KINETICS AND EQUILIBRIA OF ELECTRON-TRANSFER BETWEEN AZURIN AND HEXACYANOIRON-(II/III) COUPLE

The electron transfer reaction between the blue single copper protein azurin (from Pseudomonas aeruginosa) and the hexacyanoiron (II/III) couple was studied. Equilibrium constants for the reduction of azurin were measured spectrophotometrically in the temperature range 5-33.degree. C (K [equilibrium constant] = 1.1 .times. 10-2 at 25.degree. C, .DELTA.H.degree. [reaction enthalpy] = 10.9 kcal/mol, 0.1 M potassium phosphate, pH 7.0, I [ionic strength] = 0.22). The enthalpy change was also determined by microcalorimetry and from the analysis of chemical relaxation amplitudes. Following a temperature-jump perturbation of this equilibrium, only a single relaxation was observed. The reciprocal of the relaxation time increased linearly as oxidized azurin was reacted with increasing amounts of ferrocyanide, yet reached saturation when reduced azurin was titrated with ferricyanide. This behavior and the analysis of the relaxation amplitudes led to the following scheme for this system: .**GRAPHIC**. At 25.degree. C the rate constants for the electron transfer were k+3 = 6.4 s-1 and k-3 = 45 s-1, the association constants K1 = 54 M-1 and K2-1 = 610 M-1. The activation and overall thermodynamic parameters and the individual thermodynamic values for the different steps were combined to construct a self-consistent energy profile for the reaction.