title: 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abstract: The oxidized and reduced forms of a mutant of Pseudomonas aeruginosa azurin, in which the Cys112 has been replaced by an aspartate, have been studied by X-ray absorption spectroscopy.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 approximately 600 nm absorption feature of blue copper proteins is due to the S(Cys112) 3ppi --> 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) approximately 10(5) M(-)(1) s(-)(1) wild-type azurin vs k(ese) approximately 20 M(-)(1) s(-)(1) C112D azurin) and intramolecular electron transfer to ruthenium-labeled sites (k(et) approximately 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 (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 approximately 0.2 Å in the Cu coordination sphere trigonal plane upon reduction of the C112D mutant.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California 94305, Beckman Institute, California Institute of Technology, Pasadena, California 91125, and Stanford Synchrotron Radiation Laboratory, Stanford, California 94309.
authors.forename: Serena
authors.initials: S
authors.lastname: DeBeer
authors.forename: Cynthia N.
authors.initials: CN
authors.lastname: Kiser
authors.forename: Gary A.
authors.initials: GA
authors.lastname: Mines
authors.forename: John H.
authors.initials: JH
authors.lastname: Richards
authors.forename: Harry B.
authors.initials: HB
authors.lastname: Gray
authors.forename: Edward I.
authors.initials: EI
authors.lastname: Solomon
authors.forename: Britt
authors.initials: B
authors.lastname: Hedman
authors.forename: Keith O.
authors.initials: KO
authors.lastname: Hodgson