| Elevation in the plasma level of homocysteine, a sulfur-containing amino acid, has been identified as a risk factor for cardiovascular disease, neural tube defects, and Alzheimer's disease. Hyperhomocysteinemia can arise from defects in enzymes involved in homocysteine metabolism. In Escherichia coli, the electron-transfer protein flavodoxin is required to maintain activity of cobalamin-dependent methionine synthase, which catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine, forming tetrahydrofolate and methionine. Reduced flavodoxin donates an electron to cob(II)alamin and S-adenosylmethionine provides the methyl donor for reductive methylation of inactive methionine synthase. The reducing equivalents are provided to flavodoxin by NADPH: ferredoxin (flavodoxin) oxidoreductase.;We have identified the surfaces on flavodoxin that bind methionine synthase and flavodoxin reductase. Flavodoxin has been covalently crosslinked to methionine synthase. The crosslinking residues have been identified as glutamate 61 of flavodoxin and lysine 959 of the 38 kDa activation domain of methionine synthase. Using NMR chemical shift mapping, we find that overlapping surfaces on the flavin-containing face of flavodoxin bind to flavodoxin reductase and the 38 kDa activation domain of methionine synthase. Therefore, a functional ternary complex of these three species is unlikely. This finding suggests that flavodoxin-like domains in the homologous cytochrome P450 reductase family of proteins form mutually exclusive complexes with their electron-donating and electron-accepting partners and that these complexes will require conformational changes for interconversion.;We also examine the Ile22Met mutation in E. coli flavodoxin to provide insight into the molecular defect of a common Ile22Met variant in the flavodoxin-like domain of human methionine synthase reductase, the protein that reactivates human methionine synthase. The Ile22Met mutation perturbs resonances in the first helix of flavodoxin, which contains determinants for binding its physiological partners. Additionally, this mutation decreases the affinity of flavodoxin for methionine synthase by a factor of 6 (K d,I22Met = 4.7 muM). We propose a mechanism whereby the Ile22Met mutation alters the structure or position of this helix so that binding is no longer optimal. Thus, information on the binding interactions between E. coli flavodoxin and its physiological partners serves as a foundation for understanding homologous systems, including mutations in their human homologues. |
