Abstract
Metal-dependent formate dehydrogenases (FDHs) catalyze the reversible conversion of formate into CO2, a proton, and two electrons. Kinetic studies of FDHs provide key insights into their mechanism of catalysis, relevant as a guide for the development of efficient electrocatalysts for formate oxidation as well as for CO2 capture and utilization. Here, we identify and explain the kinetic isotope effect (KIE) observed for the oxidation of formate and deuterioformate by the Mo-containing FDH from Escherichia coli using three different techniques: steady-state solution kinetic assays, protein film electrochemistry (PFE), and pre-steady-state stopped-flow methods. For each technique, the Mo center of FDH is reoxidized at a different rate following formate oxidation, significantly affecting the observed kinetic behavior and providing three different viewpoints on the KIE. Steady-state turnover in solution, using an artificial electron acceptor, is kinetically limited by diffusional intermolecular electron transfer, masking the KIE. In contrast, interfacial electron transfer in PFE is fast, lifting the electron-transfer rate limitation and manifesting a KIE of 2.44. Pre-steady-state analyses using stopped-flow spectroscopy revealed a KIE of 3 that can be assigned to the C–H bond cleavage step during formate oxidation. We formalize our understanding of FDH catalysis by fitting all the data to a single kinetic model, recreating the condition-dependent shift in rate-limitation of FDH catalysis between active-site chemical catalysis and regenerative electron transfer. Furthermore, our model predicts the steady-state and time-dependent concentrations of catalytic intermediates, providing a valuable framework for the design of future mechanistic experiments.
Highlights
Metal-dependent formate dehydrogenases (FDHs) are paradigm electrocatalysts for the interconversion of CO2 and formate,[1,2] and play a versatile range of roles in biological systems.[3]
When fitted to the data, our model provides a conceptual framework for the rationalization of FDH catalysis in terms of both the rate of active-site catalysis and the rate of electron transfer to terminal electron acceptors
Our unifying model for FDH catalysis shows how the distinct kinetic behaviors of the solution assay kinetics, protein film electrochemistry (PFE), and stopped-flow methods can be rationalized by considering the rates of chemical catalysis and intermolecular/interfacial electron transfer, and provides predictions to guide future experimental designs
Summary
Metal-dependent formate dehydrogenases (FDHs) are paradigm electrocatalysts for the interconversion of CO2 and formate,[1,2] and play a versatile range of roles in biological systems.[3]. The stopped-flow experiment explores stoichiometric formate oxidation and terminates here (with an irreversible intramolecular kox step that combines preceding irreversible CO2 dissociation to form with the k3),[33−35] whereas in solution assays and PFE a PCET step regenerates the oxidized Mo(VI) S state to sustain the catalytic cycle In the latter two cases, electron transfer between the active site and the outside is considered as a single step, without taking into account intramolecular transfer to the [4Fe-4S]2+ cluster, which is considered fast (estimated[44] as 4 × 106 s−1, much faster than turnover, from the short ∼7 Å distance[21,45] and a ΔE of −0.3 V, in excess of the ΔE estimated for the truncated FDH from C. necator[32]). The control over the distribution of FDH states enabled by the electrode potential, and the ability to dynamically change conditions (such as varying formate concentration by titration as reported here), should allow the population of key intermediates to be promoted, increasing the ease with which they may be spectroscopically characterized by these techniques
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