Evidence That the Pi Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle.
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.
Department of Chemistry and Biochemistry, Utah State University , Logan, Utah 84322, United States.
Zhi-Yong
ZY
Yang
Department of Chemistry and Biochemistry, Utah State University , Logan, Utah 84322, United States.
Rhesa
R
Ledbetter
Department of Chemistry and Biochemistry, Utah State University , Logan, Utah 84322, United States.
Sudipta
S
Shaw
Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States.
Natasha
N
Pence
Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States.
Monika
M
Tokmina-Lukaszewska
Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States.
Brian
B
Eilers
Department of Chemistry and Biochemistry, Utah State University , Logan, Utah 84322, United States.
Qingjuan
Q
Guo
Department of Biological Sciences, Marquette University , Milwaukee, Wisconsin 53201, United States.
Nilisha
N
Pokhrel
Department of Biochemistry, Virginia Tech , Blacksburg, Virginia 24061, United States.
Valerie L
VL
Cash
Department of Biochemistry, Virginia Tech , Blacksburg, Virginia 24061, United States.
Dennis R
DR
Dean
Department of Biological Sciences, Marquette University , Milwaukee, Wisconsin 53201, United States.
Edwin
E
Antony
Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States.
Brian
B
Bothner
Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States.
John W
JW
Peters
Department of Chemistry and Biochemistry, Utah State University , Logan, Utah 84322, United States.
Lance C
LC
Seefeldt
D000255:Adenosine Triphosphate / Q000378:metabolism*; D016948:Azotobacter vinelandii / Q000378:metabolism*; D002384:Catalysis; D004579:Electron Transport; D006868:Hydrolysis; D008983:Molybdoferredoxin / Q000737:chemistry / Q000378:metabolism*; D009591:Nitrogenase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D011487:Protein Conformation
D008983:Molybdoferredoxin; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase