The oxidized and reduced forms of a mutant of Pseudomonas aeruginosa azurin, in which the Cys112 has been replaced by an aspartate, have been studied by X-ray absorption spectroscopy.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 similar to 600 nm absorption feature of blue copper proteins is due to the S(Cys112) 3p pi --> Cu 3d(x)(-y)(2)(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) approximate to 10(5) M-1 s(-1) wild-type azurin vs k(ese) approximate to 20 M-1 s(-1) C112D azurin) and intramolecular electron transfer to ruthenium-labeled sites (k(et) approximate to 10(6) s(-1) wild-type azurin vs k(et) less than or equal to 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 (H-AB) and/or in the reorganization energy (lambda). 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 similar to 0.2 Angstrom in the Cu coordination sphere trigonal plane upon reduction of the C112D mutant.