2mi7

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== Publication Abstract from PubMed ==
== Publication Abstract from PubMed ==
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Catalytically essential side-chain radicals have been recognized in a growing number of redox enzymes. Here we present a novel approach to study this class of redox cofactors. Our aim is to construct a de novo protein, a radical maquette, that will provide a protein framework in which to investigate how side-chain radicals are generated, controlled, and directed toward catalysis. A tryptophan and a tyrosine radical maquette, denoted alpha(3)W(1) and alpha(3)Y(1), respectively, have been synthesized. alpha(3)W(1) and alpha(3)Y(1) contain 65 residues each and have molecular masses of 7.4 kDa. The proteins differ only in residue 32, which is the position of their single aromatic side chain. Structural characterization reveals that the proteins fold in water solution into thermodynamically stable, alpha-helical conformations with well-defined tertiary structures. The proteins are resistant to pH changes and remain stable through the physiological pH range. The aromatic residues are shown to be located within the protein interior and shielded from the bulk phase, as designed. Differential pulse voltammetry was used to examine the reduction potentials of the aromatic side chains in alpha(3)W(1) and alpha(3)Y(1) and compare them to the potentials of tryptophan and tyrosine when dissolved in water. The tryptophan and tyrosine potentials were raised considerably when moved from a solution environment to a well-ordered protein milieu. We propose that the increase in reduction potential of the aromatic residues buried within the protein, relative to the solution potentials, is due to a lack of an effective protonic contact between the aromatic residues and the bulk solution.
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Tyrosine oxidation-reduction involves proton-coupled electron transfer (PCET) and a reactive radical state. These properties are effectively controlled in enzymes that use tyrosine as a high-potential, one-electron redox cofactor. The alpha3Y model protein contains Y32, which can be reversibly oxidized and reduced in voltammetry measurements. Structural and kinetic properties of alpha3Y are presented. A solution NMR structural analysis reveals that Y32 is the most deeply buried residue in alpha3Y. Time-resolved spectroscopy using a soluble flash-quench generated [Ru(2,2'-bipyridine)3]3+ oxidant provides high-quality Y32-O* absorption spectra. The rate constant of Y32 oxidation (kPCET) is pH dependent: 1.4 x 104 M-1 s-1 (pH 5.5), 1.8 x 105 M-1 s-1 (pH 8.5), 5.4 x 103 M-1 s-1 (pD 5.5), and 4.0 x 104 M-1 s-1 (pD 8.5). kH/kD of Y32 oxidation is 2.5 +/- 0.5 and 4.5 +/- 0.9 at pH(D) 5.5 and 8.5, respectively. These pH and isotope characteristics suggest a concerted or stepwise, proton-first Y32 oxidation mechanism. The photochemical yield of Y32-O* is 28-58% versus the concentration of [Ru(2,2'-bipyridine)3]3+. Y32-O* decays slowly, t1/2 in the range of 2-10 s, at both pH 5.5 and 8.5, via radical-radical dimerization as shown by second-order kinetics and fluorescence data. The high stability of Y32-O* is discussed relative to the structural properties of the Y32 site. Finally, the static alpha3Y NMR structure cannot explain (i) how the phenolic proton released upon oxidation is removed or (ii) how two Y32-O* come together to form dityrosine. These observations suggest that the dynamic properties of the protein ensemble may play an essential role in controlling the PCET and radical decay characteristics of alpha3Y.
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De novo proteins as models of radical enzymes.,Tommos C, Skalicky JJ, Pilloud DL, Wand AJ, Dutton PL Biochemistry. 1999 Jul 20;38(29):9495-507. PMID:10413527<ref>PMID:10413527</ref>
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Photochemical Tyrosine Oxidation in the Structurally Well-Defined alphaY Protein: Proton-Coupled Electron Transfer and a Long-Lived Tyrosine Radical.,Glover SD, Jorge C, Liang L, Valentine KG, Hammarstrom L, Tommos C J Am Chem Soc. 2014 Aug 14. PMID:25121576<ref>PMID:25121576</ref>
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>

Revision as of 05:38, 27 August 2014

Solution NMR structure of alpha3Y

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