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Role of the active-site cysteine of Pseudomonas aeruginosa azurin. Crystal structure analysis of the Cu^(II(Cys112Asp) protein

Faham, Salem and Mizoguchi, Tadashi J. and Adman, Elinor T. and Gray, Harry B. and Richards, John H. and Rees, Douglas C. (1997) Role of the active-site cysteine of Pseudomonas aeruginosa azurin. Crystal structure analysis of the Cu^(II(Cys112Asp) protein. Journal of Biological Inorganic Chemistry, 2 (4). pp. 464-469. ISSN 0949-8257. doi:10.1007/s007750050157.

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Replacement of the cysteine at position 112 of Pseudomonas aeruginosa azurin with an aspartic acid residue results in a mutant (Cys112Asp) protein that retains a strong copper-binding site. Cu^(II)(Cys112Asp) azurin can be reduced by excess [Ru^(II)(NH_3)_6]^(2+), resulting in a Cu^I protein with an electronic absorption spectrum very similar to that of wild-type Cu^I azurin. Cys112Asp azurin exhibits reversible interprotein electron-transfer reactivity with P. aeruginosa cytochrome c_(551) (μ = 0.1 M sodium phosphate (pH 7.0);E°(Cu^(II/I)) = 180 mV vs NHE); this redox activity indicates that electrons can still enter and exit the protein through the partially solvent-exposed imidazole ring of His117. The structure of Cu^(II)(Cys112Asp) azurin at 2.4-Å resolution shows that the active-site copper is five coordinate: the pseudo-square base of the distorted square-pyramidal structure is defined by the imidazole N^δ atoms of His46 and His117 and the oxygen atoms of an asymmetrically-bound bidentate carboxylate group of Asp112; the apical position is occupied by the oxygen atom of the backbone carbonyl group of Gly45. The Cu^(II)–Asp112 interaction is distinguished by an approximately 1.2-Å displacement of the metal center from the plane defined by the Asp112 carboxylate group.

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Gray, Harry B.0000-0002-7937-7876
Rees, Douglas C.0000-0003-4073-1185
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Additional Information:© 1997 SBIC. Received: 30 December 1996; Accepted: 8 May 1997. We thank Mike Hill for assistance with several electrochemical experiments, and Xiaotian Zhu, Art Chirino and Barbara Hsu for their help with the crystallographic analysis. S.F. and T.J.M. contributed equally to this work; supported by the NIH (DK19038 to H.B.G., GM16424 to J.H.R., and GM45162 to D.C.R.).
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Subject Keywords:azurin; cytochrome c(551); electron transfer; site-directed mutagenesis; protein crystallography
Issue or Number:4
Record Number:CaltechAUTHORS:20150205-135731010
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ID Code:54431
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Deposited On:08 Feb 2015 06:32
Last Modified:10 Nov 2021 20:33

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