LONG-RANGE INTRAMOLECULAR ELECTRON-TRANSFER IN AZURINS

The disulfide bridge linking cysteines-3 and -26 in the blue single copper protein azurin isolated from Alcaligenes faecalis (Alc. faec.) and Pseudomonas fluorescens B (Ps. fluor.) reacts with pulse radiolytically produced CO2- radical anions to yield the disulfide radical ion, RSSR-. This radical then decays by an intramolecular electron transfer (et) to the Cu(II) center which is at a distance of approximately 2.6 nm. At 25-degrees-C and pH 7.0, the rate constants of the intramolecular electron transfer reactions are 11 +/- 2 and 22 +/- 3 s-1 in Alc. faec. and Ps. fluor. azurin, respectively. This intramolecular reaction rate was studied over the 2.0-46.5-degrees-C temperature range. The activation enthalpies derived from the temperature dependence are DELTA-H* = 54.5 +/- 1.4 and DELTA-H* = 36.3 +/- 1.2 kJ mol-1, and the entropies of activation are DELTA-S(double dagger) = -43.9 +/- 9.5 and DELTA-S(double dagger) = -97.7 +/- 5.0 J K-1 mol-1 for Alc. faec. and Ps. fluor. azurin, respectively. Under similar conditions, the specific rate of the intramolecular et between the homologous RSSR- and the Cu(II) site in Pseudomonas aeruginosa (Ps. aer.) azurin was found to be 2-4 times higher than in the above two azurins. This difference cannot be rationalized satisfactorily by the differences in either driving force or distance between the electron donor and acceptor in these proteins. Instead the differences in activation parameters and rates may be rationalized in terms of an increased reorganization energy in Ps. fluor. and Alc. faec. as compared with Ps. aer. azurin. The other main pertinent difference among these azurins is that residue Trp 48 present in the Ps. aer. protein midway between the electron donor and acceptor is substituted in the Alc. faec. and Ps. fluor. proteins by a valine and a leucine, respectively. This suggests that the aromatic indole ring system may play a role either in enhancing the mixing of the electron donor and acceptor wave functions or by affecting the reorganization energy of the reaction.