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Published September 26, 2014 | Accepted Version
Journal Article Open

Ligand binding to the FeMo-cofactor: Structures of CO-bound and reactivated nitrogenase


The mechanism of nitrogenase remains enigmatic, with a major unresolved issue concerning how inhibitors and substrates bind to the active site. We report a crystal structure of carbon monoxide (CO)–inhibited nitrogenase molybdenum-iron (MoFe)–protein at 1.50 angstrom resolution, which reveals a CO molecule bridging Fe2 and Fe6 of the FeMo-cofactor. The μ2 binding geometry is achieved by replacing a belt-sulfur atom (S2B) and highlights the generation of a reactive iron species uncovered by the displacement of sulfur. The CO inhibition is fully reversible as established by regain of enzyme activity and reappearance of S2B in the 1.43 angstrom resolution structure of the reactivated enzyme. The substantial and reversible reorganization of the FeMo-cofactor accompanying CO binding was unanticipated and provides insights into a catalytically competent state of nitrogenase.

Additional Information

Received for publication 29 May 2014. Accepted for publication 31 July 2014. We thank J. Peters, L. Zhang, J. Rittle, C. Morrison, B. Wenke, and K. Dörner for informative discussions. This work was supported by NIH grant GM45162 (D.C.R.), Deutsche Forschungsgemeinschaft grants EI-520/7 and RTG 1976, and the European Research Council N-ABLE project (O.E.). We gratefully acknowledge the Gordon and Betty Moore Foundation, the Beckman Institute, and the Sanofi-Aventis Bioengineering Research Program at Caltech for their generous support of the Molecular Observatory at Caltech and the staff at Beamline 12–2, Stanford Synchrotron Radiation Lightsource (SSRL), for their assistance with data collection. SSRL is operated for the U.S. Department of Energy and supported by its Office of Biological and Environmental Research and by the NIH: National Institute of General Medical Sciences (P41GM103393) and the National Center for Research Resources (P41RR001209). We thank the Center for Environmental Microbial Interactions for its support of microbiology research at Caltech. Coordinates and structure factors have been deposited in the Protein Data Bank of the Research Collaboratory for Structural Bioinformatics, with IDs 4TKV (Av1-CO) and 4TKU (Av1 reactivated).

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August 20, 2023
October 26, 2023