Negative cooperativity in the nitrogenase Fe protein electron delivery cycle

Karamatullah Danyal, Sudipta Shaw, Taylor R. Page, Simon Duval, Masaki Horitani, Amy R. Marts, Dmitriy Lukoyanov, Dennis R. Dean, Simone Raugei, Brian M. Hoffman, Lance C. Seefeldt, Edwin Antony

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Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen (N2) to two ammonia (NH3) molecules through the participation of its two protein components, the MoFe and Fe proteins. Electron transfer (ET) from the Fe protein to the catalytic MoFe protein involves a series of synchronized events requiring the transient association of one Fe protein with each αβ half of the α2β2 MoFe protein. This process is referred to as the Fe protein cycle and includes binding of two ATP to an Fe protein, association of an Fe protein with the MoFe protein, ET from the Fe protein to the MoFe protein, hydrolysis of the two ATP to two ADP and two Pi for each ET, Pi release, and dissociation of oxidized Fe protein-(ADP)2 from the MoFe protein. Because the MoFe protein tetramer has two separate αβ active units, it participates in two distinct Fe protein cycles. Quantitative kinetic measurements of ET, ATP hydrolysis, and Pi release during the presteady-state phase of electron delivery demonstrate that the two halves of the ternary complex between the MoFe protein and two reduced Fe protein-(ATP)2 do not undergo the Fe protein cycle independently. Instead, the data are globally fit with a two-branch negative-cooperativity kinetic model in which ET in one-half of the complex partially suppresses this process in the other. A possible mechanism for communication between the two halves of the nitrogenase complex is suggested by normal-mode calculations showing correlated and anticorrelated motions between the two halves.

Original languageEnglish (US)
Pages (from-to)E5783-E5791
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number40
StatePublished - Oct 4 2016

Bibliographical note

Funding Information:
We thank members of all our laboratories for critical review of this work and valuable insight. We thank Dr. Martin Webb (Medical Research Council) for providing the expression construct for the phosphate-binding protein, and Dr. Andrew J. Fielding for assistance in data collection. This work is supported in part by the Biological and Electron Transfer and Catalysis program, an Energy Frontiers Research Center (EFRC) funded by the US Department of Energy (DOE), Office of Science Grant DESC0012518. It is also supported by NIH Grants HL 63203 and GM 111097 (to B.M.H.) and R15GM110671 (to E.A.); and the Division of Chemical Sciences, Geosciences, and Bio-Sciences, DOE (S.R.). The protein production, ATP hydrolysis, and stopped-flow electron transfer studies were supported by the EFRC program; phosphate release was supported by the NIH; calculations were supported by the DOE; and rapid-freeze quench and data fitting were supported by the NIH.

Publisher Copyright:
© 2016, National Academy of Sciences. All rights reserved.


  • ATP hydrolysis
  • Allosteric control
  • Conformational control
  • Half-sites reactivity


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