Mass spectrometric analysis of phosphate from β,γ-[¹⁸O]ATP hydrolyzed by Azotobacter vinelandii nitrogenase: Direct evidence for P_γOP_β bond cleavage

Nitrogenase catalyzes reduction-dependent hydrolysis of ATP to ADP and phosphate. It has been generally assumed that the catalytic process involves attack of a nucleophile at the P_γ of ATP by analogy with conventional ATPases, but no direct evidence verifying this assumption has been available. To determine which anhydride PO bond is cleaved by the enzyme, β,γ-[¹⁸O]ATP (75.4% specific enrichment; nonspecific enrichments of 6.0% nonbridging P_γ¹⁸O, 4.0% nonbridging P_β¹⁸O, and 2.0% P_β¹⁸OP_α) was incubated with purified nitrogenase proteins from Azotobacter vinelandii under turnover conditions. The phosphate produced was isolated, derivatized to trimethyl phosphate, and analyzed by high-resolution mass spectrometry. The C₃H₉O₄P C₃H₉O₃¹⁸OP mass ( 140 142) ratio found in the product phosphate derivative was 14.0 ± 2.0 (SE, n = 3). The calculated values for P_γOP_β and P_γOP_β bond breaking based on the known distribution of ¹⁸O in the substrate were 15 and 0.23, respectively. The results establish nitrogenase hydrolysis of the ATP P_γOP_β linkage.