Kinetics of MgATP-dependent iron chelation from the Fe-protein of the Azotobacter vinelandii nitrogenase complex. Evidence for two states.
Chelation of Fe from the Fe-protein component (Av2) of Azotobacter vinelandii nitrogenase has been investigated. The chelation, which requires MgATP binding by Av2, is best described as a two-exponential process. The rates for the two phases differed by approximately 10-fold and increased as the concentration of MgATP was increased. The rates for both phases were 50% of maximum at approximately 1.5 mM MgATP. At MgATP concentrations greater than 100 microM, the more rapid phase represented approximately 25% of the total Fe chelated from Av2. However, below 100 microM MgATP, the proportion of the faster phase decreased until at 20 microM MgATP, only a single phase could be detected. The properties of Av2 were studied at various stages of Fe chelation. The partially chelated protein was isolated from the reaction by gel filtration and was subjected to a second MgATP-dependent Fe chelation. Material isolated after the completion of the first phase regained biphasic kinetics in subsequent chelation reactions. However, if MgATP was present during the isolation of Av2, then only a single phase was observed in the subsequent chelation studies. In addition, the enzymatic activity of Av2 decreased concomitantly with total Fe chelation. To account for these observations, a model is presented in which Av2 exists in two conformers. Fe chelation is proposed to occur from either conformer but only when two MgATP are bound. Both conformers bind MgATP with the same affinity but are distinguished by a 10-fold difference in chelation rate. The two conformers are in equilibrium and can interconvert only in the absence of MgATP. That is, MgATP binding prevents the conversion of the two conformational states.
Deits
T L
TL
Department of Biochemistry, University of Minnesota, Minneapolis 55455.
Howard
J B
JB
D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D001395:Azotobacter / Q000201:enzymology*; D002614:Chelating Agents; D007501:Iron / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D007700:Kinetics; D009591:Nitrogenase / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases
D002614:Chelating Agents; D007506:Iron-Sulfur Proteins; D015082:2,2'-Dipyridyl; D000255:Adenosine Triphosphate; D007501:Iron; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase