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Understanding how the rate of C-H bond cleavage affects formate oxidation catalysis by a Mo-dependent formate dehydrogenase

Accepted version
Peer-reviewed

Type

Article

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Authors

Robinson, William E 
Bassegoda, Arnau 
Blaza, James N 

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 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.

Description

Keywords

Carbon Dioxide, Catalysis, Crystallography, X-Ray, Formate Dehydrogenases, Formates, Models, Molecular, Molecular Structure, Oxidation-Reduction

Journal Title

Journal of the American Chemical Society

Conference Name

Journal ISSN

0002-7863
1520-5126

Volume Title

Publisher

American Chemical Society (ACS)

Rights

All rights reserved
Sponsorship
Biotechnology and Biological Sciences Research Council (BB/J000124/1)
Engineering and Physical Sciences Research Council (EP/L015978/1)
European Research Council (682833)
Medical Research Council (MC_U105663141)
Biotechnology and Biological Sciences Research Council (BB/I026367/1)
MRC (MC_UU_00015/2)
Medical Research Council (MC_UU_00015/7)
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