Evidence on the role(s) of ATP in the mechanism of nitrogenase, from proton NMR relaxation studies on metal and nucleotide binding to the molybdenum-iron protein.

Interactions between the molybdenum-iron protein (Kp1) of nitrogenase (reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolysing,) EC 1.18.2.1) from Klebsiella pneumoniae and the divalent ions, Mn2+, Mg2+, Ca2+ and Ba2+, have been studied by monitoring the water proton NMR relaxation enhancement caused by the paramagnetism of Mn2+. We observed several binding sites for Mn2+, equivalent within experimental error (Kd = 209 +/- 23 microM), increasing in number from 1.0 to 2.9 per molecule in direct proportion to the specific activity of the protein. Metal binding sites on the MoFe protein are therefore essential to the enzymic function of nitrogenase. A maximum of four such sites is inferred for the fully active protein molecule. All manganese sites can alternatively bind the diamagnetic ions studied, the binding being one order-of-magnitude weaker (Kd = 2.2 +/- 0.3 mM for Mg2+; 1.6 +/- 0.2 mM for Ca2+; 3.4 +/- 0.3 mM for Ba2+), ATP and ADP form ternary complexes via Mn2+ with Kpl. The above data and other evidence on MgATP binding are discussed in terms of the site of hydrolysis of ATP during turnover and its possible bridging role between the two protein components of the enzyme.