INFLUENCE OF CHROMIUM BINDING ON THE KINETICS OF AQUEOUS CHROMIUM(II) REDUCTION OF THE BLUE COPPER PROTEINS AZURIN AND STELLACYANIN

The blue single copper proteins, azurin from Pseudomonas aeruginosa and stellacyanin from Rhus vernicifera can be reduced by Cr(II) aqua ions, and the Cr(III) ions formed as a result of the electron transfer to Cu(II) centers in the proteins are coordinated to specific sites on the protein surfaces. These sites have earlier been identified [Farver, O.; Pecht, 1. J. Isr. Chem. 1981, 21, 13-17; Farver, O.; Licht, A.; Pecht, I. Biochemistry 1987, 26, 7317-7321]. In order to demonstrate that the site of coordination is the result of a trapping of the Cr(III) ion close to a redox active center on the protein rather than a migration of Cr(III) to a thermodynamically more advantageous site, we have now studied the Cr(II) reduction kinetics of the Cr(III)-labeled proteins and of the native proteins. For Cr(III)-labeled azurin we find k = 300 +/- 10 M-1 s-1 (298 K), DELTAH(double dagger) = 0 +/- 2 kJ/mol, and DELTAS(double dagger) = -195 +/- 10 J/(mol.K), at 0.1 M MES and pH 5 compared with k = 450 +/- 15 M-1 s-1, DELTAH(dagger) = 8 +/- 1 kJ/mol, and DELTAS(double dagger) = -170 +/- 10 J/(mol.K) for native azurin, under the same conditions. For Cr(III)-labeled stellacyanin in 0.1 M MES pH 5 the reduction is biphasic and we find k1 almost-equal-to 5 x 10(3) M-1 s-1 (298 K), DELTAH(double dagger)1 = 0 +/- 8 kJ/mol, DELTAS(double dagger)1 = -170 +/- 30 J/(mol.K), k2 almost-equal-to 800 M-1 s-1 (298 K), DELTAH(double dagger)2 = 23 +/- 2 kJ/mol, DELTAS(double dagger)2 = -107 +/- 7 J/(mol.K), while for native stellacyanin the reaction is monophasic with k almost-equal-to 1 x 10(6) M-1 s-1 (298 K), DELTAH(double dagger) = 20 +/- 4 kJ/mol, and DELTAS(double dagger) = -60 +/- 14 J/(mol.K). The large effect of coordinated Cr(III) on the reactions demonstrate that the binding centers on azurin and stellacyanin are indeed close to redox active centers on the protein surface. Electron transfer pathways from these centers to the Cu(II) ion in azurin and stellacyanin have been calculated and consist of relatively short through-bond interactions.