{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T15:33:29Z","timestamp":1786116809185,"version":"build-2736575974"},"reference-count":43,"publisher":"American Chemical Society (ACS)","issue":"31","license":[{"start":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T00:00:00Z","timestamp":1784764800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T00:00:00Z","timestamp":1784764800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T00:00:00Z","timestamp":1784764800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/100008634","name":"University of Texas at San Antonio","doi-asserted-by":"publisher","award":["NA"],"award-info":[{"award-number":["NA"]}],"id":[{"id":"10.13039\/100008634","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,8,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The isotope composition of residual methane after microbial oxidation can provide rigorous constraints on proposed methanotrophic pathways. However, mechanistic interpretation of the abundances of naturally occurring partially substituted methane isotopomers requires accurate kinetic isotope effects (KIEs) obtained from first-principles computations, followed by kinetic calculations specific to each isotopomer. Here, we apply contemporary density functional theory methods to calculate KIEs including the quantum tunneling effects and subsequently predict kinetic isotope fractionation involving various isotopic substitutions for several possible aerobic and anaerobic methane oxidation pathways. The results successively reproduced the abundances of methane isotopomers (13CH4, 12CH3D, 13CH3D, and 12CH2D2) reported in recent experimental studies. For aerobic methanotrophy catalyzed by copper-dependent enzymes, we identify the antiferromagnetic bis(\u03bc-oxo)dicopper(II) complex as an active oxidant that exhibits substantial quantum tunneling. This pathway quantitatively reproduces not only the large D fractionation but also the large 13C fractionation and the pronounced depletion (relative to stochastic abundances) of 13CH3D and CH2D2 in residual methane observed in laboratory experiments conducted under excess copper conditions. In contrast, the mixed-valence Cu(II)+Cu(III) dicopper-dioxo complex exhibits negligible quantum tunneling and fails to reproduce the observed isotopic signatures. For anaerobic methanotrophy, the computed KIEs support methane activation by the sulfur-centered radical on coenzyme B rather than by the nickel center of the F430 cofactor. This mechanism reproduces the modest isotope effects observed experimentally and exhibits minimal quantum tunneling contributions. Overall, this work presents a computational workflow to distinguish competing reaction pathways using the isotopic compositions of multiple isotopologues in complex microbial systems under natural or engineered conditions. The results further demonstrate that the modern quantum chemical methods are sufficiently advanced to elucidate biochemical reaction mechanisms from isotope effects without requiring comprehensive isotope labeling.<\/jats:p>","DOI":"10.1021\/acs.jpcb.6c02836","type":"journal-article","created":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T04:41:38Z","timestamp":1784781698000},"page":"7845-7854","source":"Crossref","is-referenced-by-count":0,"title":["Isotopic Evidence\nfor Reaction Pathways and Quantum\nTunneling in Methanotrophy"],"prefix":"10.1021","volume":"130","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2773-2829","authenticated-orcid":true,"given":"Xinyu","family":"Xia","sequence":"first","affiliation":[{"name":"University of Texas at San Antonio , , , ,","place":["San Antonio, Texas, United States, 78249"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yongli","family":"Gao","sequence":"additional","affiliation":[{"name":"University of Texas at San Antonio , , , ,","place":["San Antonio, Texas, United States, 78249"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,7,23]]},"reference":[{"key":"2026080608125558300_cit1","doi-asserted-by":"publisher","first-page":"1287","DOI":"10.1126\/science.abm3282","article-title":"Recovery\nof Particulate Methane Monooxygenase Structure and Activity in a Lipid\nBilayer","volume":"375","author":"Koo","year":"2022","journal-title":"Science"},{"key":"2026080608125558300_cit2","doi-asserted-by":"publisher","first-page":"5840","DOI":"10.1021\/acs.jpcb.4c01807","article-title":"Mechanisms\nfor Methane and Ammonia Oxidation by Particulate Methane Monooxygenase","volume":"128","author":"Siegbahn","year":"2024","journal-title":"J. Phys. Chem. B"},{"key":"2026080608125558300_cit3","doi-asserted-by":"publisher","first-page":"1288","DOI":"10.1021\/acs.chemrev.3c00727","article-title":"Direct Methane Oxidation by Copper- and Iron-Dependent\nMethane Monooxygenases","volume":"124","author":"Tucci","year":"2024","journal-title":"Chem. Rev."