RESEARCH ARTICLE◥

ENZYME MECHANISMS

The radical mechanism of biologicalmethane synthesis by methyl-coenzyme M reductaseThanyaporn Wongnate,1 Dariusz Sliwa,1* Bojana Ginovska,2 Dayle Smith,2†Matthew W. Wolf,3 Nicolai Lehnert,3 Simone Raugei,2 Stephen W. Ragsdale1‡

Methyl-coenzyme M reductase, the rate-limiting enzyme in methanogenesis and anaerobicmethane oxidation, is responsible for the biological production of more than 1 billion tons ofmethane per year. The mechanism of methane synthesis is thought to involve either methyl-nickel(III) or methyl radical/Ni(II)-thiolate intermediates. We employed transient kinetic,spectroscopic, and computational approaches to study the reaction between the active Ni(I)enzyme and substrates. Consistent with the methyl radical–based mechanism, there was noevidence for a methyl-Ni(III) species; furthermore, magnetic circular dichroism spectroscopyidentified the Ni(II)-thiolate intermediate. Temperature-dependent transient kinetics alsoclosely matched density functional theory predictions of the methyl radical mechanism.Identifying the key intermediate in methanogenesis provides fundamental insights to developbetter catalysts for producing and activating an important fuel and potent greenhouse gas.

Methanogenic archaea produce more than90% of Earth’s atmospheric methane (1),totalingmore than 1 billion tons ofmeth-ane per year globally (2). Furthermore,methanogens living in microbial com-

munities containing sulfate- or nitrate-reducingbacteria are responsible for the annual anaerobicoxidation of 0.1 billion tons of methane (3–6).The enzyme that catalyzes the chemical step ofmethane synthesis (Eq. 1) or oxidation (the re-verse of Eq. 1) is methyl-coenzyme M reductase(MCR), which contains a nickel hydrocorphinateF430 at its active site (4, 7–9). This reaction in-volves conversion of the methyl donor, methyl-coenzyme M (methyl-SCoM), and the electrondonor, coenzyme B (CoBSH, N-7-mercaptohepta-noylthreonine phosphate) (10), to methane andthe mixed disulfide CoBS-SCoM (11) (Eq. 1). Thesubstrates bind inside a deep substrate channelwith CoBSH nearer to the surface, stretching to-ward methyl-SCoM, which is close to F430 (12).

Methyl-SCoM + CoBSH → CH4 + CoBS-SCoMDG0′ = –30 kJ/mol (1)

The mechanism of methane formation is notfully resolved, mainly because intermediates inthe catalytic cycle have not been identified. Un-

covering the MCR mechanism is critical becauseof the important biogeochemical and environ-mental roles of this enzyme in generating (andmetabolizing) a Janus-like compound that servesas a key energy source and is a potent greenhousegas. Furthermore, the chemical principles under-lying both synthesis and activation ofmethanemayinform the development of catalysts that mimicthe structure and/or function of the key enzymaticintermediate(s) or transition state(s). The two pro-posed mechanisms for how methane is generateddiffer in whether the first step involves an orga-nometallic methyl-Ni(III) [mechanism I (13–15)]or a methyl radical intermediate [mechanism II(16)] (Fig. 1). In both mechanisms, the nickelcenter of F430must be in the Ni(I) oxidation statefor the enzyme to initiate catalysis (17, 18).Support for mechanism I is based on exper-

iments using F430 model complexes (19, 20), en-zymatic studies involving isotope exchange (21),and the reaction of the active form of MCR(MCRred1) with various activated alkyl donorssuch as alkyl halides (22–24). These substrateanalogs react with Ni(I) to generate alkyl-Ni(III)species that undergo reduction to the alkane (asin the forward direction of Eq. 1) or conversion tothioethers—e.g., methyl-SCoM upon reactionwith organic thiolates like CoM (as in the reversereaction) (22–24). Mechanism II is supported bydensity functional theory (DFT) computations inwhich it was argued that formation of themethyl-Ni(III) intermediate is not energetically feasible,being endoergic by 91 kJ/mol (with an activationfree energy of 94 kJ/mol), whereas the forma-tion of a methyl radical and Ni(II)-thiolate isexoergic by 10 kJ/mol (with an activation freeenergy of 63 kJ/mol) (16, 25–27).

