A reinvestigation of the pre-steady-state ATPase activity of the nitrogenase from Azotobacter vinelandii.
The pre-steady-state ATPase activity of nitrogenase has been reinvestigated. The exceptionally high burst in the hydrolysis of MgATP by the nitrogenase from Azotobacter vinelandii communicated by Cordewener et al. (1987) [Cordewener J., ten Asbroek A., Wassink H., Eady R. R., Haaker H. & Veeger C. (1987) Eur. J. Biochem. 162, 265-270] was found to be caused by an apparatus artefact. A second possible artefact in the determination of the stoichiometry of the pre-steady-state ATPase activity of nitrogenase was observed. Acid-quenched mixtures of dithionite-reduced MoFe or Fe protein of Azotobacter vinelandii nitrogenase and MgATP contained phosphate above the background level. It is proposed that due to this reaction, quenched reaction mixtures of nitrogenase and MgATP may contain phosphate in addition to the phosphate released by the ATPase activity of the nitrogenase complex. It was feasible to monitor MgATP-dependent pre-steady-state proton production by the absorbance change at 572 nm of the pH indicator o-cresolsulfonaphthalein in a weakly buffered solution. At 5.6 degrees C, a pre-steady-state phase of H+ production was observed, with a first-order rate constant of 2.2 s-1, whereas electron transfer occurred with a first-order rate constant of 4.9 s-1. At 20.0 degrees C, MgATP-dependent H+ production and electron transfer in the pre-steady-state phase were characterized by observed rate constants of 9.4 s-1 and 104 s-1, respectively. The stopped-flow technique failed to detect a burst in the release of protons by the dye-oxidized nitrogenase complex. It is concluded that the hydrolysis rate of MgATP, as judged by proton release, is lower than the rate of electron transfer from the Fe protein to the MoFe protein.
Mensink
R E
RE
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Wassink
H
H
Haaker
H
H
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000201:enzymology*; D003124:Colorimetry; D004227:Dithionite; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007501:Iron; D007700:Kinetics; D008982:Molybdenum; D009591:Nitrogenase / Q000378:metabolism*; D010637:Phenolsulfonphthalein / Q000031:analogs & derivatives; D010648:Phenylacetates / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011522:Protons; D013053:Spectrophotometry
D010648:Phenylacetates; D010710:Phosphates; D011522:Protons; D004227:Dithionite; C026869:2,4-dinitrophenylacetic acid; D008982:Molybdenum; C009743:Cresol red; D000255:Adenosine Triphosphate; D007501:Iron; D009591:Nitrogenase; D000251:Adenosine Triphosphatases; D010637:Phenolsulfonphthalein