Covalent modification of nitrogenase MoFe protein by ADP.
MgADP- reacted with the nitrogenase molybdenum-iron (MoFe) protein of Klebsiella pneumoniae (Kp1) over a period of 2 h to yield a stable, catalytically active conjugate. The isolated protein exhibited a new, broad 31P NMR resonance at -1 p.p.m. lacking phosphorus J coupling. The adenine ring of [8-14C]ADP remained associated with the conjugate. A covalently bound nucleotide was identified as AMP by NMR and TLC. Extended dialysis of Kp1 against MgADP- resulted in further AMP binding 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 course of the formation of the Kp1-AMP was altered by the nitrogenase iron protein (Kp2) and was dependent on redox potential. Kp1-AMP was stable to concentration and oxidation with ferricyanide ion at -350 mV. Slow hydrolysis of Kp1-AMP over a period of 6 h yielded AMP and unaltered Kp1. The adenine ring of ADP exchanged with adenine of MgATP2- during reductant-limited turnover of nitrogenase under N2, indicating reversibility of ATP hydrolysis at 15 degrees C. [32P]Pi exchanged with the terminal phosphate group of both ADP and ATP on incubation with Kp1. 32P exchange and the catalytic activity of Kp1 were inhibited by a 20-fold molar excess of the lysine-modifying reagent, o-phthalaldehyde (OPT). Preincubation with MgADP- protected against OPT inactivation. Two potentially reactive lysine residues on the alpha chain of the MoFe protein near a putative hydrophobic docking site for the nitrogenase 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 counterpart in known protein-nucleotide interactions.
Miller
R W
RW
The Nitrogen Fixation Laboratory, The Joseph Chatt Building, John Innes Centre, Colney Lane, Norwich NR4 7UH, U.K.
Eady
R R
RR
Gormal
C
C
Fairhurst
S A
SA
Smith
B E
BE
D000244:Adenosine Diphosphate / Q000737:chemistry*; D007711:Klebsiella pneumoniae / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008983:Molybdoferredoxin / Q000737:chemistry*; D009591:Nitrogenase / Q000737:chemistry*; D011485:Protein Binding
D008983:Molybdoferredoxin; D000244:Adenosine Diphosphate; D009591:Nitrogenase