Abstract
Electron and proton transfer reactions in enzymes are enigmatic and have attracted a great deal of theoretical, experimental, and practical attention. The oxidoreductases provide model systems for testing theoretical predictions, applying experimental techniques to gain insight into catalytic mechanisms, and creating industrially important bio(electro)conversion processes. Most previous and ongoing research on enzymatic electron transfer has exploited a theoretically and practically sound but limited approach that uses a series of structurally similar (“homologous”) substrates, measures reaction rate constants and Gibbs free energies of reactions, and analyses trends predicted by electron transfer theory. This approach, proposed half a century ago, is based on a hitherto unproved hypothesis that pre-exponential factors of rate constants are similar for homologous substrates. Here, we propose a novel approach to investigating electron and proton transfer catalysed by oxidoreductases. We demonstrate the validity of this new approach for elucidating the kinetics of oxidation of “non-homologous” substrates catalysed by compound II of Coprinopsis cinerea and Armoracia rusticana peroxidases. This study – using the Marcus theory – demonstrates that reactions are not only limited by electron transfer, but a proton is transferred after the electron transfer event and thus both events control the reaction rate of peroxidase-catalysed oxidation of substrates.
Highlights
The reduced form of enzyme Ered is oxidized by hydrogen peroxide
At 25 °C, the rate constants ranged from about 2 × 106 to 5 × 108 M−1 s−1 for CIP and from about 1 × 105 to 3 × 108 M−1 s−1 for horseradish peroxidase (HRP)
We demonstrated a general approach for the investigation of electron and proton transfers in oxidoreductase-catalysed reactions
Summary
The reduced form of enzyme Ered is oxidized by hydrogen peroxide. The resultant intermediate form compound I (CpdI) is reduced back to Ered via CpdII with two reduction steps (each consisting of one electron and one proton transfer). Semi-parabolic dependencies between both k1 and k2 and ΔG0 were found for both CpdI and CpdII of HRP and CIP and a variety of substrates, which led to the conclusion that both k1 and k2 are ET6–10 This conclusion appears surprising because the substrate oxidation steps II and III in Eq 1 involve both ET and proton transfer (PT). Due to the greater weight of a proton, the PT is generally much slower than an ET, suggesting that k1 and k2 are PT rather than ET limited, which calls the previous research on HP catalysis into question We believe that this contradiction arises from the use of homologous series of compounds, which limits the range of ΔG0 and makes it impossible to investigate the entire range of catalytic properties of HPs and other oxidoreductases. To provide a picture of the CpdII reduction mechanism that is as complete as possible, we measured the temperature dependencies of k2 for each reaction, calculated the quantum chemical self-exchange solvent and inner reorganisation energies (λs), estimated the change of λs for compounds in enzyme substrate complexes, and measured the kinetic isotope effects for the oxidation rate of the chosen compounds
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