Evidence that MgATP accelerates primary electron transfer in a Clostridium pasteurianum Fe protein-Azotobacter vinelandii MoFe protein nitrogenase tight complex.
The nitrogenase catalytic cycle involves binding of the iron (Fe) protein to the molybdenum-iron (MoFe) protein, transfer of a single electron from the Fe protein to the MoFe protein concomitant with the hydrolysis of at least two MgATP molecules, followed by dissociation of the two proteins. Earlier studies found that combining the Fe protein isolated from the bacterium Clostridium pasteurianum with the MoFe protein isolated from the bacterium Azotobacter vinelandii resulted in an inactive, nondissociating Fe protein-MoFe protein complex. In the present work, it is demonstrated that primary electron transfer occurs within this nitrogenase tight complex in the absence of MgATP (apparent first-order rate constant k = 0.007 s-1) and that MgATP accelerates this electron transfer reaction by more than 10,000-fold to rates comparable to those observed within homologous nitrogenase complexes (k = 100 s-1). Electron transfer reactions were confirmed by EPR spectroscopy. Finally, the midpoint potentials (Em) for the Fe protein [4Fe-4S]2+/+ cluster and the MoFe protein P2+/N cluster were determined for both the uncomplexed and complexed proteins and with or without MgADP. Calculations from electron transfer theory indicate that the measured changes in Em are not likely to be sufficient to account for the observed nucleotide-dependent rate accelerations for electron transfer.
Chan
J M
JM
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, USA.
Ryle
M J
MJ
Seefeldt
L C
LC
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D016948:Azotobacter vinelandii / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry; D003013:Clostridium / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D007700:Kinetics; D008983:Molybdoferredoxin / Q000737:chemistry*; D009591:Nitrogenase / Q000737:chemistry*; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D013053:Spectrophotometry
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D008983:Molybdoferredoxin; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase