COVALENT MODIFICATION OF NITROGENASE MOFE PROTEIN BY ADP

MgADP(-) reacted with the nitrogenase molybdenum-iron (MoFe) protein o f Klebsiella pneumoniae (Kp1) over a period of 2 h to yield a stable, catalytically active conjugate. The isolated protein exhibited a new, broad P-31 NMR resonance at -1 p.p.m, lacking phosphorus J coupling, T he adenine ring of [8-C-14]ADP remained associated with the conjugate. A covalently bound nucleotide was identified as AMP by NMR and TLC. E xtended dialysis of Kp1 against MgADP(-) resulted in further AMP bindi ng at the protein surface. ADP was initially bound tightly to Kp1 at a site distinct from the AMP sites. ATP did not replace ADP. The time c ourse of the formation of the Kp1-AMP was altered by the nitrogenase i ron protein (Kp2) and was dependent on redox potential. Kp1-AMP was st able to concentration and oxidation with ferricyanide ion at -350 mV, Slow hydrolysis of Kp1-AMP over a period of 6 h yielded AMP and unalte red Kp1. The adenine ring of ADP exchanged with adenine of MgATP(2-) d uring reductant-limited turnover of nitrogenase under N-2, indicating reversibility of ATP hydrolysis at 15 degrees C. [P-32]P-i exchanged w ith the terminal phosphate group of both ADP and ATP on incubation wit h Kpl1 P-32 exchange and the catalytic activity of Kp1 were inhibited by a 20-fold molar excess of the lysine-modifying reagent, o-phthalald ehyde (OPT). Preincubation with MgADP(-) protected against OPT inactiv ation. Two potentially reactive lysine residues on the alpha chain of the MoFe protein near a putative hydrophobic docking site for the nitr ogenase Fe protein are proposed as sites of OPT and nucleotide binding . Azotobacter vinelandii MoFe protein (Av1) also formed an AMP adduct but Kp2 did not. Catalase did not interact with ADP. The reactions of the nitrogenase MoFe protein with adenine nucleotides have no counterp art in known protein-nucleotide interactions.