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The electron-transfer kinetics between three different mediators and the hexahemic enzyme nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774) were investigated by cyclic voltammetry and by chronoamperometry. The mediators, methyl viologen, Desulfovibrio vulgaris (Hildenborough) cytochrome c3 and D. desulfuricans (ATCC 27774) cytochrome c3 differ in structure, redox potential and charge. The reduced form of each mediator exchanged electrons with nitrite reductase. Second-order rate constants, k, were calculated on the basis of the theory for a simple catalytic mechanism and the results, obtained by cyclic voltammetry, were compared with those obtained by chronoamperometry. Values for k are in the range 106-108 M-1 s-1 and increase in the direction D. desulfuricans cytochrome c3→D. vulgaris cytochrome c3→methyl viologen. An explanation is advanced on the basis of electrostatic interactions and relative orientation between the partners involved. Chronoamperometry (computer controlled) offers advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate and better reproducibility). Direct, unmediated electrochemical responses of the hexaheme nitrite reductase were also reported. [ABSTRACT FROM AUTHOR]

The gene for pseudoazurin was isolated from Paracoccus pantotrophus LMD 52.44 and expressed in a heterologous system with a yield of 54.3 mg of pure protein per liter of culture. The gene and protein were shown to be identical to those from P. pantotrophus LMD 82.5. The extinction coefficient of the protein was re-evaluated and was found to be 3.00 mM-1 cm-1 at 590 nm. It was confirmed that the oxidized protein is in a weak monomer/dimer equilibrium that is ionic-strength-dependent. The pseudoazurin was shown to be a highly active electron donor to cytochrome c peroxidase, and activity showed an ionic strength dependence consistent with an electrostatic interaction. The pseudoazurin has a very large dipole moment, the vector of which is positioned at the putative electron-transfer site, His81, and is conserved in this position across a wide range of blue copper proteins. Binding of the peroxidase to pseudoazurin causes perturbation of a set of NMR resonances associated with residues on the His81 face, including a ring of lysine residues. These lysines are associated with acidic residues just back from the rim, the resonances of which are also affected by binding to the peroxidase. We propose that these acidic residues moderate the electrostatic influence of the lysines and so ensure that specific charge interactions do not form across the interface with the peroxidase. [ABSTRACT FROM AUTHOR]