Faculty Collaboration Database

Evidence That the Pi Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle. Biochemistry 2016 Jul 05;55(26):3625-35

Date

06/14/2016

Pubmed ID

27295169

DOI

10.1021/acs.biochem.6b00421

Scopus ID

2-s2.0-84978081750 (requires institutional sign-in at Scopus site)   102 Citations

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, Pi 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 Pi 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.

Author List

Yang ZY, Ledbetter R, Shaw S, Pence N, Tokmina-Lukaszewska M, Eilers B, Guo Q, Pokhrel N, Cash VL, Dean DR, Antony E, Bothner B, Peters JW, Seefeldt LC

Author

Edwin Antony PhD Assistant Professor in the Biology department at Marquette University

MESH terms used to index this publication - Major topics in bold

Adenosine Triphosphate
Azotobacter vinelandii
Catalysis
Electron Transport
Hydrolysis
Molybdoferredoxin
Nitrogenase
Oxidation-Reduction
Oxidoreductases
Protein Conformation