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Solvent Deuterium Isotope Effects of Substrate Reduction by Nitrogenase from Azotobacter vinelandii

MacArdle, Siobhán G. and Rees, Douglas C. (2022) Solvent Deuterium Isotope Effects of Substrate Reduction by Nitrogenase from Azotobacter vinelandii. Journal of the American Chemical Society, 144 (46). pp. 21125-21135. ISSN 0002-7863. doi:10.1021/jacs.2c07574.

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The mechanism of nitrogenase, the enzyme responsible for biological nitrogen fixation, has been of great interest for understanding the catalytic strategy utilized to reduce dinitrogen to ammonia under ambient temperatures and pressures. The reduction mechanism of nitrogenase is generally envisioned as involving multiple cycles of electron and proton transfers, with the known substrates requiring at least two cycles. Solvent kinetic isotope effect experiments, in which changes of reaction rates or product distribution are measured upon enrichment of solvent with heavy atom isotopes, have been valuable for deciphering the mechanism of complex enzymatic reactions involving proton or hydrogen transfer. We report the distribution of ethylene, dihydrogen, and methane isotopologue products measured from nitrogenase-catalyzed reductions of acetylene, protons, and cyanide, respectively, performed in varying levels of deuterium enrichment of the solvent. As has been noted previously, the total rate of product formation by nitrogenase is largely insensitive to the presence of D₂O in the solvent. Nevertheless, the incorporation of H/D into products can be measured for these substrates that reflect solvent isotope effects on hydrogen atom transfers that are faster than the overall rate-determining step for nitrogenase. From these data, a minimal isotope effect is observed for acetylene reduction (1.4 ± 0.05), while the isotope effects for hydrogen and methane evolution are significantly higher at 4.2 ± 0.1 and 4.4 ± 0.1, respectively. These results indicate that there are pronounced differences in the sensitivity to isotopic substitution of the hydrogen atom transfer steps associated with the reduction of these substrates by nitrogenase.

Item Type:Article
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URLURL TypeDescription ItemSiobhán MacArdle’s PhD thesis
MacArdle, Siobhán G.0000-0001-7843-5977
Rees, Douglas C.0000-0003-4073-1185
Additional Information:The authors thank Dr. Nathan Dalleska for guidance in GC–MS methods and data analysis, Dr. Jay Winkler for FTIR quantification and analysis of ethylene isotopologues, and Dr. David Vander Velde for guidance with NMR analysis. This project benefited from the use of instrumentation made available by the Resnick Sustainability Institute’s Water and Environment Lab at the California Institute of Technology. FTIR spectra were recorded in the Beckman Institute Laser Resource Center at Caltech. The authors thank Dr. Trixia Buscagan, Dr. Rebeccah Warmack, Ailiena Maggiolo, and Dr. Stephanie Threatt for nitrogenase discussions. Discussions with Prof. Ralf Thauer on unpublished proton inventory studies with methyl coenzyme M reductase and Prof. James Howard (1942–2022) on nitrogenase, and enzyme mechanisms generally, are gratefully acknowledged. This work was generously funded by grants from the National Institute of Health (NIH GM045162) and the Howard Hughes Medical Institute to DCR and the Parsons Graduate Fellowship to SGM. This paper was adapted from Siobhán MacArdle’s PhD thesis from the California Institute of Technology.
Group:Resnick Sustainability Institute
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Ralph M. Parsons FoundationUNSPECIFIED
Issue or Number:46
Record Number:CaltechAUTHORS:20221128-494241100.48
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:118086
Deposited By: Research Services Depository
Deposited On:21 Dec 2022 17:17
Last Modified:21 Dec 2022 17:17

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