A third mechanism is also possible in whichnucleophilic attack ofNi(I) onmethyl-SCoMgen-erates a Ni(III)-SCoM species and, formally, ananionic methyl group that undergoes protona-tion to generatemethane (mechanism III) (Fig. 1).A Ni(III)-thiolate species known asMCRox1 is oth-erwise formed when growing cells are exposed tosodium sulfide (18) or to an oxidizing gas mixture(80% N2/20% CO2) (28). MCRox1 is also called the“ready” state of the enzyme because it can be ac-tivated to the active MCRred1 state (17, 18). Boththemethyl-Ni(III) (23, 29) and theNi(III)-thiolate(MCRox1) (30) states have been generated in highyield, are relatively stable, and exhibit distinctiveelectron paramagnetic resonance (EPR) spectra.Actually, spectroscopic and computational studiesindicate that MCRox1 is best described as ahigh-spin Ni(II)-thiyl radical in resonance with aNi(III)-thiolate species (30, 31). Conversely, theNi(II)-MCRox1-silent state is EPR-silent. TheMCRox1,MCRox1-silent, and MCRred1 states also display dis-tinct magnetic circular dichroism (MCD) spectra(31, 32). Thus, performing rapid mixing experi-ments and monitoring the accumulation of an in-termediate exhibiting the spectroscopic featuresof themethyl-Ni(III),MCRox1-silent, orMCRox1 statesassociated with decay of MCRred1 should provideunambiguous evidence supporting one of thethreemechanisms.However, onlyminor spectro-scopic changes are observed when MCR reactswith methyl-SCoM and the natural substrateCoBSH (33).We performed transient kinetic, spectroscopic

[ultraviolet-visible (UV-Vis), EPR, and MCD], andcomputational studies of the first step in theMCRcatalytic mechanism to trap and identify the keyintermediates that differ between mechanisms Iand II.MCR contained a sufficiently high amount(70 to 80%) of the active Ni(I)-MCRred1 state tomonitor changes in its spectroscopic propertiesduring the reaction and identify intermediates.We rapidly mixed MCR with methyl-SCoM andCoB6SH, containing a hexanoyl instead of hepta-noyl side chain, which sufficiently slows downthe first step in the MCR reaction (34, 35) toallow accumulation and detection of the first in-termediate in the MCR mechanism.

Rapid kinetic studies rule out methyl-Ni(III)and trap the MCRox1-silent intermediate

We performed stopped-flow studies by rapidlymixing a solution containing MCR and methyl-SCoMwith the slow substrate CoB6SH (Fig. 2A).We tracked the reaction at 385 nm to followNi(I)decay and at 420 nm to measure the rate atwhich the Ni(II) or Ni(III) intermediate forms.Although the steady-state and presteady-staterate constants are slower by factors of 1000 and440 with CoB6SH than with CoBSH, no spectro-scopic changes are observed upon addition ofmethyl-SCoM alone; in fact, even for a singleturnover, both substrates must be present beforeany reaction can occur (34). This strongly sug-gests thatwith the slow (CoB6SH) substrate,MCRemploys the same strict ternary-complex mecha-nismaswith the native (CoBSH) substrate (33, 34).The spectroscopic features at both 385 and420nm

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1Department of Biological Chemistry, University of Michigan,Ann Arbor, MI 48109-0606, USA. 2Physical Sciences Division,Pacific Northwest National Laboratory, Post Office Box 999,K1-83, Richland, WA 99352, USA. 3Department of Chemistryand Department of Biophysics, University of Michigan, AnnArbor, MI 48109-1055, USA.*Present address: Applied Photophysics Inc., 100 Cummings Center,Suite 440C, Beverly, MA 01915, USA. †Present address: IntelCorporation, 2111 NW 25th Avenue, JF5-202, Hillsboro, OR 97124,USA. ‡Corresponding author. Email: sragsdal@umich.edu

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exhibited monophasic kinetics, with 60% of thestarting MCRred1 state undergoing conversion ata rate constant of 0.35 ± 0.01 s−1. Two additionalslow phases [observed rate constant 2 (kobs2) =0.05 ± 0.01 s−1 and kobs3 = 0.008 ± 0.001 s−1] withgreatly reduced amplitudes are observed thatalso occur in control reactions lacking substrate,indicating that these phases correspond to thenoncatalytic Ni(I) oxidation and are not rel-evant to the catalytic mechanism. Over a longertime frame, the spectrumof activeMCRred1 returns(kobs = 0.011 ± 0.001 min−1), validating that MCRremains active during these spectroscopic trans-formations with methyl-SCoM and CoB6SH, asrecently shown for the reaction with CoBSH (33).To further study the conversion of methyl-

SCoM to methane, we used the rapid chemical-quenchmethod under conditions similar to thoseof the stopped-flowexperiments.A solutioncontain-ingMCRred1 (20 mM, after mixing) and [14C]methyl-SCoM (20 mM, after mixing) was rapidly mixedwith CoB6SH (500 mM, after mixing), and incu-bated for various time points between 0.62 and90 s. The reaction was quenched by mixing with0.2 M perchloric acid and analyzed by liquidscintillation counting. The amount of remaining[14C]methyl-SCoM was plotted versus time (Fig.

2B) and fit to a single-exponential curve, reveal-ing a limiting rate constant of 0.31 ± 0.04 s−1. Theresults demonstrated that conversion of themeth-yl group ofmethyl-SCoM tomethane is limited bythe same rate constant (~0.30 s−1) as conversionof Ni(I) to Ni(II)/Ni(III) in the stopped-flow ex-periment. These results supportmechanism II (orIII), because the reactivemethyl radical (andmeth-yl anion) would have very transient existence andwould immediately abstract a hydrogen atom orproton, respectively, fromCoBSHtogeneratemeth-ane with the same rate constant as that of Ni(I)decay. However, because methyl-Ni(III) is rela-tively stable, methane formation bymechanism Irequires another step and, thus, would occurmoreslowly than Ni(I) decay. For example, the alkyl-Ni(III) state ofMCR reacts slowlywith thiolates.Themethyl-Ni(III) state ofMCRreactswithCoMSHto generate methyl-SCoM and MCRred1 at a rateconstant of 0.04 s−1 (at 25°C) (23). Similarly, thealkyl-Ni(III) state generated from bromopropa-nesulfonate or various brominated acids reactslowly (kmax ~ 0.005 s−1) with small thiolates andeven slower with CoBSH (and analogs) to gen-erate MCRred1 and the thioether (22, 24, 36).To identify and characterize any EPR-detectable

intermediates formed during the MCR reaction,

we performed rapid freeze-quench (RFQ) EPRexperiments under similar conditions as thosefor the stopped-flow and rapid chemical-quenchexperiments. We observed a dominant (~90%)decrease in intensity of the characteristic Ni(I)EPR spectrum of MCRred1 at g values of 2.249,2.084, and 2.061 (Fig. 2C). Comparable results areobserved in two similar FQ-EPR experiments (fig.S1). The decay curve fits to a single-exponentialequation with a limiting rate constant of 0.53 ±0.25 s−1 (circles in Fig. 2D). We did not observeany EPR-active species [e.g., methyl-Ni(III)] thataccumulate to an amplitude similar to that ofMCRred1 decay (~43 mM), suggesting that the Ni-based product of this reaction is a Ni(II) EPR-silent species. However, two other EPR signalsformed at low levels during the time course ofNi(I) decay (insets, Fig. 2C and fig. S1). A rhombicsignal (g = 2.212, 2.183, and 2.150) identical tothat of MCRox1 (30), present at very low levels inthe initial sample, slightly increased in intensityto 3% of the initial MCRred1 according to a rateconstant of 0.69 ± 0.24 s−1. A radical-type species(g~ 2.0), observed earlier (35), formedwith a rateconstant of 0.52 ± 0.32 s−1 to an amplitude thatreached 6 to 7% of the initial Ni(I)-MCRred1

signal (fig. S1). The low level of accumulation of

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Fig. 1. Initial steps in three mechanisms of MCR catalysis. Mechanism I involves nucleophilic attack of Ni(I)-MCRred1 on the methyl group of methyl-SCoMto 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 amethyl 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 methylanion and Ni(III)-SCoM (MCRox1).

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this radical speciesmay be due to the difficulty ofobserving thiyl radicals due to their short life-times and large spin-orbit coupling with the sul-fur atom (37). Regardless, a sulfur radical(s) ispredicted to be an intermediate in all threemech-anisms and, thus, is not diagnostic of which oneis correct.The above results suggest that, when active

MCRred1 is incubated with methyl-SCoM andCoB6SH, it converts nearly quantitatively to anEPR-silent Ni(II)-thiolate species, consistent withthe predictions of mechanism II. Therefore, weturned our attention to a spectroscopic methodthat could positively identify that intermediate. TheMCD spectra of MCRred1, MCRox1, and MCRox1-silent

are distinct (31). Figure S2 shows the MCD dataof MCRred1 and MCRox1-silent, prepared for com-parison. The temperature dependence of the datashowed that both species are paramagnetic, withMCRred1 andMCRox1-silent having ground states ofS = ½ and S = 1, respectively. MCRred1 has acharacteristic negative feature (at 21,620 cm−1)that can be used to monitor the reaction and de-cay of this species. The kinetic studies indicatedthat the first intermediate in methanogenesisforms at a rate constant of 0.35 s−1 (t1/2 = 2 s)and remains for at least 70 s; thus, to directlyobserve this intermediate by MCD, we mixed asolution containing MCRred1 and methyl-SCoMwith CoB6SH, rapidly froze this mixture in liquidnitrogen within ~10 s, and performed MCD ex-periments of the samples prepared before andafter reaction with substrates. The dominantchanges in the MCD spectrum indicate an al-

most quantitative conversion of MCRred1 to aspecies nearly identical to MCRox1-silent (Fig. 3).For example, the negative band at 21,620 cm−1

disappeared, direct evidence of the consump-tion of MCRred1, as all of the characteristic bandsassociated with MCRox1-silent appear, positivelyidentifying that Ni(II) species as the reactionproduct. Although there is some difference in ab-solute intensity between the data sets (which islikely due to problems with depolarization of thecircular polarized light by the frozen glass MCDsample), the spectra are almost identical in bandshape—i.e., the number of features and their relativeintensities—confirming formation of MCRox1-silent

as the major reaction product. The CD spectro-meter also records a single-channel voltage curvethat can be converted into a low-resolutionUV-Visabsorption spectrum. These data demonstratethat the MCD samples were initially in the Ni(I)state and underwent quantitative conversionafter reaction (fig. S2D). Altogether, our spectro-scopic results provide direct evidence that theEPR-silent Ni(II)-thiolate intermediate proposedin mechanism II is the key intermediate inmethanogenesis.Mechanisms I, II, and III propose the forma-

tion of distinct intermediates. We found no evi-dence for a methyl-Ni(III) species in our RFQEPR studies. This [and other alkyl-Ni(III)] speciesis relatively stable when bound to MCR (23, 38)and should have been observed if it had formed.Moreover, there was no evidence for theMCRred2a

or MCRred2r states (g values of 2.273, 2.077, 2.073and 2.288, 2.231, 2.175, respectively) (39) during

the transient kinetic reaction, strongly indicatingthat neither of these species, assigned as aNi(III)-hydride or a Ni(I)-thiolate, respectively,serve as an intermediate in theMCRmechanism.A recent computational study suggested that theNi(III)-H should be reassigned to a catalyticallyinactive species in which the proton of CoMSHbinds near the Ni(I) of MCRred1 (27). Regardless,the data do not support a mechanism involvinga methyl-Ni(III), Ni(III)-hydride or side-on C-Scoordination to the Ni (39).We further exploredwhether theminorMCRox1

signal could have any catalytic relevance formeth-ane formation, perhaps as a parallel pathway viamechanism III (fig. S3). If MCRox1 forms with arate constant of 0.5 s−1 and accumulates to 3% ofthe initial amount of MCRred1, it must decay toanother Ni-based intermediate with a rate con-stant (k2) of ~ 15 s−1 (fig. S4). However, the con-centration of MCRox1 did not change for at least70 s, strongly suggesting that the slight increasein the MCRox1 EPR signal occurred by an off-pathway process that is unrelated to catalysis.

Computational studies rule out the Ni(III)-thiolate MCRox1-like intermediate

DFT calculations were undertaken to assess therelative energetics of the initial steps inmechanismsI, II and III involving methyl-Ni(III), Ni(II)-MCRox1-silent, or the Ni(III)-MCRox1 species, respec-tively (fig. S5).We used truncated forms of the F430cofactor, CoBSH, and CoM and performed thesecomputations as previously described (25–27, 40).The formation of a MCRox1-silent-like intermediate

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Fig. 2. Rapid kinetic studies of the reaction ofthe MCR:methyl-SCoM complex with CoB6SH.(A) Stopped-flow. Kinetic traces of the reaction ofa premixed solution containing MCRred1 (20 mM, af-ter mixing) and methyl-SCoM (20 mM, after mixing)with CoB6SH (500 mM, after mixing) in 50mM Tris-HCl, pH 7.6. The reactions were performed underanaerobic conditions using the stopped-flow spec-trophotometer at 18°C and monitored by followingthe decay of Ni(I) at 385 nm (blue line) and theformation of Ni(II)/Ni(III) at 420 nm (red line).Thereaction showed monophasic kinetics with a rateconstant of 0.35 ± 0.01 s−1. (B) Rapid chemical-quench. Reactions of a premixed solution containingequimolar MCRred1 (20 mM, after mixing) and [14C]methyl-SCoM (20 mM, after mixing) with CoB6SH(500 mM, after mixing) were quenched with 0.2 Mperchloric acid at various times using the rapidchemical-quench apparatus. Volatile methane pro-duct was lost from the solution, and the percentageconversion of [14C]methyl-SCoM was determinedby comparing the remaining concentration of [14C]methyl-SCoM to the initial concentration. Plottingthe percentage conversion versus time yielded asingle-exponential curve with a rate constant of 0.31 ± 0.04 s−1. The verticalbrackets at each point indicate the standard deviation of the measurement.(C and D) RFQ EPR. A solution containing MCRred1 (48 mM) and methyl-SCoM (600 mM) was reacted with CoB6SH (120 mM) and freeze-quenched atvarious times using an RFQ apparatus. Representative time-dependent EPRspectra are shownon (C).The inset shows the g~2.2 region near theS-shapedfeature of MCRox1. The percentage decay of MCRred1 (blue) and formationof MCRox1 (red) were determined by comparing their doubly integrated sig-

