Reversible voltammograms and a Pourbaix diagram for a protein tyrosine radical.
ABSTRACT: 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 ?(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 ± 2 mV versus the normal hydrogen electrode at pH 8.40 ± 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 tyrosine-radical 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 square-wave net potential closely approximates the formal potential of the Y32-O(•)/Y32-OH redox couple to 1,070 ± 1 mV versus the normal hydrogen electrode at pH 5.52 ± 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.
Project description: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 ?(3)Y three-helix bundle. ?(3)Y 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 ?(3)Y at both acidic (pH 5.6) 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. ?(3)Y was re-engineered with the specific aim to place the imidazole group of a histidine close to the Y32 phenol ring. ?(3)Y-K29H and ?(3)Y-K36H each contain a histidine residue whose 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.
Project description:The reversible Y-O•/Y-OH redox properties of the ?3Y model protein allow access to the electrochemical and thermodynamic properties of 3,5-difluorotyrosine. The unnatural amino acid has been incorporated at position 32, the dedicated radical site in ?3Y, by in vivo nonsense codon suppression. Incorporation of 3,5-difluorotyrosine gives rise to very minor structural changes in the protein scaffold at pH values below the apparent pK (8.0±0.1) of the unnatural residue. Square-wave voltammetry on ?3(3,5)F2Y provides an E°'(Y-O•/Y-OH) of 1026±4 mV versus the normal hydrogen electrode (pH 5.70±0.02) and shows that the fluoro substitutions lower the E°' by -30±3 mV. These results illustrate the utility of combining the optimized ?3Y tyrosine radical system with in vivo nonsense codon suppression to obtain the formal reduction potential of an unnatural aromatic residue residing within a well-structured protein. It is further observed that the protein E°' values differ significantly from peak potentials derived from irreversible voltammograms of the corresponding aqueous species. This is notable because solution potentials have been the main thermodynamic data available for amino acid radicals. The findings in this paper are discussed relative to recent mechanistic studies of the multistep radical-transfer process in Escherichia coli ribonucleotide reductase site-specifically labeled with unnatural tyrosine residues.
Project description:Proton-coupled electron transfer (PCET) from tyrosine produces a neutral tyrosyl radical (Y•) that is vital to many catalytic redox reactions. To better understand how the protein environment influences the PCET properties of tyrosine, we have studied the radical formation behavior of Y32 in the ?3Y model protein. The previously solved ?3Y solution NMR structure shows that Y32 is sequestered ?7.7 ± 0.3 Å below the protein surface without any primary proton acceptors nearby. Here we present transient absorption kinetic data and molecular dynamics (MD) simulations to resolve the PCET mechanism associated with Y32 oxidation. Y32• was generated in a bimolecular reaction with [Ru(bpy)3]3+ formed by flash photolysis. At pH > 8, the rate constant of Y32• formation (kPCET) increases by one order of magnitude per pH unit, corresponding to a proton-first mechanism via tyrosinate (PTET). At lower pH < 7.5, the pH dependence is weak and shows a previously measured KIE ? 2.5, which best fits a concerted mechanism. kPCET is independent of phosphate buffer concentration at pH 6.5. This provides clear evidence that phosphate buffer is not the primary proton acceptor. MD simulations show that one to two water molecules can enter the hydrophobic cavity of ?3Y and hydrogen bond to Y32, as well as the possibility of hydrogen-bonding interactions between Y32 and E13, through structural fluctuations that reorient surrounding side chains. Our results illustrate how protein conformational motions can influence the redox reactivity of a tyrosine residue and how PCET mechanisms can be tuned by changing the pH even when the PCET occurs within the interior of a protein.
Project description: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 ?3Y model protein contains Y32, which can be reversibly oxidized and reduced in voltammetry measurements. Structural and kinetic properties of ?3Y are presented. A solution NMR structural analysis reveals that Y32 is the most deeply buried residue in ?3Y. 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 × 10(4) M(-1) s(-1) (pH 5.5), 1.8 × 10(5) M(-1) s(-1) (pH 8.5), 5.4 × 10(3) M(-1) s(-1) (pD 5.5), and 4.0 × 10(4) M(-1) s(-1) (pD 8.5). k(H)/k(D) 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 ?3Y 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 ?3Y.
Project description:Protein-based "hole" hopping typically involves spatially arranged redox-active tryptophan or tyrosine residues. Thermodynamic information is scarce for this type of process. The well-structured ?3W model protein was studied by protein film square wave voltammetry and transient absorption spectroscopy to obtain a comprehensive thermodynamic and kinetic description of a buried tryptophan residue. A Pourbaix diagram, correlating thermodynamic potentials (E°') with pH, is reported for W32 in ?3W and compared to equivalent data recently presented for Y32 in ?3Y ( Ravichandran , K. R. ; Zong , A. B. ; Taguchi , A. T. ; Nocera , D. G. ; Stubbe , J. ; Tommos , C. J. Am. Chem. Soc. 2017 , 139 , 2994 - 3004 ). The ?3W Pourbaix diagram displays a pKOX of 3.4, a E°'(W32(N•+/NH)) of 1293 mV, and a E°'(W32(N•/NH); pH 7.0) of 1095 ± 4 mV versus the normal hydrogen electrode. W32(N•/NH) is 109 ± 4 mV more oxidizing than Y32(O•/OH) at pH 5.4-10. In the voltammetry measurements, W32 oxidation-reduction occurs on a time scale of about 4 ms and is coupled to the release and subsequent uptake of one full proton to and from bulk. Kinetic analysis further shows that W32 oxidation likely involves pre-equilibrium electron transfer followed by proton transfer to a water or small water cluster as the primary acceptor. A well-resolved absorption spectrum of W32• is presented, and analysis of decay kinetics show that W32• persists ?104 times longer than aqueous W• due to significant stabilization by the protein. The redox characteristics of W32 and Y32 are discussed relative to global and local protein properties.
Project description:3-Aminotyrosine (NH2Y) has been a useful probe to study the role of redox active tyrosines in enzymes. This report describes properties of NH2Y of key importance for its application in mechanistic studies. By combining the tRNA/NH2Y-RS suppression technology with a model protein tailored for amino acid redox studies (?3X, X = NH2Y), the formal reduction potential of NH2Y32(O•/OH) ( E°' = 395 ± 7 mV at pH 7.08 ± 0.05) could be determined using protein film voltammetry. We find that the ? E°' between NH2Y32(O•/OH) and Y32(O•/OH) when measured under reversible conditions is ?300-400 mV larger than earlier estimates based on irreversible voltammograms obtained on aqueous NH2Y and Y. We have also generated D6-NH2Y731-?2 of ribonucleotide reductase (RNR), which when incubated with ?2/CDP/ATP generates the D6-NH2Y731•-?2/?2 complex. By multifrequency electron paramagnetic resonance (35, 94, and 263 GHz) and 34 GHz 1H ENDOR spectroscopies, we determined the hyperfine coupling (hfc) constants of the amino protons that establish RNH2• planarity and thus minimal perturbation of the reduction potential by the protein environment. The amount of Y in the isolated NH2Y-RNR incorporated by infidelity of the tRNA/NH2Y-RS pair was determined by a generally useful LC-MS method. This information is essential to the utility of this NH2Y probe to study any protein of interest and is employed to address our previously reported activity associated with NH2Y-substituted RNRs.
Project description:Redox-active tyrosines (Ys) play essential roles in enzymes involved in primary metabolism including energy transduction and deoxynucleotide production catalyzed by ribonucleotide reductases (RNRs). Thermodynamic characterization of Ys in solution and in proteins remains a challenge due to the high reduction potentials involved and the reactive nature of the radical state. The structurally characterized ?3Y model protein has allowed the first determination of formal reduction potentials (E°') for a Y residing within a protein (Berry, B. W.; Mart??nez-Rivera, M. C.; Tommos, C. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 9739-9743). Using Schultz's technology, a series of fluorotyrosines (FnY, n = 2 or 3) was site-specifically incorporated into ?3Y. The global protein properties of the resulting ?3(3,5)F2Y, ?3(2,3,5)F3Y, ?3(2,3)F2Y and ?3(2,3,6)F3Y variants are essentially identical to those of ?3Y. A protein film square-wave voltammetry approach was developed to successfully obtain reversible voltammograms and E°'s of the very high-potential ?3FnY proteins. E°'(pH 5.5; ?3FnY(O•/OH)) spans a range of 1040 ± 3 mV to 1200 ± 3 mV versus the normal hydrogen electrode. This is comparable to the potentials of the most oxidizing redox cofactors in nature. The FnY analogues, and the ability to site-specifically incorporate them into any protein of interest, provide new tools for mechanistic studies on redox-active Ys in proteins and on functional and aberrant hole-transfer reactions in metallo-enzymes. The former application is illustrated here by using the determined ?3FnY ?E°'s to model the thermodynamics of radical-transfer reactions in FnY-RNRs and to experimentally test and support the key prediction made.
