Evidence That the P_i Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle

Authors

Zhi-Yong Yang, Utah State University
Rhesa Ledbetter, Utah State University
Sudipta Shaw, Utah State UniversityFollow
Natasha Pence, Montana State University-Bozeman
Monika Tokmina-Lukaszewska, Montana State University-Bozeman
Brian Eilers, Montana State University-Bozeman
Qingjuan Guo, Utah State University
Nilisha Pokhrel, Marquette University
Valerie L. Cash, Virginia Polytechnic Institute and State University
Dennis R. Dean, Virginia Polytechnic Institute and State UniversityFollow
Edwin Antony, Marquette UniversityFollow
Brian Bothner, Montana State University-Bozeman
John W. Peters, Montana State University-BozemanFollow
Lance C. Seefeldt, Utah State University

Document Type

Article

Language

eng

Publication Date

7-5-2016

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

Abstract

Nitrogenase reduction of dinitrogen (N₂) to ammonia (NH₃) involves a sequence of events that occur upon the transient association of the reduced Fe protein containing two ATP molecules with the MoFe protein that includes electron transfer, ATP hydrolysis, P_i release, and dissociation of the oxidized, ADP-containing Fe protein from the reduced MoFe protein. Numerous kinetic studies using the nonphysiological electron donor dithionite have suggested that the rate-limiting step in this reaction cycle is the dissociation of the Fe protein from the MoFe protein. Here, we have established the rate constants for each of the key steps in the catalytic cycle using the physiological reductant flavodoxin protein in its hydroquinone state. The findings indicate that with this reductant, the rate-limiting step in the reaction cycle is not protein–protein dissociation or reduction of the oxidized Fe protein, but rather events associated with the P_i release step. Further, it is demonstrated that (i) Fe protein transfers only one electron to MoFe protein in each Fe protein cycle coupled with hydrolysis of two ATP molecules, (ii) the oxidized Fe protein is not reduced when bound to MoFe protein, and (iii) the Fe protein interacts with flavodoxin using the same binding interface that is used with the MoFe protein. These findings allow a revision of the rate-limiting step in the nitrogenase Fe protein cycle.

Comments

Accepted version. Biochemistry, Vol. 55, No. 26 (July 5, 2016): 3625-3635. DOI. © 2016 American Chemical Society. Used with permission.

Recommended Citation

Yang, Zhi-Yong; Ledbetter, Rhesa; Shaw, Sudipta; Pence, Natasha; Tokmina-Lukaszewska, Monika; Eilers, Brian; Guo, Qingjuan; Pokhrel, Nilisha; Cash, Valerie L.; Dean, Dennis R.; Antony, Edwin; Bothner, Brian; Peters, John W.; and Seefeldt, Lance C., "Evidence That the P_i Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle" (2016). Biological Sciences Faculty Research and Publications. 549.
https://epublications.marquette.edu/bio_fac/549