nal intensities at various quenching times to their initial intensities.The datawere plotted and fit to single-exponential curves in (D). The MCRred1 signaldecayed by 90% during the first phase of the reaction with a rate constantof 0.53 ± 0.25 s−1, whereas the MCRox1-like signal increased by ~3% (relativeto the initial MCRred1), with a rate constant of 0.69 ± 0.24 s−1. A radicalformed with a rate constant of 0.52 ± 0.32 s−1 and reached ~7% of the initialMCRred1 (see fig. S1). A vertical line at each point indicates the standarddeviation of each measurement.

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via the homolytic process corresponding tomechanism II is thermodynamically favored(DG0 = –3.5 kJ/mol) and has an activation en-ergy (DG‡) of 72 kJ/mol, whereas generation ofthe methyl-Ni(III) state (by mechanism I) ishighly endoergic (DG0 = 100 kJ/mol) with a DG‡

of 102 kJ/mol.We were unable to identify an MCRox1-like

state, specifically a F430-Ni(III)-SCoM/CoBS– inter-mediate, from direct DFT calculations; however,performing a wave function optimization (41) im-posing a –1 e charge on the CoBS fragment, wefound this MCRox1-like state residing at 136.0 kJ/mol above theMCRox1-silent baseline. A similar com-putational strategy applied to identify an anionictransition state connecting MCRred1 to MCRox1

failed. Nevertheless, starting from the homolytictransition state of mechanism II, we identifiedan excited state with a strong anionic CH3

– char-acter, residing at 134.6 kJ/mol above theMCRred1

baseline. The inability to optimize any transitionstate corresponding to MCRox1 and the corre-sponding methyl anion could be due to thereduced size of the model; however, the largeestimated energetic difference between theMCRox1

and MCRox1-silent states is unlikely to be substan-tially affected by increasing the model size.Additional calculations suggested that the

MCRox1-like state is inaccessible because of thevery positive reduction potential of the Ni(III)-SCoM intermediate species, E0′ = 1.4 V versus thenormal hydrogen electrode (NHE) (see the sup-plementary materials). Indeed, with a calculatedredox potential for the CoBS•/CoBS– couple inthe enzyme cavity of E0′ = 0.0 V versus NHE,Ni(III)-SCoMwouldpromptly oxidizeCoBS–duringturnover. Taken together, these computationalfindings, along with the kinetic simulations de-scribed above, rule out the possibility of aMCRox1-like intermediate (and of mechanismIII) in methane synthesis.

Temperature-dependence studiesmap the MCR reaction profile

One way to discriminate among different mech-anisms is to compare experimental activationenergy barriers with the transition-state barriersobtained by DFT computations. To obtain theexperimental thermodynamic values, a premixed

solution containing equimolar MCRred1 andmethyl-SCoM was rapidly mixed with a saturat-ing concentration of CoB7SH or CoB6SH at var-ious temperatures between 10° and 50°C, andthe reactionwasmonitored by stopped-flow spec-trophotometry (Fig. 4 and fig. S6).For the reactionswithbothCoB7SHandCoB6SH,

kobs increased as the temperature increased (Fig.4A), giving a biphasic Arrhenius plot with a tran-sition temperature at ~30°C (Fig. 4B). For CoB7SH,the activation energies (Ea) above and below thetransition temperature were calculated to be 51and 84 kJ/mol, respectively (Table 1), indicatingthat there is a temperature-dependent structuraltransition that alters the MCR mechanism. TheEyring plot (Fig. 4C) also showed a similar non-linear response with a transition temperature atthe same temperature. The enthalpy of activa-tion (DH‡) and the entropy of activation (DS‡)above the transition temperature are 48 kJ/moland –56 J/(mol K), respectively (Table 1). Between30° and 50°C, the Gibbs energy of activation(DG‡) increased due to a more negative entropyof activation (DS‡). The DH‡ and DS‡ values belowthe transition temperature were 82 kJ/mol and54 J/(mol K), respectively.The experimental values of Ea (51 kJ/mol), DH‡

