Functional Studies on Oligotropha carboxidovorans Molybdenum–Copper CO Dehydrogenase Produced in Escherichia coli

Abstract

The Mo/Cu-dependent CO dehydrogenase (CODH) from Oligotropha carboxidovorans is an enzyme that is able to catalyze both the oxidation of CO to CO and the oxidation of H to protons and electrons. Despite the close to atomic resolution structure (1.1 Å), significant uncertainties have remained with regard to the reaction mechanism of substrate oxidation at the unique Mo/Cu center, as well as the nature of intermediates formed during the catalytic cycle. So far, the investigation of the role of amino acids at the active site was hampered by the lack of a suitable expression system that allowed for detailed site-directed mutagenesis studies at the active site. Here, we report on the establishment of a functional heterologous expression system of O. carboxidovorans CODH in Escherichia coli. We characterize the purified enzyme in detail by a combination of kinetic and spectroscopic studies and show that it was purified in a form with characteristics comparable to those of the native enzyme purified from O. carboxidovorans. With this expression system in hand, we were for the first time able to generate active-site variants of this enzyme. Our work presents the basis for more detailed studies of the reaction mechanism for CO and H oxidation of Mo/Cu-dependent CODHs in the future.

A thiocarbonate sink on the enzymatic energy landscape of aerobic CO oxidation? Answers from DFT and QM/MM models of Mo Cu CO-dehydrogenases

“…Moreover, mutation of Phe390 to Tyr was reported to partially impair CO-oxidation catalysis [38]. The latter experimental evidence further illustrates the importance of going beyond the first coordination sphere when details on CO binding and processing need to be modeled.…”

Theoretical Insights into the Aerobic Hydrogenase Activity of Molybdenum–Copper CO Dehydrogenase

“…This process is favoured by the presence of a highly delocalised redox-active orbital over the Mo-µS-Cu moiety [10]. The catalytic activity of MoCu-CODH is also promoted by the presence of highly conserved amino acids in the metal second coordination sphere [11]. A glutamate residue (Glu763) positioned in proximity of the equatorial oxygen ligand of molybdenum is believed to promote deprotonation events [9,12].…”

The Challenging in silico Description of Carbon Monoxide Oxidation as Catalyzed by Molybdenum-Copper CO Dehydrogenase

“…This is due not only to the relevance of the reactions it catalyzes, but also to its resistance to atmospheric O 2 exposure—a rare feature in the case of enzymes expressing carbon monoxide dehydrogenase and hydrogenase activities (Choi et al, 2017; Gourlay et al, 2018; Groysman et al, 2018). Notably, the recent establishment of a functional heterologous expression system for the MoCu-CODH enzyme (Kaufmann et al, 2018) together with developments in the computational chemistry field will hopefully boost the positive feedback among biochemical, biomimetic and quantum chemical studies, opening new perspectives for a deeper understanding of this interesting metalloenzyme.…”