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X-ray Absorption Spectra of the Oxidized and Reduced Forms of C112D Azurin from Pseudomonas aeruginosa

DeBeer, Serena and Kiser, Cynthia N. and Mines, Gary A. and Richards, John H. and Gray, Harry B. and Solomon, Edward I. and Hedman, Britt and Hodgson, Keith O. (1999) X-ray Absorption Spectra of the Oxidized and Reduced Forms of C112D Azurin from Pseudomonas aeruginosa. Inorganic Chemistry, 38 (3). pp. 433-438. ISSN 0020-1669. doi:10.1021/ic9804622.

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The oxidized and reduced forms of a mutant of Pseudomonas aeruginosa azurin, in which the Cys112 has been replaced by an aspartate, have been studied by X-ray absorption spectroscopy.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

Item Type:Article
Related URLs:
URLURL TypeDescription
DeBeer, Serena0000-0002-5196-3400
Kiser, Cynthia N.0000-0002-0102-996X
Gray, Harry B.0000-0002-7937-7876
Solomon, Edward I.0000-0003-0291-3199
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.
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Department of Energy (DOE)UNSPECIFIED
Issue or Number:3
Record Number:CaltechAUTHORS:20150424-155359723
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Official Citation:X-ray Absorption Spectra of the Oxidized and Reduced Forms of C112D Azurin from Pseudomonas aeruginosa Serena DeBeer, Cynthia N. Kiser, Gary A. Mines, John H. Richards,‡, Harry B. Gray, Edward I. Solomon, Britt Hedman, and Keith O. Hodgson Inorganic Chemistry 1999 38 (3), 433-438 DOI: 10.1021/ic9804622
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:56983
Deposited By: George Porter
Deposited On:27 Apr 2015 15:17
Last Modified:10 Nov 2021 21:06

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