Resolution of two distinct electron transfer sites on azurin

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Abstract
Pseudomonas aeruginosa azurin is stoichiometrically and specifically labeled on reduction by Cr(II)aq ions, yielding a substitution-inert Cr(III) adduct on the protein surface. The effect of this chemical modification on the reactivity of azurin with 2 of its presumed partners in the redox system of the bacterium was investigated. The Pseudomonas cytochrome oxidase catalyzed oxidation of reduced native and Cr(III)-labeled azurin by O2 was unaffected by the modificaiton. The kinetics of the electron exchange reaction between native or Cr(III)-labeled azurin and cytochrome c551 were studied by the temperature-jump method. Though similar chemical relaxation spectra were observed for native and modified systems, they differ quantitatively. Analysis of the concentration dependences of the relaxation times and amplitudes showed that both obey the same mechanism but that the specific reaction rates of the Cr(III)-modified protein are attenuated. This decreased reactivity of Cr(III)-labeled azurin toward one of its physiological partners suggests the involvement of the labeled region in the electron transfer reaction with cytochrome c551. The presence of a 2nd active site, involved in the reduction of cytochrome oxidase, is suggested by the results.