},{"key":"2026080608125558300_cit4","doi-asserted-by":"publisher","first-page":"824","DOI":"10.1021\/acs.accounts.4c00730","article-title":"Structural\nand Mechanistic Advances in the Chemistry of Methyl-Coenzyme M Reductase\n(MCR)","volume":"58","author":"Ginovska","year":"2025","journal-title":"Acc. Chem. Res."},{"key":"2026080608125558300_cit5","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1007\/978-3-031-51552-1_5","article-title":"Radical Processes","volume-title":"Enzyme Catalysis Today\nand the Chemistry of the 21st Century","author":"Likhtenshtein","year":"2025"},{"key":"2026080608125558300_cit6","doi-asserted-by":"publisher","first-page":"6309","DOI":"10.1002\/chem.201200274","article-title":"How\nIs Methane Formed and Oxidized\nReversibly When Catalyzed by Ni-Containing Methyl-Coenzyme M Reductase?","volume":"18","author":"Chen","year":"2012","journal-title":"Chem.\u2500Eur. J."},{"key":"2026080608125558300_cit7","doi-asserted-by":"publisher","first-page":"5198","DOI":"10.1021\/acs.biochem.9b00164","article-title":"Methyl\n(Alkyl)-Coenzyme M Reductases: Nickel F430-Containing Enzymes Involved\nin Anaerobic Methane Formation and Oxidation of Methane or Short-Chain\nAlkanes","volume":"58","author":"Thauer","year":"2019","journal-title":"Biochemistry"},{"key":"2026080608125558300_cit8","doi-asserted-by":"publisher","first-page":"1783","DOI":"10.1021\/acs.biochem.4c00168","article-title":"Structural Dynamics\nof the Methyl-Coenzyme\nM Reductase Active Site Are Influenced by Coenzyme F430 Modifications","volume":"63","author":"Pole\u0302to","year":"2024","journal-title":"Biochemistry"},{"key":"2026080608125558300_cit9","doi-asserted-by":"publisher","first-page":"2059","DOI":"10.1021\/acs.chemrev.6b00636","article-title":"Copper\u2013Oxygen\nComplexes Revisited:\nStructures, Spectroscopy, and Reactivity","volume":"117","author":"Elwell","year":"2017","journal-title":"Chem.\nRev."},{"key":"2026080608125558300_cit10","doi-asserted-by":"publisher","first-page":"7907","DOI":"10.1021\/ic400417d","article-title":"Role of Tyrosine Residue in Methane\nActivation at the Dicopper Site of Particulate Methane Monooxygenase:\nA Density Functional Theory Study","volume":"52","author":"Shiota","year":"2013","journal-title":"Inorg. Chem."},{"key":"2026080608125558300_cit11","doi-asserted-by":"publisher","first-page":"2771","DOI":"10.1021\/acs.inorgchem.5b02603","article-title":"Possible Peroxo\nState of the Dicopper Site of Particulate Methane Monooxygenase from\nCombined QM\/MM Calculations","volume":"55","author":"Itoyama","year":"2016","journal-title":"Inorg. Chem."},{"key":"2026080608125558300_cit12","doi-asserted-by":"publisher","first-page":"307","DOI":"10.1007\/s00775-016-1419-y","article-title":"A Tale\nof Two Methane Monooxygenases","volume":"22","author":"Ross","year":"2017","journal-title":"J. Biol. Inorg.\nChem."},{"key":"2026080608125558300_cit13","doi-asserted-by":"publisher","first-page":"9357","DOI":"10.1021\/ja00141a030","article-title":"High-precision simultaneous\ndetermination of multiple\nsmall kinetic isotope effects at natural abundance","volume":"117","author":"Singleton","year":"1995","journal-title":"J. Am. Chem. 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Adv."},{"key":"2026080608125558300_cit20","doi-asserted-by":"publisher","first-page":"2742","DOI":"10.1021\/acs.jcim.3c00531","article-title":"Determining Reaction\nPaths by Evaluating Kinetic Isotope Effects\nwith Density Functional Theory: Ex-ample of Methane Thermogenesis","volume":"63","author":"Xia","year":"2023","journal-title":"J. Chem. Inf. Model."},{"key":"2026080608125558300_cit21","doi-asserted-by":"publisher","first-page":"50","DOI":"10.1016\/j.gca.2024.05.001","article-title":"Compound-Specific,\nIntramolecular, and Clumped 13C Fractionations\nin the Thermal Generation and De-composition of Ethane and Propane:\nA DFT and Kinetic Investigation","volume":"375","author":"Xia","year":"2024","journal-title":"Geochim. Cosmochim.\nActa"},{"key":"2026080608125558300_cit22","doi-asserted-by":"publisher","first-page":"70","DOI":"10.1016\/j.gca.2020.10.015","article-title":"Clumped Isotopologue\nFractionation by Microbial Cultures\nPerforming the Anaerobic Oxidation of Methane","volume":"293","author":"Ono","year":"2021","journal-title":"Geochim. Cosmochim. Acta"},{"key":"2026080608125558300_cit23","doi-asserted-by":"publisher","first-page":"223","DOI":"10.1016\/j.gca.2024.06.032","article-title":"The Evolution of\nMulti-ply Substituted Isotopologues of Methane during Microbial Aerobic\nOxidation","volume":"381","author":"Li","year":"2024","journal-title":"Geochim. Cosmochim. Acta"},{"key":"2026080608125558300_cit24","doi-asserted-by":"publisher","first-page":"252","DOI":"10.1016\/j.gca.2019.01.004","article-title":"Kinetic Clumped Isotope\nFractionation during the Thermal Generation\nand Hydrogen Exchange of Methane","volume":"248","author":"Xia","year":"2019","journal-title":"Geochim. 