Structural Superimposition

Abstact

Flavodiiron proteins (FDPs) are NO and/or O(2) reductases which contain a di-iron catalytic center. Interestingly, they exhibit different selectivities towards each one of these substrates, despite having the same ligands of the iron ions. Escherichia coli FDP is a selective NO reductase that protects this bacterium against nitric oxide by catalyzing two-electron reduction to the nontoxic N(2)O. Previously, based on kinetic studies, we explored the possible role of two amino acids located in the di-iron second coordination sphere, Lys53 and Tyr271, in modulation of the substrate selectivity of Entamoeba histolytica FDP, a selective O(2) reductase. In this work, we replaced the structurally equivalent residues in E. coli FDP, Asp52 and Ser262, by those present in the O(2)-selective FDP and determined their crystal structures in both oxidized and reduced states. Furthermore, the molecular-substrate tunnels were experimentally identified using krypton pressurization of the crystals. The data obtained corroborated previous molecular-dynamics calculations on this FDP. The side chains of residues in both positions 52 and 262 of E. coli FDP variants and wild type are in the vicinity of the shorter intramolecular tunnel, which is suggested to be the exit route for the reaction products N(2)O and H(2)O. The E. coli FDP S262Y variant shows photoreduction of the di-iron center and partial loss of electron density in some of its coordinating ligands after X-ray exposure, and these effects are consistent with increased radiation sensitivity. The kinetic properties of the variants towards NO and O(2) were not significantly different from the wild type, contrary to what was observed previously for E. histolytica FDP.