The radical mechanism of biological methane synthesis by methyl-coenzyme M reductase by Thanyaporn Wongnate, Dariusz Sliwa, Bojana Ginovska, Dayle Smith, Matthew W. Wolf, Nicolai Lehnert, Simone Raugei, and Stephen W. Ragsdale Science Volume 352(6288):953-958 May 20, 2016 Published by AAAS

Fig. 1 Initial steps in three mechanisms of MCR catalysis. Initial steps in three mechanisms of MCR catalysis. Mechanism I involves nucleophilic attack of Ni(I)-MCRred1 on the methyl group of methyl-SCoM to generate a methyl-Ni(III) intermediate (34). This mechanism is similar to that of B12-dependent methyltransferases (48), which generate a methyl-cob(III)alamin intermediate. In mechanism II, Ni(I) attack on the sulfur atom of methyl-SCoM promotes the homolytic cleavage of the methyl-sulfur bond to produce a methyl radical (•CH3) and a Ni(II)-thiolate. Mechanism III involves nucleophilic attack of Ni(I) on the sulfur of methyl-SCoM to form a highly reactive methyl anion and Ni(III)-SCoM (MCRox1). Thanyaporn Wongnate et al. Science 2016;352:953-958 Published by AAAS

Fig. 2 Rapid kinetic studies of the reaction of the MCR:methyl-SCoM complex with CoB6SH. Rapid kinetic studies of the reaction of the MCR:methyl-SCoM complex with CoB6SH. (A) Stopped-flow. Kinetic traces of the reaction of a premixed solution containing MCRred1 (20 μM, after mixing) and methyl-SCoM (20 μM, after mixing) with CoB6SH (500 μM, after mixing) in 50 mM Tris-HCl, pH 7.6. The reactions were performed under anaerobic conditions using the stopped-flow spectrophotometer at 18°C and monitored by following the decay of Ni(I) at 385 nm (blue line) and the formation of Ni(II)/Ni(III) at 420 nm (red line). The reaction showed monophasic kinetics with a rate constant of 0.35 ± 0.01 s−1. (B) Rapid chemical-quench. Reactions of a premixed solution containing equimolar MCRred1 (20 μM, after mixing) and [14C]methyl-SCoM (20 μM, after mixing) with CoB6SH (500 μM, after mixing) were quenched with 0.2 M perchloric acid at various times using the rapid chemical-quench apparatus. Volatile methane product was lost from the solution, and the percentage conversion of [14C]methyl-SCoM was determined by comparing the remaining concentration of [14C]methyl-SCoM to the initial concentration. Plotting the percentage conversion versus time yielded a single-exponential curve with a rate constant of 0.31 ± 0.04 s−1. The vertical brackets at each point indicate the standard deviation of the measurement. (C and D) RFQ EPR. A solution containing MCRred1 (48 μM) and methyl-SCoM (600 μM) was reacted with CoB6SH (120 μM) and freeze-quenched at various times using an RFQ apparatus. Representative time-dependent EPR spectra are shown on (C). The inset shows the g ~ 2.2 region near the S-shaped feature of MCRox1. The percentage decay of MCRred1 (blue) and formation of MCRox1 (red) were determined by comparing their doubly integrated signal intensities at various quenching times to their initial intensities. The data were plotted and fit to single-exponential curves in (D). The MCRred1 signal decayed by 90% during the first phase of the reaction with a rate constant of 0.53 ± 0.25 s−1, whereas the MCRox1-like signal increased by ~3% (relative to the initial MCRred1), with a rate constant of 0.69 ± 0.24 s−1. A radical formed with a rate constant of 0.52 ± 0.32 s−1 and reached ~7% of the initial MCRred1 (see fig. S1). A vertical line at each point indicates the standard deviation of each measurement. Thanyaporn Wongnate et al. Science 2016;352:953-958 Published by AAAS

Fig. 3 MCD studies of the reaction of the MCR:methyl-SCoM complex with CoB6SH. MCD studies of the reaction of the MCR:methyl-SCoM complex with CoB6SH. MCD spectra were taken at 2 K of MCRred1 before and after the reaction with methyl-SCoM and CoB6SH. Samples were prepared in 50 mM GPT buffer [(50 mM glycine, 50 mM phosphate, and 50 mM Tris), pH 7.6, containing 0.05 mM Ti(III) citrate] with 50% glycerol for MCRred1 and 73% glycerol for the reaction with CoB6SH. Thanyaporn Wongnate et al. Science 2016;352:953-958 Published by AAAS

Fig. 4 Effect of temperature on the presteady-state reaction of the MCR:methyl-SCoM complex with CoB6SH and CoB7SH. Effect of temperature on the presteady-state reaction of the MCR:methyl-SCoM complex with CoB6SH and CoB7SH. Reactions of a premixed solution containing equimolar MCRred1 (20 μM, after mixing) and methyl-SCoM (20 μM, after mixing) with the saturating concentration of CoB7SH (1 mM, after mixing) (circle blue) or CoB6SH (500 μM, after mixing) (diamond red) at various temperature (10° to 50°C) were investigated using stopped-flow spectrophotometry (A). (B) is the Arrhenius plot and (C) is the Eyring plot for formation of a Ni(II)-thiolate showing a transition temperature (arrow). A vertical line at each point indicates a standard deviation of the measurement. The thermodynamic values (Ea, ΔH‡, and ΔS‡) and kobs from rapid kinetics are summarized in Table 1. Thanyaporn Wongnate et al. Science 2016;352:953-958 Published by AAAS

Fig. 5 Hydrogen bonding interactions among MCR active site residues. Hydrogen bonding interactions among MCR active site residues. Red sticks indicate hydrogen bonds at 25°C. The dashed line indicates the weak hydrogen bond between Ser399 and Tyr333 above 30°C. Residues are numbered according to MCR from Methanothermobacter marburgensis. See also table S1. Thanyaporn Wongnate et al. Science 2016;352:953-958 Published by AAAS

Fig. 6 Proposed steps of mechanism II. Proposed steps of mechanism II. In the first step, Ni(I) attack on the sulfur of methyl-SCoM leads to homolytic cleavage of the C-S bond and generation of a methyl radical and a Ni(II)-thiolate (MCRox1-silent). Next, H-atom abstraction from CoBSH generates methane and the CoBS• radical, which in the third step combines with the Ni-bound thiolate of CoM to generate the Ni(II)-disulfide anion radical. Then, one-electron transfer to Ni(II) generates MCRred1 and the heterodisulfide (CoBSSCoM) product, which dissociates leading to ordered binding of methyl-SCoM and CoBSH and initiation of the next catalytic cycle. Thanyaporn Wongnate et al. Science 2016;352:953-958 Published by AAAS