(48 kJ/mol), and calculated DG‡ at 30°C (65 kJ/

mol) were congruent with previous (25) and cur-rent DFT-calculated transition-state barriers forthe formation of a Ni(II)-thiolate (about 63 kJ/mol and 71 kJ/mol, respectively) via mechanismII, given that only the enzyme’s active site andtruncated substrates were considered in the DFTmodel. In contrast, the DFT-calculated transitionbarrier (DG‡) for formation of the methyl-Ni(III)intermediate was 102 kJ/mol, which is muchhigher than the experimental value. In additionto the high kinetic barrier, formation of methyl-Ni(III) from MCRred1 is computed to be highlyunfavorable thermodynamically (DG0 = 100 kJ/mol). Thus, our temperature-dependent studiesstrongly support the formation of the Ni(II)-MCRox1-silent-like species as the key intermediatein the MCR reaction, consistent with our spec-troscopic data.Similar temperature-dependence studies mea-

sured the thermodynamic values for the reactionwith CoB6SH. As with CoB7SH, the Arrheniusand Eyring plots were biphasic with a transitiontemperature at ~29°C. The Ea values above andbelow the transition temperature were 47 and127 kJ/mol, respectively (Fig. 4B). The Eyringanalysis resulted in DH‡ of 45 and 125 kJ/moland DS‡ of –90 and 176 J/mol.K, above and belowthe transition temperature, respectively (Fig. 4C).

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Fig. 3. MCD studies of thereaction of the MCR:methyl-SCoM complex withCoB6SH. MCD spectra weretaken at 2 K of MCRred1 beforeand after the reaction withmethyl-SCoM and CoB6SH.Samples were prepared in50 mM GPT buffer [(50 mMglycine, 50 mM phosphate,and 50 mM Tris), pH 7.6,containing 0.05 mM Ti(III)citrate] with 50% glycerolfor MCRred1 and 73% glycerolfor the reaction with CoB6SH.

Fig. 4. Effect of temperature on the presteady-state reaction of theMCR:methyl-SCoM complexwith CoB6SH and CoB7SH. Reactions of a premixed solutioncontaining equimolar MCRred1 (20 mM, after mixing) and methyl-SCoM (20 mM, after mixing) with the saturating concentration of CoB7SH (1 mM, after mixing) (circleblue) or CoB6SH (500 mM, after mixing) (diamond red) at various temperature (10° to 50°C) were investigated using stopped-flow spectrophotometry (A). (B) is theArrhenius 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 standarddeviation of the measurement. The thermodynamic values (Ea, DH

‡, and DS‡) and kobs from rapid kinetics are summarized in Table 1.

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Between 30° and 50°C, the Ea (47 kJ/mol) andDH‡ (45 kJ/mol) for the CoB6SH reaction are inthe same range as those for CoB7SH (Ea = 51 kJ/mol and DH‡ = 48 kJ/mol). These results indicatethat the same type of temperature-dependenttransition occurs in the MCR reaction withCoB6SH as with the natural CoB7SH substrate.The DS‡ value between 30° and 50°C for thereaction with CoB6SH was 1.6 times lower thanthat for CoB7SH, suggesting that the slow sub-strate has a higher degree of freedom in theMCRactive site than the longer natural substrate. Asexpected, the DG‡ at 30°C for the reaction ofCoB6SH is 7 kJ/mol higher than that for CoB7SH(72 kJ/mol versus 65 kJ/mol), in agreement withthe kobs at 30°C for CoB6SH being lower by afactor of 15 than that with CoB7SH (2 s−1 versus31 s−1). These results strongly indicate that MCRemploys the same rate-limiting chemical step inits reactionmechanism, whether the second sub-strate is CoB6SH or CoB7SH. Furthermore, it ap-pears that themajor reason for the factor of 1000slower ratewith CoB6SH is the increased entropyof the slowly reacting substrate, CoB6SH, at theactive site and in the substrate channel relativeto that of CoB7SH.

