X-ray Absorption Spectra of the Oxidized and Reduced Forms of C112D Azurin from Pseudomonas aeruginosa

Abstract

The oxidized and reduced forms of a mutant of Pseudomonas aeruginosa azurin, in which the Cys112 has been replaced by an aspartate, have been studied by X-ray absorption spectroscopy. It is well established that the characteristic ∼600 nm absorption feature of blue copper proteins is due to the S(Cys112) 3pπ → Cu 3d_(x^2-y^2) charge-transfer transition. While other mutagenesis studies have involved the creation of an artificial blue copper site, the present work involves a mutant in which the native blue copper site has been destroyed, thus serving as a direct probe of the importance of the copper−thiolate bond to the spectroscopy, active site structure, and electron-transfer function of azurin. Of particular interest is the dramatic decrease in electron-transfer rates, both electron self-exchange (k_(ese) ≈ 10^5 M^(-1) s^(-1) wild-type azurin vs k_(ese) ≈ 20 M^(-1) s^(-1) C112D azurin) and intramolecular electron transfer to ruthenium-labeled sites (k_(et) ≈ 10^6 s^(-1) wild-type azurin vs k_(et) ≤ 10^3 s^(-1) C112D azurin), which is observed in the mutant. These changes may be a reflection of significant differences in electronic coupling into the protein matrix (HAB) and/or in the reorganization energy (λ). These effects can be probed by the use of Cu K-edge X-ray absorption spectroscopy, the results of which indicate both a decrease in the covalency of the active site and an expansion of ∼0.2 Å in the Cu coordination sphere trigonal plane upon reduction of the C112D mutant.