Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor
Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32-1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.
© 2015, Spatzal et al. This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited. Received September 15, 2015. Accepted December 15, 2015. Published December 16, 2015. We thank J. Rittle, J.C. Peters, B. Wenke, H. Segal, C. Morrison and O. Einsle for informative discussions. This work was supported by NIH grant GM45162 (D.C.R.). 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 DOE and supported by its OBER and by the NIH, NIGMS (P41GM103393) and the NCRR (P41RR001209). We thank the Center for Environmental Microbial Interactions and the William T. Gimbel Discovery fund for their support of microbiology research at Caltech. These authors contributed equally to this work: Thomas Spatzal, Kathryn A Perez. Author contributions: TS, Purified, crystallized and biochemically characterized the enzyme samples, and collected the X-ray crystallographic data; Processed, refined and analyzed the X-ray data; Contributed equally to the study; Discussed the results and participated in writing the manuscript; Initiated and directed this research. KAP, Purified, crystallized and biochemically characterized the enzyme samples, and collected the X-ray crystallographic data; Processed, refined and analyzed the X-ray data; Contributed equally to the study; Discussed the results and participated in writing the manuscript. JBH, Contributed to the experimental design and data interpretation; Discussed the results and participated in writing the manuscript. DCR, Discussed the results and participated in writing the manuscript; Initiated and directed this research, Conception and design, Analysis and interpretation of data, Drafting or revising the article. The authors declare that no competing interests exist. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Reviewing editor: Wilfred A van der Donk, Reviewing editor, University of Illinois at Urbana-Champaign, United States
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