STUDIES ON THE INCORPORATION OF A COVALENTLY BOUND DISUBSTITUTED PHOSPHATE RESIDUE INTO AZOTOBACTER-VINELANDII FLAVODOXIN INVIVO

Previous studies have shown the flavodoxin from Azotobacter vinelandii (strain OP, Berkeley) to contain a covalently bound disubstituted phosphate residue [Edmondson and James (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3786-3789]. Phosphorylation of the protein in vivo was investigated by the addition of [32P]phosphate to cells grown under N2-fixing conditions, under conditions of nif-gene repression and under conditions of nif-gene de-repression. Rocket immunoelectrophoresis of cell extracts showed an approx. 5-fold decrease in the concentration of flavodoxin expressed in cells grown in the presence of NH4+ as compared with those grown under N2-fixing conditions. A similar increase in flavodoxin concentration was observed on nif-gene de-repression. Incorporation of [32P]phosphate occurs only into newly synthesized flavodoxin, as observed on SDS/PAGE of immunoprecipitates of cell extracts. Western blots demonstrated no observable precursor forms of flavodoxin. These data provide conclusive evidence for the phosphorylation of Azotobacter strain OP flavodoxin in vivo and suggest that the covalently bound phosphate residue does not exchange with cellular phosphate pools. Thus the role of this phosphodiester cross-link is proposed to be structural rather than regulatory.