Development of Structural and Synthetic Tools to Determine the Structure of the Active Ni(I) State of Methyl-Coenzyme M Reductase:Nature's Catalyst for Methane Synthesis, Activation, and Oxidation

Abstract

Methyl-Coenzyme M Reductase (MCR) catalyzes the forward and reverse reactions of methane biosynthesis and anaerobic methane oxidation, respectively. MCR employs radical chemistry via the catalytic Ni cofactor F430, where in the low Ni(I) valence state leads to the CH3-S bond cleavage of methyl-coenzyme M (CH3SCoM) generating a methyl radical. The methyl radical abstracts a hydrogen atom from coenzyme-B (HSCoB) to form the heterodisulfide (CoBSSCoM) and the inert, high-energy molecule, methane. Due to MCR’s low redox-potential of the Ni(I)/Ni(II) states, MCR is one of the most oxygen sensitive enzymes on the planet as well as one of the hardest enzymes to harvest and purify in the active-Ni(I) state. In the Ragsdale lab, we can purify and activate MCR. Therefore, we can monitor the catalysis of this enzyme in two strategies, structural biology and synthetic radical trapping. Structural biology in MCR has been hindered due to MCR’s oxygen sensitivity, as only inhibited inactive-Ni(II) structures have been found. Due to plausible radiation damage of the CH3-S bond of CH3SCoM, only inhibitor bound coenzyme M (CoM) forms of the Ni(II) have been elucidated. Also, the radical mechanism proposed for MCR has lacked evidence due to the reactivity of the proposed methyl radical H-atom abstraction resulting in CoBSᐧ, which has never been seen. Herein, we have solved these gaps of knowledge by (1) utilizing synthetic chemistry to make radical trapping HSCoB analogues and characterize the reaction with spectroscopy and (2) elucidating the structure of active-Ni(I) using X-ray Free Electron Laser (XFEL) femtosecond serial crystallography. In a synthetic approach, we synthesized a HSCoB analogue containing an aryl thiol to delocalize the radical intermediate, thus, sharpening the thiyl radical peak via EPR. Furthermore, we employ XFEL serial crystallography to obtain what may be the first Ni(I) active structure revealing a very dynamic structure with high b-factors as well as novel Ni coordination distances. This knowledge will further the fields of methane mitigation with an understanding of the active Ni(I) MCR structure and biofuel production, and biocatalysis by further proving the radical mechanism by which this enzyme participates in CH functionalization.