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Published February 8, 1999 | public
Journal Article

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The oxidized and reduced forms of a mutant of Pseudomonas aeruginosa azurin, in which the Cys112 has been replaced by an aspartate, have been studied by X-ray absorption spectroscopy.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.

Additional Information

© 1999 American Chemical Society. Received April 23, 1998. This research was supported by the NSF [CHE-9217628 (E.I.S.); CHE-9423181 (K.O.H.)] and the NIH [RR-01209 (K.O.H.); DK-19038 (H.B.G.)]. SSRL operations are funded by the Department of Energy, Office of Basic Energy Sciences. The Biotechnology Program is supported by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program and by the Department of Energy, Office of Biological and Environmental Research.

Additional details

Created:
August 19, 2023
Modified:
October 23, 2023