Positive cooperativity during Azotobacter vinelandii nitrogenase-catalyzed acetylene reduction.

ABSTRACT

The MoFe protein component of the nitrogenase enzyme complex is the substrate reducing site and contains two sets of symmetrically arrayed metallo centers called the P (Fe8S7) and the FeMoco (MoFe7S9-C-homocitrate) centers. The ATP-binding Fe protein is the specific reductant for the MoFe protein. Both symmetrical halves of the MoFe protein are thought to function independently during nitrogenase catalysis. Forming [AlF4]- transition-state complexes between the MoFe protein and the Fe protein of Azotobacter vinelandii ranging from 0 to 2 Fe protein/MoFe protein produced a series of complexes whose specific activity decreases with increase in bound Fe protein/MoFe protein ratio. Reduction of 2H+ to H2 was inhibited in a linear manner with an x-intercept at 2.0 with increasing Fe protein binding, whereas acetylene reduction to ethylene decreased more rapidly with an x-intercept near 1.5. H+ reduction is a distinct process occurring independently at each half of the MoFe protein but acetylene reduction decreases more rapidly than H+ reduction with increasing Fe protein/MoFe protein ratio, suggesting that a response is transmitted between the two αß halves of the MoFe protein for acetylene reduction as Fe protein is bound. A mechanistic model is derived to investigate this behavior. The model predicts that each site functions independently for 2H+ reduction to H2. For acetylene reduction, the model predicts positive (synchronous) not negative cooperativity arising from acetylene binding to both sites before substrate reduction occurs. When this model is applied to inhibition by Cp2 and modified Av2 protein (L127∆) that form strong, non-dissociable complexes, positive cooperativity is absent and each site acts independently. The results suggest a new paradigm for the catalytic function of the MoFe protein during nitrogenase catalysis.