Theoretical analysis of the unusual temperature dependence of the kinetic isotope effect in quinol oxidation.
ABSTRACT: In this paper we present theoretical calculations on model biomimetic systems for quinol oxidation. In these model systems, an excited-state [Ru(bpy)(2)(pbim)](+) complex (bpy = 2,2'-dipyridyl, pbim = 2-(2-pyridyl)benzimidazolate) oxidizes a ubiquinol or plastoquinol analogue in acetonitrile. The charge transfer reaction occurs via a proton-coupled electron transfer (PCET) mechanism, in which an electron is transferred from the quinol to the Ru and a proton is transferred from the quinol to the pbim(-) ligand. The experimentally measured average kinetic isotope effects (KIEs) at 296 K are 1.87 and 3.45 for the ubiquinol and plastoquinol analogues, respectively, and the KIE decreases with temperature for plastoquinol but increases with temperature for ubiquinol. The present calculations provide a possible explanation for the differences in magnitudes and temperature dependences of the KIEs for the two systems and, in particular, an explanation for the unusual inverse temperature dependence of the KIE for the ubiquinol analogue. These calculations are based on a general theoretical formulation for PCET reactions that includes quantum mechanical effects of the electrons and transferring proton, as well as the solvent reorganization and proton donor-acceptor motion. The physical properties of the system that enable the inverse temperature dependence of the KIE are a stiff hydrogen bond, which corresponds to a high-frequency proton donor-acceptor motion, and small inner-sphere and solvent reorganization energies. The inverse temperature dependence of the KIE may be observed if the 0/0 pair of reactant/product vibronic states is in the inverted Marcus region, while the 0/1 pair of reactant/product vibronic states is in the normal Marcus region and is the dominant contributor to the overall rate. In this case, the free energy barrier for the dominant transition is lower for deuterium than for hydrogen because of the smaller splittings between the vibronic energy levels for deuterium, and the KIE increases with increasing temperature. The temperature dependence of the KIE is found to be very sensitive to the interplay among the driving force, the reorganization energy, and the vibronic coupling in this regime.
Project description:The rate constants for typical concerted proton-coupled electron transfer (PCET) reactions depend on the vibronic coupling between the diabatic reactant and product states. The form of the vibronic coupling is different for electronically adiabatic and nonadiabatic reactions, which are associated with hydrogen atom transfer (HAT) and electron-proton transfer (EPT) mechanisms, respectively. Most PCET rate constant expressions rely on the Condon approximation, which assumes that the vibronic coupling is independent of the nuclear coordinates of the solute and the solvent or protein. Herein we test the Condon approximation for PCET vibronic couplings. The dependence of the vibronic coupling on molecular geometry is investigated for an open and a stacked transition state geometry of the phenoxyl-phenol self-exchange reaction. The calculations indicate that the open geometry is electronically nonadiabatic, corresponding to an EPT mechanism that involves significant electronic charge redistribution, while the stacked geometry is predominantly electronically adiabatic, corresponding primarily to an HAT mechanism. Consequently, a single molecular system can exhibit both HAT and EPT character. The dependence of the vibronic coupling on the solvent or protein configuration is examined for the soybean lipoxygenase enzyme. The calculations indicate that this PCET reaction is electronically nonadiabatic with a vibronic coupling that does not depend significantly on the protein environment. Thus, the Condon approximation is shown to be valid for the solvent and protein nuclear coordinates but invalid for the solute nuclear coordinates in certain PCET systems. These results have significant implications for the calculation of rate constants, as well as mechanistic interpretations, of PCET reactions.
Project description:Proton-coupled electron transfer (PCET) plays a vital role in many biological and chemical processes. PCET rate constant expressions are available for various well-defined regimes, and determining which expression is appropriate for a given system is essential for reliable modeling. Quantitative diagnostics have been devised to characterize the vibronic nonadiabaticity between the electron-proton quantum subsystem and the classical nuclei, as well as the electron-proton nonadiabaticity between the electrons and proton(s) within the quantum subsystem. Herein these diagnostics are applied to a model of the active site of the enzyme soybean lipoxygenase, which catalyzes a PCET reaction that exhibits unusually high deuterium kinetic isotope effects at room temperature. Both semiclassical and electronic charge density diagnostics illustrate vibronic and electron-proton nonadiabaticity for this PCET reaction, supporting the use of the Golden rule nonadiabatic rate constant expression with a specific form of the vibronic coupling. This type of characterization will be useful for theoretical modeling of a broad range of PCET processes.
Project description:Developing new strategies to activate and cleave C-H bonds is important for a broad range of applications. Recently a new approach for C-H bond activation using multi-site concerted proton-coupled electron transfer (PCET) involving intermolecular electron transfer to an oxidant coupled to intramolecular proton transfer was reported. For a series of oxidants reacting with 2-(9 H-fluoren-9-yl)benzoate, experimental studies revealed an atypical Brønsted ?, defined as the slope of the logarithm of the PCET rate constant versus the logarithm of the equilibrium constant or the scaled driving force. Herein this reaction is modeled with a vibronically nonadiabatic PCET theory. Hydrogen tunneling, thermal sampling of the proton donor-acceptor mode, solute and solvent reorganization, and contributions from excited vibronic states are found to play important roles. The calculations qualitatively reproduce the experimental observation of a Brønsted ? significantly less than 0.5 and explain this shallow slope in terms of exoergic processes between pairs of electron-proton vibronic states. These fundamental mechanistic insights may guide the design of more effective strategies for C-H bond activation and cleavage.
Project description:The driving force dependence of the rate constants for nonadiabatic electron transfer (ET), proton transfer (PT), and proton-coupled electron transfer (PCET) reactions is examined. Inverted region behavior, where the rate constant decreases as the reaction becomes more exoergic (i.e., as DeltaG(0) becomes more negative), has been observed experimentally for ET and PT. This behavior was predicted theoretically for ET but is not well understood for PT and PCET. The objective of this Letter is to predict the experimental conditions that could lead to observation of inverted region behavior for PT and PCET. The driving force dependence of the rate constant is qualitatively different for PT and PCET than for ET because of the high proton vibrational frequency and substantial shift between the reactant and product proton vibrational wave functions. As a result, inverted region behavior is predicted to be experimentally inaccessible for PT and PCET if only the driving force is varied. This behavior may be observed for PT over a limited range of rates and driving forces if the solvent reorganization energy is low enough to cause observable oscillations. Moreover, this behavior may be observed for PT or PCET if the proton donor-acceptor distance increases as DeltaG(0) becomes more negative. Thus, a plausible explanation for experimentally observed inverted region behavior for PT or PCET is that varying the driving force also impacts other properties of the system, such as the proton donor-acceptor distance.
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:The suitability of ubiquinol(1) and duroquinol as pulse reductants for initiating respirationdriven proton translocation by aerobic ox heart mitochondria was investigated. At 25 degrees C the V(max.) for oxidation was close to 280nmol of quinol oxidized/min per mg of protein, and the K(m) values were 8mum for ubiquinol(1) and 28mum for duroquinol. Pulses of ubiquinol(1) and duroquinol were rapidly and completely oxidized by aerobic mitochondria with a simultaneous acidification of the suspending medium as detected with a glass electrode. The -->H(+)/2e(-) ratios (Mitchell, 1966) calculated from the observed extent of acidification and the amount of quinol added were 3.62 for ubiquinol(1) and 2.98 for duroquinol. These values are underestimates of the true value owing to proton back-flow across the membrane. An analogue computer model was used to correct the observed extent of respirationdriven acidification for proton back-flow. The corrected -->H(+)/2e(-) values were 4.01 for ubiquinol and 3.86 for duroquinol oxidation. Attempts to measure the rate of proton translocation with a pH-measuring system with a response time of 0.4s were not entirely satisfactory, owing to the relative slowness of the electrode response. Nevertheless the maximal rate of proton generation during ubiquinol(1) oxidation was about 1200ng-ions of H(+)/min per mg of mitochondrial protein. It is concluded, contrarily to Chance & Mela (1967), that mitochondria exhibit a proton-translocating ubiquinol oxidase activity with a -->H(+)/2e(-) ratio of 4.0.
