of low molecular weight. Typically, the EPR spectrum of blue copper also is quite different from that observed for "normal" copper; the values of AzCu, and ^iso0*, the isotropic coupling, all show unusually small values.7Near infrared, visible absorption, circular dichroism, and pulsed EPR spectroscopy7 are consistent with a distorted tetrahedral coordination geometry for blue copper site. It also has been suggested, in part from model compound studies,8 that the intense blue color is due to RS -* Cu charge-transfer excitation6,9 and that the unpaired electron is substantially delocalized onto a sulfur

The nickel(II) derivative of Rhus vernicifera stellacyanin has been prepared. The near‐infrared, visible, and near‐ultraviolet absorption spectra of the nickel(II) derivatives of stellacyanin and Pseudomonas aeruginosa azurin are reported. Charge transfer bands in the visible and near‐ultraviolet regions are assigned as follows: nickel(II)‐stellacyanin, 335 nm (29,900 cnr−1) (πN(his)→Ni(II)), 410 nm (24,400 cm−1) (σS(cys) → Ni(II)), and 470 nm (21,300 cm−1)(πS(cys)→ Ni(II)); nickel(II)‐azurin, 355 nm (28,200 cm−1) (πN(his)→Ni(II)), 440 nm (22,700 cm−1) (σS(cys)→ Ni(II)), and 500 nm (20,000 cm−1) (πS(cys)→ Ni(II)). Relatively weak bands at 550 (18,200) and 590 nm (16,900 cm−1) (Ni(II)St) and 540 (18,500) and 565 nm (17,700 cm−1) (Ni(II)Az) are attributed to d–d excitations. No evidence for lower energy d‐d bands has been found for either Ni(II)–protein. The data now in hand raise the possibility that the Ni(II) site structure differs from the flattened tetrahedral geometry exhibited by Cu(II) in these proteins.

The carbamate ester N-(phenoxycarbonyl)-L-phenylalanine binds well to carboxypeptidase A in the manner of peptide substrates. The ester exhibits linear competitive inhibition toward carboxypeptidase A catalyzed hydrolysis of the amide hippuryl-L-phenylalanine (Ki = 1.0 X 10(-3) M at pH 7.5) and linear noncompetitive inhibition toward hydrolysis of the specific ester substrate O-hippuryl-L-beta-phenyllactate (Ki = 1.4 X 10(-3) M at pH 7.5). Linear inhibition shows that only one molecule of inhibitor is bound per active site at pH 7.5. The hydrolysis of the carbamate ester is not affected by the presence of 10(-8)-10(-9) M enzyme (the concentrations employed in inhibition experiments), but at an enzyme concentration of 3 X 10(-6) M catalysis can be detected. The value of kcat at 30 degrees C, mu = 0.5 M, and pH 7.45 is 0.25 s-1, and Km is 1.5 X 10(-3) M. The near identity of Km and Ki shows that Km is a dissociation constant. Substrate inhibition can be detected at pH less than 7 but not at pH values above 7, which suggests that a conformational change is occurring near that pH. The analogous carbonate ester O-(phenoxycarbonyl)-L-beta-phenyllactic acid is also a substrate for the enzyme. The Km is pH independent from pH 6.5 to 9 and has the value of 7.6 X 10(-5) M in that pH region. The rate constant kcat is pH independent from pH 8 to 10 at 30 degrees C (mu = 0.5 M) with a limiting value of 1.60 s-1. Modification of the carboxyl group of glutamic acid-270 to the methoxyamide strongly inhibits the hydrolysis of O-(phenoxycarbonyl)-L-beta-phenyllactic acid. Binding of beta-phenyllactate esters and phenylalanine amides must occur in different subsites, but the ratios of kcat and kcat/Km for the structural change from hippuryl to phenoxy in each series are closely similar, which suggests that the rate-determining steps are mechanistically similar.

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