Mechanism of N2 Reduction Catalyzed by Fe-Nitrogenase Involves Reductive Elimination of H2

Derek F. Harris, Dmitriy A. Lukoyanov, Sudipta Shaw, Phil Compton, Monika Tokmina-Lukaszewska, Brian Bothner, Neil Kelleher, Dennis R. Dean, Brian M. Hoffman, Lance C. Seefeldt

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Of the three forms of nitrogenase (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase), Fe-nitrogenase has the poorest ratio of N2 reduction relative to H2 evolution. Recent work on the Mo-nitrogenase has revealed that reductive elimination of two bridging Fe-H-Fe hydrides on the active site FeMo-cofactor to yield H2 is a key feature in the N2 reduction mechanism. The N2 reduction mechanism for the Fe-nitrogenase active site FeFe-cofactor was unknown. Here, we have purified both component proteins of the Fe-nitrogenase system, the electron-delivery Fe protein (AnfH) plus the catalytic FeFe protein (AnfDGK), and established its mechanism of N2 reduction. Inductively coupled plasma optical emission spectroscopy and mass spectrometry show that the FeFe protein component does not contain significant amounts of Mo or V, thus ruling out a requirement of these metals for N2 reduction. The fully functioning Fe-nitrogenase system was found to have specific activities for N2 reduction (1 atm) of 181 ± 5 nmol NH3 min-1 mg-1 FeFe protein, for proton reduction (in the absence of N2) of 1085 ± 41 nmol H2 min-1 mg-1 FeFe protein, and for acetylene reduction (0.3 atm) of 306 ± 3 nmol C2H4 min-1 mg-1 FeFe protein. Under turnover conditions, N2 reduction is inhibited by H2 and the enzyme catalyzes the formation of HD when presented with N2 and D2. These observations are explained by the accumulation of four reducing equivalents as two metal-bound hydrides and two protons at the FeFe-cofactor, with activation for N2 reduction occurring by reductive elimination of H2.

Original languageEnglish (US)
Pages (from-to)701-710
Number of pages10
Issue number5
StatePublished - Feb 6 2018

Bibliographical note

Funding Information:
*E-mail: Phone: +1-847-491-3104 (B.M.H.). *E-mail: Phone: +1-435-797-3964 (L.C.S.). ORCID Derek F. Harris: 0000-0003-4277-2976 Neil Kelleher: 0000-0002-8815-3372 Brian M. Hoffman: 0000-0002-3100-0746 Lance C. Seefeldt: 0000-0002-6457-9504 Funding Support for the cell growth, protein purification, and kinetic analysis was provided by the U.S. Department of Energy, Office of Science, Basic Energy Sciences (BES) under awards to L.C.S. and D.R.D. (DE-SC0010687 and DE-SC0010834). Support for HD exchange measurements was provided by a grant from the National Institutes of Health to B.M.H. (GM111097). Protein mass spectrometry work was done as part of the Biological Electron Transfer and Catalysis (BETCy) Energy Frontiers Research Center (EFRC) supported by the Department of Energy (DE-SC0012518). The Mass Spectrometry Facility at MSU is supported in part by the Murdock Charitable Trust and an NIH IDEA program grant P20GM103474. Notes The authors declare no competing financial interest. Data sets in comma delimited format for Figures 2−5 are available at DOI: 10.5281/zenodo.1040952.

Publisher Copyright:
© 2017 American Chemical Society.


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