K-050. Physical and Kinetic Evidence for Direct Reduced Flavin Transfer in the Alkanesulfonate Monooxygenase System

K. Abdurachim1, H. R. Ellis2;
1Yale Univ., New Haven, CT, 2Auburn Univ., Auburn, AL.

The reaction mechanism involving reduced flavin transfer in two-component enzyme systems has currently only been identified in bacteria. In E. coli, the two-component alkanesulfonate monooxygenase is responsible for the liberation of sulfite from alkanesulfonate during sulfur starvation [1]. In this enzyme system, the NAD(P)H-dependent FMN reductase (SsuE) provides reduced flavin to the monooxygenase (SsuD) for the desulfonation of alkanesulfonates in the presence of molecular oxygen [2]. The mechanism of reduced flavin transfer could occur either by free diffusion or direct transfer from SsuE to SsuD requiring the interactions between the two proteins. In this study, the mechanism of reduced flavin transfer between SsuE and SsuD has been investigated through different physical and kinetic approaches. Previous studies have shown that SsuE and SsuD were able to form stable protein-protein interactions as detected by affinity chromatography, chemical cross-linking, and fluorescence spectroscopy [3]. Rapid reaction kinetic analyses were performed to determine if FMN reduction and charge transfer formation by SsuE were altered due to protein-protein interactions with SsuD. Data analyses showed that the rate constant of FMN reduction at 450 nm dramatically increased in the presence of SsuD. Results from data analyses at 550 nm showed that the rate of charge transfer formation of FMN reduction significantly increased in coupled-enzyme reactions. There were no significant spectral and rate constants changes for FMN reduction and charge transfer formation if SsuD or either of its substrates (oxygen or octanesulfonate) was absent. These results suggest that the interactions between SsuE and SsuD affect FMN reduction, which further indicate that the reduced flavin is directly transferred from SsuE to SsuD. Interestingly, the rate of charge transfer formation by SsuE is affected only when SsuD and its biological substrates are present in the reaction, suggesting that there is allosteric communication between the active sites of SsuE and SsuD. Current studies are focused on identifying the amino acid residues involved in protein-protein interactions by chemical cross-linking and mutagenesis.