Project description:Tyramine ?-monooxygenase (T?M) belongs to a family of physiologically important dinuclear copper monooxygenases that function with a solvent-exposed active site. To accomplish each enzymatic turnover, an electron transfer (ET) must occur between two solvent-separated copper centers. In wild-type T?M, this event is too fast to be rate limiting. However, we have recently shown [Osborne, R. L.; et al. Biochemistry 2013, 52, 1179] that the Tyr216Ala variant of T?M leads to rate-limiting ET. In this study, we present a pH-rate profile study of Tyr216Ala, together with deuterium oxide solvent kinetic isotope effects (KIEs). A solvent KIE of 2 on kcat is found in a region where kcat is pH/pD independent. As a control, the variant Tyr216Trp, for which ET is not rate determining, displays a solvent KIE of unity. We conclude, therefore, that the observed solvent KIE arises from the rate-limiting ET step in the Tyr216Ala variant, and show how small solvent KIEs (ca. 2) can be fully accommodated from equilibrium effects within the Marcus equation. To gain insight into the role of the enzyme in the long-range ET step, a temperature dependence study was also pursued. The small enthalpic barrier of ET (Ea = 3.6 kcal/mol) implicates a significant entropic barrier, which is attributed to the requirement for extensive rearrangement of the inter-copper environment during PCET catalyzed by the Tyr216Ala variant. The data lead to the proposal of a distinct inter-domain pathway for PCET in the dinuclear copper monooxygenases.
Project description:Three phenols with pendant, hydrogen-bonded bases (HOAr-B) have been oxidized in MeCN with various one-electron oxidants. The bases are a primary amine (-CPh(2)NH(2)), an imidazole, and a pyridine. The product of chemical and quasi-reversible electrochemical oxidations in each case is the phenoxyl radical in which the phenolic proton has transferred to the base, (*)OAr-BH(+), a proton-coupled electron transfer (PCET) process. The redox potentials for these oxidations are lower than for other phenols, predominately from the driving force for proton movement. One-electron oxidation of the phenols occurs by a concerted proton-electron transfer (CPET) mechanism, based on thermochemical arguments, isotope effects, and DeltaDeltaG(++)/DeltaDeltaG degrees . The data rule out stepwise paths involving initial electron transfer to form the phenol radical cations [(*)(+)HOAr-B] or initial proton transfer to give the zwitterions [(-)OAr-BH(+)]. The rate constant for heterogeneous electron transfer from HOAr-NH(2) to a platinum electrode has been derived from electrochemical measurements. For oxidations of HOAr-NH(2), the dependence of the solution rate constants on driving force, on temperature, and on the nature of the oxidant, and the correspondence between the homogeneous and heterogeneous rate constants, are all consistent with the application of adiabatic Marcus theory. The CPET reorganization energies, lambda = 23-56 kcal mol(-)(1), are large in comparison with those for electron transfer reactions of aromatic compounds. The reactions are not highly non-adiabatic, based on minimum values of H(rp) derived from the temperature dependence of the rate constants. These are among the first detailed analyses of CPET reactions where the proton and electron move to different sites.
Project description:In view of the considerably high activation energy barrier of the O-O bond formation photocatalytic step in water oxidation, it is essential to understand if and how nonadiabatic factors can accelerate the proton-coupled electron transfer (PCET) rate in this process to find rational design strategies facilitating this step. Herein, constrained ab initio molecular dynamics simulations are performed to investigate this rate-limiting step in a series of catalyst-dye supramolecular complexes functionalized with different alkyl groups on the catalyst component. These structural modifications lead to tunable thermodynamic driving forces, PCET rates, and vibronic coupling with specific resonant torsional modes. These results reveal that such resonant coupling between electronic and nuclear motions contributes to crossing catalytic barriers in PCET reactions by enabling semiclassical coherent conversion of a reactant into a product. Our results provide insight on how to engineer efficient catalyst-dye supramolecular complexes by functionalization with steric substituents for high-performance dye-sensitized photoelectrochemical cells.
Project description:Ubiquinol-cytochrome c reductase (Complex III), cytochrome c and cytochrome c oxidase can be combined to reconstitute antimycin-sensitive ubiquinol oxidase activity. In 25 mM-acetate/Tris, pH 7.8, cytochrome c binds at high-affinity sites (KD = 0.1 microM) and low-affinity sites (KD approx. 10 microM). Quinol oxidase activity is 50% of maximal activity when cytochrome c is bound to only 25% of the high affinity sites. The other 50% of activity seems to be due to cytochrome c bound at low-affinity sites. Reconstitution in the presence of soya-bean phospholipids prevents aggregation of cytochrome c oxidase and gives rise to much higher rates of quinol oxidase. The cytochrome c dependence was unaltered. Antimycin curves have the same shape regardless of lipid/protein ratio, Complex III/cytochrome c oxidase ratio or cytochrome c concentration. Proposals on the nature of the interaction between Complex III, cytochrome c and cytochrome c oxidase are considered in the light of these results.