The Influence of pH on Long-Range Electron Transfer and Proton-Coupled Electron Transfer in Ruthenium-Modified Azurin

Long-range electron transfer (ET) is an essential component of all biological systems. Reactions of metalloproteins are important in this context. Recent work on protein "charge ladders" has revealed how the redox state of embedded metal ions can influence the ionization of amino acid residues at protein surface sites. Inspired by these observations, we carried out a variable pH investigation of intramolecular ET reactions in a ruthenium-modified protein system built on azurin from Pseudomonas aeruginosa. We also generate a Pourbaix diagram that describes the variable pH redox behavior of a Ru model complex, Ru(2,2'-bipyridyl)₂(imidazole)₂(PF₆)₂. The intramolecular ET rate constants for the oxidation of azurin-Cu⁺ by flash-quench-generated Ru³⁺ oxidants do not follow the predictions of the semi-classical ET rate expression with fixed values of reorganization energy (λ) and electronic coupling (H_DA). Based on the pH dependence of the Ru^3+/2+ redox couple, we propose a model where pure ET is operative at acidic pH values (≤ 7) and the mechanism changes to proton-coupled electron transfer at pH ≥ 7.5. The implications of this mechanistic proposal are discussed in the context of biological redox reactions and with respect to other examples of intramolecular ET reactions in the literature.