Reversible voltammograms and a voltammetry half-wave potential versus solution pH diagram are described for a protein tyrosine radical. This work required a de novo designed tyrosine-radical protein displaying a unique combination of structural and electrochemical properties. The α 3 Y protein is structurally stable across a broad pH range. The redox-active tyrosine Y32 resides in a desolvated and well-structured environment. Y32 gives rise to reversible square-wave and differential pulse voltammograms at alkaline pH. The formal potential of the Y32-O • ∕Y32-OH redox couple is determined to 918 AE 2 mV versus the normal hydrogen electrode at pH 8.40 AE 0.01. The observation that Y32 gives rise to fully reversible voltammograms translates into an estimated lifetime of ≥30 ms for the Y32-O • state. This illustrates the range of tyrosineradical stabilization that a structured protein can offer. Y32 gives rise to quasireversible square-wave and differential pulse voltammograms at acidic pH. These voltammograms represent the Y32 species at the upper edge of the quasirevesible range. The squarewave net potential closely approximates the formal potential of the Y32-O • ∕Y32-OH redox couple to 1,070 AE 1 mV versus the normal hydrogen electrode at pH 5.52 AE 0.01. The differential pulse voltammetry half-wave potential of the Y32-O • ∕Y32-OH redox pair is measured between pH 4.7 and 9.0. These results are described and analyzed.protein voltammetry | proton-coupled electron transfer P roton-coupled electron transfer (PCET) represents a fundamental component of catalytic and long range radical-transfer processes involving tyrosine radicals (1-4). The thermodynamic and kinetic effects of coupled tyrosine oxidation/reduction and acid/base chemistry at the protein matrix are likely to play a key role in activating the aromatic residue for redox chemistry and for fine tuning its functional properties. This prediction is largely based on three sets of experimental observations that include the thermodynamic properties of aqueous tyrosine (5-7), the hydrogen-bonding properties of the kinetically well-characterized Y Z and Y D radicals in photosystem II (PSII) (7-10), and data derived from small-molecule model systems designed to delineate the PCET processes associated with tyrosine/phenol oxidation (4, 11-16). Tyrosine redox chemistry involves three redox couples and two pK a values. The cation Y-OH •þ ∕Y-OH redox pair exists at pH below the pK a of the oxidized state (pK oY ) whereas the tyrosinate Y-O • ∕Y-O − couple is observed at pH above the pK a of the reduced state (pK rY ). The neutral tyrosine Y-O • ∕Y-OH redox couple operates in the pK oY < pH < pK rY region. With pK oY and pK rY values of −2 and 10 for aqueous tyrosine, respectively (5), Y-O • ∕Y-OH is predicted to be the dominant protein redox pair. Consequently, long-range electron transfer involving protein tyrosine residues is coupled to shortrange proton motions between the radical species and the protein matrix. Indeed, studies on Y Z and Y D suggested early on t...
This report describes a model protein specifically tailored to electrochemically study the reduction potential of protein tyrosine radicals as a function of pH. The model system is based on the 67-residue α3Y three-helix bundle. α3Y contains a single buried tyrosine at position 32 and displays structural properties inherent to a protein. The present report presents differential pulse voltammograms obtained from α3Y at both acidic (pH 5.4) and alkaline (pH 8.3) conditions. The observed Faradaic response is uniquely associated with Y32, as shown by site-directed mutagenesis. This is the first time voltammetry is successfully applied to detect a redox-active tyrosine residing in a structured protein environment. Tyrosine is a proton coupled electron-transfer cofactor making voltammetry-based pH titrations a central experimental approach. A second set of experiments was performed to demonstrate that pH-dependent studies can be conducted on the redox-active tyrosine without introducing large-scale structural changes in the protein scaffold. α3Y was re-engineered with the specific aim to place the imidazole group of a histidine close to the Y32 phenol ring. α3Y-K29H and α3Y-K36H each contain a histidine residue which protonation perturbs the fluorescence of Y32. We show that these variants are stable and well-folded proteins whose helical content, tertiary structure, solution aggregation state and solvent-sequestered position of Y32 remain pH insensitive across a range of at least 3–4 pH units. These results confirm that the local environment of Y32 can be altered and the resulting radical site studied by voltammetry over a broad pH range without interference from long-range structural effects.
Amino-acid radical enzymes are often highly complex structures containing multiple protein subunits and cofactors. These properties have in many cases hampered the detailed characterization of their amino-acid redox cofactors. To address this problem, a range of approaches has recently been developed in which a common strategy is to reduce the complexity of the radical-containing system. This work will be reviewed and it includes the light-induced generation of aromatic radicals in small-molecule and peptide systems. Natural redox proteins, including the blue copper protein azurin and a bacterial photosynthetic reaction center, have been engineered to introduce amino-acid radical chemistry. The redesign strategies to achieve this remarkable change in the properties of these proteins will be described. An additional approach to gain insights into the properties of amino-acid radicals is to synthesize de novo designed model proteins in which the redox chemistry of these species can be studied. Here we describe the design, synthesis and characteristics of monomeric three-helix bundle and four-helix bundle proteins designed to study the redox chemistry of tryptophan and tyrosine. This work demonstrates that de novo protein design combined with structural, electrochemical and quantum chemical analyses can provide detailed information on how the protein matrix tunes the thermodynamic properties of tryptophan.
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