Molecular dynamics simulations highlightthe presence of temperature-dependentstructural changes at the active site

Long time-scale molecular dynamics simulationsover a wide range of temperatures (from roomtemperature to 50°C) were performed to identifyresidues at the active site that could be respon-sible for the observed biphasic temperature de-pendence of the kinetics (Table 1 and Fig. 4). Thesimulations suggested that above 25°C, Tyr333,which is one of the two tyrosine residues that ishydrogen-bonded to the thioether sulfur ofmethyl-CoM, moves away from CoM, establishing a weakhydrogen bond with Ser399 and allowingmethyl-SCoM to approach the Ni center of F430 (Fig. 5).This conformational change lengthens the Tyr-OH-(S)CoM hydrogen bond distance from 2.5 ±0.4 Å (25°C) to 3.1 ± 0.5 Å at around 30°C as theCoM(S)-Ni average distance decreases from 3.7 ±0.2 Å to 3.2 ± 0.3 Å (table S1 and Fig. 5).Shortening the CoM(S)-Ni distance is expectedto facilitate cleavage of the CoM(S)-CH3 bond.None of the other hydrogen bond interactions atthe active site changed as the temperatureincreased (table S1 and fig. S7).

Implications

In the first step of mechanism II (Fig. 6), attackof Ni(I) on the sulfur of methyl-SCoM leads tohomolytic cleavage of the C-S bond and gener-ation of a methyl radical and a Ni(II)-thiolate,known asMCRox1-silent. Carbon (1.04) and second-ary deuterium (1.19) isotope effect studies (42, 43)indicate that the transition state for the rate-limiting C-S bond cleavage involves a trigonalplanar carbon, consistent with formation of amethyl radical in the first step in methane syn-thesis. Because anaerobic methane oxidation oc-curs by direct reversal of its synthesis (3), themethyl radicalwill also be formed in the transition

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Fig. 6. Proposed stepsof mechanism II. In thefirst step, Ni(I) attack onthe sulfur of methyl-SCoMleads to homolytic cleav-age of the C-S bond andgeneration of a methylradical and a Ni(II)-thiolate(MCRox1-silent). Next, H-atomabstraction from CoBSHgenerates methane and theCoBS• radical, which inthe third step combineswith the Ni-bound thiolateof CoM to generate theNi(II)-disulfide anion radical.Then, one-electron transferto Ni(II) generates MCRred1

and the heterodisulfide(CoBSSCoM) product,which dissociates leading toordered binding of methyl-SCoM and CoBSH and initiation of the next catalytic cycle.

Fig. 5. Hydrogenbonding interactionsamong MCR activesite residues. Redsticks indicate hydrogenbonds at 25°C. Thedashed line indicatesthe weak hydrogenbond between Ser399

and Tyr333 above 30°C.Residues are numberedaccording to MCR fromMethanothermobactermarburgensis. See alsotable S1.

Table 1. Activation barriers and observed rate constants for the first step in the MCR reaction.Activation barriers and rate constants were determined by presteady-state kinetic experiments (Fig. 4).

These experiments were performed at the transition temperature, 30°C.

Substrate Temp. rangeEa*

(kJ/mol)

DH†

(kJ/mol)

DS†

(J/mol.K)

kobs (s−1),

30°C§

CoB7SH 10–30°C 84 82 5431.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... ..

30–50°C 51 48 –56.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. .

CoB6SH 10–29°C 127 125 1762.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... ..

29–50°C 47 45 –90.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. .

*The activation energy was calculated from the Arrhenius plot. †Enthalpy and entropy of the transitionstate were determined from the Eyring plot. §kobs values at the temperature break.

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state for the final step in reversemethanogenesis.The second step involves H-atom abstractionfromCoBSH, generatingmethane and the CoBS•radical, which in the third step combines with theNi-bound thiolate of CoM to generate a Ni(II)-disulfide anion radical, poised for one-electrontransfer to Ni(II) to generate Ni(I)-MCRred1 andthe heterodisulfide (CoBSSCoM) product. Thefinal mechanistic step involves dissociation ofthe heterodisulfide to allow the ordered bindingofmethyl-SCoM andCoBSH and initiate the nextcatalytic cycle.Understanding the mechanism of these reac-

tions has large implications for developing tech-nologies to catalytically generate and activatemethane (44, 45). The latter process is one of themost challenging chemical reactions because acatalyst must selectively break the first carbon-hydrogen bond of methane at a huge free-energycost (438.9 kJ/mol) without cleaving any of theremainingweaker carbon-hydrogen bonds. Exploi-tation of methane for energy and chemistry istimely because natural gas reserves are predictedto increase by >40% in the United States over thenext 30 years (46) and because anthropogenicsources ofmethane contribute a large proportion(20%) of the world’s annual greenhouse gas war-ming potential (48).

