S. Debeer et al., X-ray absorption spectra of the oxidized and reduced forms of C112D azurinfrom Pseudomonas aeruginosa, INORG CHEM, 38(3), 1999, pp. 433-438
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, in which the Cys112 has been replaced by an aspartate, have been studied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 character
istic similar to 600 nm absorption feature of blue copper proteins is due t
o the S(Cys112) 3p pi --> Cu 3d(x)(-y)(2)(2) charge-transfer transition. Wh
ile other mutagenesis studies have involved the creation of an artificial b
lue copper site, the present work involves a mutant in which the native blu
e copper site has been destroyed, thus serving as a direct probe of the imp
ortance of the copper-thiolate bond to the spectroscopy, active site struct
ure, and electron-transfer function of azurin. Of particular interest is th
e 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) approxim
ate to 20 M-1 s(-1) C112D azurin) and intramolecular electron transfer to r
uthenium-labeled sites (k(et) approximate to 10(6) s(-1) wild-type azurin v
s 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 reorga
nization energy (lambda). These effects can be probed by the use of Cu K-ed
ge X-ray absorption spectroscopy, the results of which indicate both a decr
ease 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 th
e C112D mutant.