Takematsu, Kana and Williamson, Heather and Blanco-Rodríguez, Ana María and Sokolová, Lucie and Nikolovski, Pavle and Kaiser, Jens T. and Towrie, Michael and Clark, Ian P. and Vlček, Antonín, Jr. and Winkler, Jay R. and Gray, Harry B. (2013) Tryptophan-Accelerated Electron Flow Across a Protein−Protein Interface. Journal of the American Chemical Society, 135 (41). pp. 15515-15525. ISSN 0002-7863. http://resolver.caltech.edu/CaltechAUTHORS:20131105-131815168
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We report a new metallolabeled blue copper protein, Re126W122Cu^I Pseudomonas aeruginosa azurin, which has three redox sites at well-defined distances in the protein fold: Re^I(CO)_3(4,7-dimethyl-1,10-phenanthroline) covalently bound at H126, a Cu center, and an indole side chain W122 situated between the Re and Cu sites (Re-W122(indole) = 13.1 Å, dmp-W122(indole) = 10.0 Å, Re-Cu = 25.6 Å). Near-UV excitation of the Re chromophore leads to prompt Cu^I oxidation (<50 ns), followed by slow back ET to regenerate Cu^I and ground-state Re^I with biexponential kinetics, 220 ns and 6 μs. From spectroscopic measurements of kinetics and relative ET yields at different concentrations, it is likely that the photoinduced ET reactions occur in protein dimers, (Re126W122CuI)2 and that the forward ET is accelerated by intermolecular electron hopping through the interfacial tryptophan: ^*Re//←W122←Cu^I, where // denotes a protein–protein interface. Solution mass spectrometry confirms a broad oligomer distribution with prevalent monomers and dimers, and the crystal structure of the Cu^(II) form shows two Re126W122Cu^(II) molecules oriented such that redox cofactors Re(dmp) and W122-indole on different protein molecules are located at the interface at much shorter intermolecular distances (Re-W122(indole) = 6.9 Å, dmp-W122(indole) = 3.5 Å, and Re-Cu = 14.0 Å) than within single protein folds. Whereas forward ET is accelerated by hopping through W122, BET is retarded by a space jump at the interface that lacks specific interactions or water molecules. These findings on interfacial electron hopping in (Re126W122Cu^I)^2 shed new light on optimal redox-unit placements required for functional long-range charge separation in protein complexes.
|Additional Information:||© 2013 American Chemical Society. Publication Date (Web): September 13, 2013. Received: July 4, 2013. Author Contributions K.T. and H.W. contributed equally to this work. The authors declare no competing financial interest. We thank Yuling Shen, Crystal Shih, and Jeff Warren (Caltech), respectively, for azurin mutant preparation, preliminary laser measurements, and helpful discussions. Prof. B. Brutschy (Frankfurt) is thanked for discussions on LILBID-MS, and Hana Kvapilová and Jan Sýkora (JH Institute) for their help with some of the TRIR and TCSPC emission experiments. Research at Caltech was supported by NIH (DK019038 to HBG, JRW) and the Arnold and Mabel Beckman Foundation. The TRIR experiments were funded by the STFC Rutherford Appleton Laboratory, Queen Mary University of London, and the Ministry of Education of the Czech Republic grant LH13015. Crystal data were collected on the SSRL Beamline 12-2 through the support of the Caltech Molecular Observatory, funded by the Gordon and Betty Moore Foundation, the Sanofi-Aventis Bioengineering Research Program.|
|Official Citation:||Tryptophan-Accelerated Electron Flow Across a Protein–Protein Interface Kana Takematsu, Heather Williamson, Ana María Blanco-Rodríguez, Lucie Sokolová, Pavle Nikolovski, Jens T. Kaiser, Michael Towrie, Ian P. Clark, Antonín Vlček, Jr., Jay R. Winkler, and Harry B. Gray Journal of the American Chemical Society 2013 135 (41), 15515-15525|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Ruth Sustaita|
|Deposited On:||06 Nov 2013 00:15|
|Last Modified:||06 Nov 2013 00:15|
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