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ACKNOWLEDGMENTS

Data presented here are available through the Ragsdale LabGuruportal (https://my.labguru.com/knowledge/projects/361/milestones/711). This work was supported by U.S. Department

of Energy (DOE), Office of Science, Office of Basic EnergySciences, under award DE-FG02-08ER15931 and by U.S. DOE,Advanced Research Projects Agency – Energy, under award numberDE-AR0000426. Computer resources were provided by theW. R. Wiley Environmental Molecular Sciences Laboratory, aDOE Office of Science User Facility located at Pacific NorthwestNational Laboratory and sponsored by DOE’s Office of Biologicaland Environmental Research. Computer resources were alsoprovided by the National Energy Research Computing Center atthe Lawrence Berkeley National Laboratory. Author contributions:T.W. generated the data, performed the data analysis, andprepared Figs. 2, A and B, and 4, and Table 1; and performed thedata analysis and generated Fig. 2, C and D, and figs. S1, S4, andS6. D. Sliwa prepared CoBSH substrates and enzyme; generatedthe data and performed preliminary data analysis for Fig. 2,C and D, and fig. S1; and prepared the samples for the MCDmeasurements described in Fig. 3 and fig. S2. B.G. and S.R.performed and interpreted the computational studies describedin Fig. 5; figs. S5, S7, S8, and S9; and tables S1 to S3.D. Smith performed initial computational experiments on theMCR mechanisms. M.W. and N.L. performed, analyzed, andinterpreted the MCD spectroscopic experiments and preparedFig. 3 and fig. S2. S.W.R. conceptualized and refined the researchidea; prepared Figs. 1 and 6 and fig. S3; and coordinated thevarious collaborations and guided analysis and interpretation ofthe biochemical experiments. All authors were involved tovarying degrees in writing and/or editing the manuscript.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/352/6288/953/suppl/DC1Materials and MethodsFigs. S1 to S8Tables S1 to S3References (49–79)

11 December 2015; accepted 5 April 201610.1126/science.aaf0616

REPORTS◥

SUPERCONDUCTIVITY

Ubiquitous signatures of nematicquantum criticality in optimallydoped Fe-based superconductorsHsueh-Hui Kuo,1,2* Jiun-Haw Chu,1,3*† Johanna C. Palmstrom,1,3

Steven A. Kivelson,1,4 Ian R. Fisher1,3†

A key actor in the conventional theory of superconductivity is the induced interactionbetween electrons mediated by the exchange of virtual collective fluctuations (phonons inthe case of conventional s-wave superconductors). Other collective modes that can playthe same role, especially spin fluctuations, have been widely discussed in the context ofhigh-temperature and heavy Fermion superconductors. The strength of such collectivefluctuations is measured by the associated susceptibility. Here we use differentialelastoresistance measurements from five optimally doped iron-based superconductors toshow that divergent nematic susceptibility appears to be a generic feature in the optimaldoping regime of these materials. This observation motivates consideration of the effectsof nematic fluctuations on the superconducting pairing interaction in this family ofcompounds and possibly beyond.

Agrowing body of evidence suggests the pos-sibility of an intimate connection betweenelectronic nematic phases (1) and high-temperature superconductivity. However,it is currently unclear to what extent there

is any causal relationship between nematic fluc-tuations and superconductivity. Strongly aniso-tropic electronic phases have been found in theunderdoped regime of both cuprate (2–6) andFe-based (7–12) high-temperature superconductors.

958 20 MAY 2016 • VOL 352 ISSUE 6288 sciencemag.org SCIENCE

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The radical mechanism of biological methane synthesis by methyl-coenzyme M reductase

Stephen W. RagsdaleThanyaporn Wongnate, Dariusz Sliwa, Bojana Ginovska, Dayle Smith, Matthew W. Wolf, Nicolai Lehnert, Simone Raugei and

DOI: 10.1126/science.aaf0616 (6288), 953-958.352Science 

, this issue p. 953;, see also p. 892Sciencethrough Ni(II)-thiolate and methyl radical intermediates rather than an organometallic methyl-Ni(III) mechanism.cofactor F430 contained Ni(II), consistent with computational results. The final step of methanogenesis thus proceedsmethyl-coenzyme M reductase (see the Perspective by Lawton and Rosenzweig). Spectroscopy demonstrated that

used stopped-flow and rapid freeze-quench experiments to trap a methyl radical in the active site ofet al.Wongnate methane production has an ambiguous mechanism because it involves difficult-to-isolate reaction intermediates.

Microorganisms are the main drivers of Earth's methane cycle. The enzyme ultimately responsible for biologicalA radical route to making methane

ARTICLE TOOLS http://science.sciencemag.org/content/352/6288/953

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2016/05/18/352.6288.953.DC1

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