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 degrees 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 10(4) M-1 X s-1, 4.3 X 10(3) M-1 X s-1, and 6.0 X 10(3) M-1 X 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.
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D001400:Azurin / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D004579:Electron Transport; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011550:Pseudomonas aeruginosa / Q000378:metabolism*
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D001400:Azurin; C017167:cytochrome C(551)