Paramagnetic properties of the halide-bound derivatives of oxidised tyrosinase investigated by H-1 NMR spectroscopy

The (1)H NMR relaxation characteristics of the histidines in the oxidised type-3 copper site of tyrosinase (Ty(met)) from the bacterium Streptomyces antibioticus in the halide-bound forms (Ty(met)X with X = F(-), Cl(-), Br(-)) have been determined and analysed. The (1)H NMR spectra of the Ty(met)X species display remarkably sharp, well-resolved, paramagnetically shifted (1)H signals, which originate from the protons of the six His residues coordinated to the two Cu(II) ions in the type-3 centre. From the temperature-dependence of the (1)H paramagnetic shifts the following values for the exchange-coupling parameter -2J were determined: 260 (Ty(met)F), 200 (Ty(met)Cl) and 162 cm(-1) (Ty(met)Br). The (1)H T(1) relaxation is dipolar in origin and correlates with the Cu--H distances. Electronic relaxation times tau(S) derived from the (1)H T(1) data amount to about 10(-11) s and follow the order Ty(met)F>Ty(met)Cl>Ty(met)Br. They are two orders of magnitude shorter than the tau(S) values reported for mononuclear copper systems, in accordance with the sharpness of the (1)H signals. The results corroborate the Cu(2) bridging mode of the halide ions. On the basis of the measured hyperfine interaction constants for the ligand histidine nuclei, it is concluded that 70-80 % of the spin density in the excited triplet state resides on the two copper ions and the bridging atoms

Stopped-flow fluorescence studies of inhibitor binding to tyrosinase from streptomyces antibioticus

Tyrosinase (Ty) is a type 3 copper protein involved in the rate-limiting step of melanin synthesis. It is shown that the endogenous Trp fluorescence of tyrosinase from Streptomyces antibioticus is remarkably sensitive to the redox state. The fluorescence emission intensity of the [(Cu(I) Cu(I)] reduced species is more than twice that of the oxygen-bound [Cu(II)-O(2)(2-)-Cu(II)] form. The emission intensity of the oxidized [Cu(II)-OH(-)-Cu(II)] protein (Ty(met)) appears to be dependent on an acid-base equilibrium with a pK(a) value of 4.5 +/- 0.1. The binding of fluoride was studied under pseudo first-order conditions using stopped-flow fluorescence spectroscopy. The kinetic parameters k(on), K(d), and the fraction of fluorescence emission quenched upon fluoride binding show a similar pH dependence as above with an average pK(a) value of 4.62 +/- 0.05. Both observations are related to the dissociation of Cu(2)-bridging hydroxide at low pH. It is further shown that Ty is rapidly inactivated at low pH and that halide protects the enzyme from this inactivation. All results support the hypothesis that halide displaces hydroxide as the Cu(2)-bridging ligand in Ty(met). The relevance of the experimental findings for the catalytic cycle is discussed. The data are consistent with the data obtained from other techniques, validating the use of fluorescence quenching as a sensitive and effective tool in studying ligand binding and substrate conversion

Bidirectional catalysis by copper-containing nitrite reductase

The copper-containing nitrite reductase from Alcaligenes faecalis S-6 was found to catalyze the oxidation of nitric oxide to nitrite, the reverse of its physiological reaction. Thermodynamic and kinetic constants with the physiological electron donor pseudoazurin were determined for both directions of the catalyzed reaction in the pH range of 6-8. For this, nitric oxide was monitored by a Clark-type electrode, and the redox state of pseudoazurin was measured by optical spectroscopy. The equilibrium constant (K-eq) depends on the reduction potentials of pseudoazurin and nitrite/nitric oxide, both of which vary with pH. Above pH 6.2 the formation of NiR substrates (nitrite and reduced pseudoazurin) is favored over the products (NO and oxidized pseudoazurin). At pH 8 the K-eq amounts to 103. The results show that dissimilatory nitrite reductases catalyze an unfavorable reaction at physiological pH (pH = 7-8). Consequently, nitrous oxide production by copper-containing nitrite reductases is unlikely to occur in vivo with a native electron donor. With increasing pH, the rate and specificity constant of the forward reaction decrease and become lower than the rate of the reverse reaction. The opposite occurs for the rate of the reverse reaction; thus the catalytic bias for nitrite reduction decreases. At pH 6.0 the kat for nitrite reduction was determined to be 1.5 x 10(3) s(-1), and at pH 8 the rate of the reverse reaction is 125 s(-1)

Genetic modulation of metalloprotein electron transfer at bare gold.

Engineered metalloproteins and enzymes can be self assembled on pristine gold electrodes in robust, electrochemically-addressable, arrays

A random-sequential mechanism for nitrite binding and active site reduction in copper-containing nitrite reductase.

The homotrimeric copper-containing nitrite reductase (NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of pH and nitrite on the turnover rate in the presence of three different electron donors at saturating concentrations were measured. The activity of NiR was also measured electrochemically by exploiting direct electron transfer to the enzyme immobilized on a graphite rotating disk electrode. In all cases, the steady-state kinetics fitted excellently to a random-sequential mechanism in which electron transfer from the type-1 to the type-2 site is rate-limiting. At low [NO(-)(2)] reduction of the type-2 site precedes nitrite binding, at high [NO(-)(2)] the reverse occurs. Below pH 6.5, the catalytic activity diminished at higher nitrite concentrations, in agreement with electron transfer being slower to the nitrite-bound type-2 site than to the water-bound type-2 site. Above pH 6.5, substrate activation is observed, in agreement with electron transfer to the nitrite-bound type-2 site being faster than electron transfer to the hydroxyl-bound type-2 site. To study the effect of slower electron transfer between the type-1 and type-2 site, NiR M150T was used. It has a type-1 site with a 125-mV higher midpoint potential and a 0.3-eV higher reorganization energy leading to an approximately 50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism

Design of novel molecular wires for realizing long-distance electron transfer

Novel heteroarene oligomers, consisting of two pyridinium groups, linked by thiophene units of variable length, thienoviologens, are described as promising candidates for molecular wires. Two representative thienoviologens were coated by adsorption from micromolar concentrations in ethanol onto octadecylmercaptan (ODM)-coated gold electrodes and induced a gradual restoration of the electrochemistry with hexacyanoferrate as a function of molecular wire concentration. Glucose oxidase and choline oxidase showed strong adsorption to these conductive layers, but showed striking differences in adsorption to the different thienoviologen layers. The measurements support the hypothesis that the molecules are incorporated in the ODM layer in a different fashion. Also the complex formation of an engineered azurin redox protein with water-soluble pyridyl ligands is presented in relation to a possible application of the thienoviologens as conductive spacers, in which the contact with the redox protein is achieved via complex formation with a free pyridine nitrogen. (C) 1997 Elsevier Science S.A.</p