The proposed work will focus on the structure and function of protein-bound covalent phosophorus residues in two proteins: Azotobacter vinelandii (strain OP) flavodoxin and bovine milk xanthine oxidase. In the case of flavodoxin, a novel phosphodiester crosslink between a seryl and a threonyl residue has been identified. Work over the past project period has shown this crosslink appears to have a structural function and that cloning the flavodoxin gene and expressing it in E. coli results in formation of the flavodoxin without this covalent phosphorus residue. The PI plans to extend these studies to 1) identify the sequence location of the phosphate-bridged seryl and threonyl residues, 2) examine the comparative physical and chemical properties of the "dephospho" cloned flavodoxin with that of the phospho wild-type form, 3) initiate studies to characterize the enzyme system responsible for incorporation of the phosphodiester linkage into the protein, 4) initiate studies using site-directed mutagenesis as a probe of protein structure and function, 5) identify non-phospho crosslinks suggested to occur in Azotobacter vinelandii (strain 478) flavodoxin and maybe present in flavodoxin isolated from other organisms, and 6) prepare a polyclonal antibody against a synthetic peptide containing a ser-thr phosphodiester linkage to use as a probe to test whether this linkage occurs in proteins in other organisms. Previous work in this laboratory (supported by NSF) has shown bovine milk xanthine oxidase to contain a phosphoseryl residue near the active site Mo center of the enzyme. Preliminary 31P NMR and mass spectroscopic data suggest substrate turnover results in the incorporation of solvent oxygen into this phosphorus residue. Work is proposed to extend these findings to determine if this phosphoseryl residue is the active site nucleophile involved in the mechanism of xanthine oxidation. Evidence for covalent 32P incorporation in the related enzymes: chicken liver xanthine dehydrogenase and rabbit liver aldehyde oxidase will be obtained by cell growth in the presence of 32Pi and using antibodies to probe whether synthesized xanthine dehydrogenase and aldehyde oxidase also contain covalently-bound phosphorus residues. From this laboratory ENDOR data and recent ENDOR data published by Bray; evidence is presented to suggest a phosphorus ligand to the functional Mo center of xanthine oxidase. Further experiments will be done to further determine whether this signal is indeed due to phosphorus and whether that phosphorus is due to the phosphoseryl residue. The results of these studies should expand our understanding of the role of phospho residues in protein structure and function and may have relevance to a wide range of problems in biochemistry.
