X-ray Absorption Spectra of the Oxidized and Reduced Forms of C112D Azurin from Pseudomonas aeruginosa.
The oxidized and reduced forms of a mutant of Pseudomonas aeruginosa azurin, in which the Cys112 has been replaced by an aspartate, have been studied by X-ray absorption spectroscopy.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.
DeBeer
Serena
S
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.
Kiser
Cynthia N.
CN
Mines
Gary A.
GA
Richards
John H.
JH
Gray
Harry B.
HB
Solomon
Edward I.
EI
Hedman
Britt
B
Hodgson
Keith O.
KO