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Phys."},{"key":"2026080608125558300_cit33","doi-asserted-by":"publisher","first-page":"1995","DOI":"10.1021\/jp9716997","article-title":"Quantum Calculation\nof Molecular Energies and Energy Gradients in\nSolution by a Conductor Solvent Model","volume":"102","author":"Barone","year":"1998","journal-title":"J. Phys.\nChem. A"},{"key":"2026080608125558300_cit34","doi-asserted-by":"publisher","first-page":"922","DOI":"10.1002\/jcc.26139","article-title":"Effect of the Solute\nCavity on the Solvation Energy and Its Derivatives\nwithin the Framework of the Gaussian Charge Scheme","volume":"41","author":"Garcia-Rate\u0301s","year":"2020","journal-title":"J. Comput. Chem."},{"key":"2026080608125558300_cit35","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1186\/1758-2946-4-17","article-title":"Avogadro:\nAn Advanced Semantic Chemical\nEditor, Visualization, and Analysis Platform","volume":"4","author":"Hanwell","year":"2012","journal-title":"J. Cheminf."},{"key":"2026080608125558300_cit36","doi-asserted-by":"publisher","first-page":"9955","DOI":"10.1002\/chem.201200497","article-title":"Supramolecular\nBinding Thermodynamics by Dispersion-Corrected Density Functional\nTheory","volume":"18","author":"Grimme","year":"2012","journal-title":"Chem.\u2500Eur. J."},{"key":"2026080608125558300_cit37","doi-asserted-by":"publisher","first-page":"580","DOI":"10.1002\/jcc.22885","article-title":"Multiwfn: A Multifunctional Wavefunction Analyzer","volume":"33","author":"Lu","year":"2012","journal-title":"J. Comput. Chem."},{"key":"2026080608125558300_cit38","doi-asserted-by":"publisher","first-page":"082503","DOI":"10.1063\/5.0216272","article-title":"A comprehensive\nelectron wavefunction analysis toolbox for chemists, Multiwfn","volume":"161","author":"Lu","year":"2024","journal-title":"J. Chem. Phys."},{"key":"2026080608125558300_cit39","doi-asserted-by":"publisher","first-page":"6378","DOI":"10.1021\/jp810292n","article-title":"Universal\nSolvation Model Based on\nSolute Electron Density and a Continuum Model of the Solvent Defined\nby the Bulk Dielectric Constant and Atomic Surface Tensions","volume":"113","author":"Marenich","year":"2009","journal-title":"J. Phys. Chem. B"},{"key":"2026080608125558300_cit40","doi-asserted-by":"publisher","first-page":"624","DOI":"10.1021\/j150606a003","article-title":"Parabolic\nTunneling Calculations","volume":"85","author":"Skodje","year":"1981","journal-title":"J. Phys. Chem."},{"key":"2026080608125558300_cit41","doi-asserted-by":"publisher","first-page":"1303","DOI":"10.1103\/PhysRev.35.1303","article-title":"The Penetration\nof a Potential Barrier by Electrons","volume":"35","author":"Eckart","year":"1930","journal-title":"Phys. Rev."},{"key":"2026080608125558300_cit42","doi-asserted-by":"publisher","first-page":"357","DOI":"10.6028\/jres.086.014","article-title":"A Method\nof Calculating Tunneling Corrections for Eckart Potential Barriers","volume":"86","author":"Brown","year":"1981","journal-title":"J. Res. Natl. Bur. Stand."},{"key":"2026080608125558300_cit43","doi-asserted-by":"publisher","first-page":"813","DOI":"10.1007\/s00214-007-0328-0","article-title":"Symmetry\nNumbers and Chemical Reaction Rates","volume":"118","author":"Ferna\u0301ndez-Ramos","year":"2007","journal-title":"Theor.\nChem. Acc."}],"container-title":["The Journal of Physical\nChemistry B"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jpcb.6c02836","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jpcbfk\/article-pdf\/130\/31\/7845\/66190721\/acs.jpcb.6c02836.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jpcbfk\/article-pdf\/130\/31\/7845\/66190721\/acs.jpcb.6c02836.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T12:19:08Z","timestamp":1786018748000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jpcbfk\/article\/130\/31\/7845\/5229080\/Isotopic-Evidence-for-Reaction-Pathways-and"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,23]]},"references-count":43,"journal-issue":{"issue":"31","published-online":{"date-parts":[[2026,7,23]]},"published-print":{"date-parts":[[2026,8,6]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jpcb.6c02836","relation":{},"ISSN":["1520-6106","1520-5207"],"issn-type":[{"value":"1520-6106","type":"print"},{"value":"1520-5207","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,23]]}}}