Covalently bound non-coenzyme phosphorus residues in flavoproteins: 31 P nuclear magnetic resonance studies of Azotobacter flavodoxin

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Abstract
In addition to the 5''-phosphate ester on its FMN moiety, flavodoxin from A. vinelandii contains 2 mol of tightly bound phosphate. One non-coenzyme phosphate group is covalently bound to the protein, as it remains with the protein on acid precipitation, whereas the other phosphate is released. The invariance of the 31P NMR chemical shift of the covalently bound phosphate (-0.8 ppm relative to 85% phosphoric acid) with pH, even in the presence of protein denaturants, implies it is in a diester linkage to the protein. Because no evidence could be found for the presence of covalently bound sugars, nucleotides or phospholipids, the phosphate residue probably forms a diester linkage with 2 hydroxyl amino acids in the protein. The only other suggestion of a phosphodiester linkage in proteins is from previous studies on pepsin and pepsinogen. The observed changes in 31P chemical shift with pH show that the covalent P in pepsinogen has ionization properties of a monoester rather than a diester. The 31P resonance of the FMN phosphate occurs at -5.6 ppm in native Azobacter flavodoxin. No ionization of the protein-bound FMN phosphate is observed since the chemical shift does not change appreciably in the pH range of 5.5-9.5. The chemical shift data suggest, but do not prove, that the coenzyme phosphate in its protein-bound form is dianionic. Chemical analysis of several other flavoenzymes from a variety of sources shows the presence of covalently bound P in quantities stoichiometric with the flavin content in most of the enzymes tested. Thus, the presence of covalent P in flavoenzymes may be a general phenomenon with currently unknown catalytic significance.