pH dependence of the redox reaction of azurin with cytochrome c551: role of His-35 of azurin in electron transfer

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Abstract
A fluorescence quenching experiment confirms that in the redox reaction between cytochrome c-551 and azurin, protein complexing is negligible. Azurin-pH indictor T-jump experiments show that Pseudomonas aeruginosa azurin exhibits a slow time constant, .tau., in its return to pH equilibrium but Alkcaligenes faecalis azurin does not. The decrease of 1/.tau. with increasing pH shows that the rate-determining process is a slow transformation of the imidazolium form of His-35 from a conformation where it cannot ionize to one in which it can. The fast relaxation time constant of the redox reaction varies little with pH, but the slow time constant increased by a factor of .apprx. 2.5 with increasing pH between 5 and 8. The corresponding amplitudes, especially the slow one, vary with pH. While some differences of redox reactivity do occur on prontonation, these differences apparently are not major. In general, the 2 proteins cytochrome c-551 and azurin react with each other with rates only weakly dependent upon pH. A classicial pH titration was carried out on the reduced and oxidized form of P. aeruginosa and A. faecalis azurin with the result that 2 protons were released between pH 6 and pH 8, in the former from His-35 and -83 and in the latter from His-83 and Ala-1.