NMR study of structure and electron transfer mechanism of Pseudomonas aeruginosa azurin.

The nuclear spin-spin and spin-lattice relaxation times of the C epsilon 1-proton of His-35 and the C delta 2-proton of His-46 of reduced Pseudomonas aeruginosa azurin have been determined at 298 and 320 K and at pH 4.5 and 9.0 at various concentrations of total azurin and in the presence of varying amounts of oxidized azurin. The relaxation times appear strongly influenced by the electron self-exchange reaction between oxidized and reduced protein. The T1 data of the His-35 proton have been analyzed according to the "fast-exchange limit," while the "slow-exchange limit" appears to obtain for the T2 data of the His-46 proton. Analysis of the proton relaxation data yields values of the electron self-exchange rate constants of (9.6 +/- 0.7) X 10(5) M-1 S-1 (pH 4.5) and (7.0 +/- 1.3) X 10(5) M-1 S-1 (pH 9.0) at 298 K. The dipolar correlation time amounts to 1-2.5 ns in the temperature range of 298-320 K. A Fermi-contact interaction of about 100 mG for the C delta 2-proton of His-46 is compatible with the experimental observations. The pH-induced conformational changes lead to variations on the order of about 1 A in the distance from the copper to the His-35 protons. The data implicate the "hydrophobic patch" around His-117 as the site of electron transfer in the self-exchange reaction of the azurin.