Project description:Successive one-electron reductions of molecular oxygen yield the superoxide radical (O2-) H2O2, the hydroxyl radical (OH) and water. Redox potentials at pH 7 for one-, two- and four-electron couples involving these states are presented as a potential diagram. The significance of each of these potentials is explained. The complete potential diagram enables complex systems to be rationalized, such as production of OH by H2O2 plus Fe3+.
Project description:2-Mercaptophenol-??C serves as a biomimetic model for enzymes that use tyrosine residues in redox catalysis and multistep electron transfer. This model protein was tailored for electrochemical studies of phenol oxidation and reduction with specific emphasis on the redox-driven protonic reactions occurring at the phenol oxygen. This protein contains a covalently modified 2-mercaptophenol-cysteine residue. The radical site and the phenol compound were specifically chosen to bury the phenol OH group inside the protein. A solution nuclear magnetic resonance structural analysis (i) demonstrates that the synthetic 2-mercaptophenol-??C model protein behaves structurally as a natural protein, (ii) confirms the design of the radical site, (iii) reveals that the ligated phenol forms an interhelical hydrogen bond to glutamate 13 (phenol oxygen-carboxyl oxygen distance of 3.2 ± 0.5 Å), and (iv) suggests a proton-transfer pathway from the buried phenol OH (average solvent accessible surface area of 3 ± 5%) via glutamate 13 (average solvent accessible surface area of the carboxyl oxygens of 37 ± 18%) to the bulk solvent. A square-wave voltammetry analysis of 2-mercaptophenol-??C further demonstrates that (v) the phenol oxidation-reduction cycle is reversible, (vi) formal phenol reduction potentials can be obtained, and (vii) the phenol-O(•) state is long-lived with an estimated lifetime of ?180 millisecond. These properties make 2-mercaptophenol-??C a unique system for characterizing phenol-based proton-coupled electron transfer in a low-dielectric and structured protein environment.
Project description:Arterial stiffening may lead to hypertension, greater left ventricular after-load and adverse clinical outcomes. The underlying mechanisms influencing arterial elasticity may involve oxidative injury to the vessel wall. We sought to examine the relationship between novel markers of oxidative stress and arterial elastic properties in healthy humans.We studied 169 subjects (mean age 42.6 ± 14 years, 51.6% male) free of traditional cardiovascular risk factors. Indices of arterial stiffness and wave reflections measured included carotid-femoral Pulse Wave Velocity (PWV), Augmentation Index (Aix) and Pulse Pressure Amplification (PPA). Non-free radical oxidative stress was assessed as plasma oxidized and reduced amino-thiol levels (cysteine/cystine, glutathione/GSSG) and their ratios (redox potentials), and free radical oxidative stress as derivatives of reactive oxygen metabolites (dROMs). Inflammation was assessed as hsCRP and interleukin-6 levels. The non-free radical marker of oxidative stress, cystine was significantly correlated with all arterial indices; PWV (r=0.38, p<0.001), Aix (r=0.35, p<0.001) and PPA (r=-0.30, p<0.001). Its redox potential, was also associated with PWV (r=0.22, p=0.01), while the free radical marker of oxidative stress dROMS was associated with Aix (r=0.25, p<0.01). After multivariate adjustment for age, gender, arterial pressure, height, weight, heart rate and CRP, of these oxidative stress markers, only cystine remained independently associated with PWV (p=0.03), Aix (p=0.01) and PPA (p=0.05).In healthy subjects without confounding risk factors or significant systemic inflammation, a high cystine level, reflecting extracellular oxidant burden, is associated with increased arterial stiffness and wave reflections. This has implications for understanding the role of oxidant burden in pre-clinical vascular dysfunction.