¹H NMR studies of electron exchange rate of Pseudomonas aeruginosa azurin

T₁ values of the His-35 C-2 proton resonance of reduced Pseudomonas aeruginosa azurin were determined at 25°C and pH values 4.5, 7.3, and 9.0 in the presence of different fractional amounts of oxidized azurin. The C-2 proton of His-35 undergoes very rapid spin relaxation in oxidized azurin because of its close proximity to the paramagnetic copper. In the presence of oxidized protein, the T₁ values of this proton in reduced azurin depend on the lifetime of the reduced protein. From the T₁ data, the electron self-exchange rate constant for azurin was calculated to be 1.4 x 10⁴ M^-1.s^-1, 4.3 x 10³ M^-1.s^-1, and 6.0 x 10³ M^-1.s^-1 at pH values 4.5, 7.3, and 9, respectively. At pH 7.3, the C-2 proton of His-35 is in slow exchange between the imidazole and imidazolium forms and gives rise to two separate resonances at 9.39 and 8.00 ppm. By using these two resonances, the electron self-exchange rate constants were determined separately for the two species of azurin for which the His-35 residue is in the imidazole or the imidazolium forms; results showed that both species participate in self-exchange of electrons with equal efficiency.