Studies to Elucidate the Mechanism of Reduced Flavin Transfer in the Alkanesulfonate Monooxygenase System from Escherichia coli

Abstract

The two-component alkanesulfonate monooxygenase system utilizes reduced flavin as a substrate to catalyze a unique desulfonation reaction during times of sulfur starvation. The flavin reductase (SsuE) provides reduced flavin for the monooxygenase (SsuD) reaction. The mechanism of reduced flavin transfer was analyzed in these studies. The results from affinity chromatography and cross-linking experiments support the formation of a stable complex between SsuE and SsuD enzymes. Interactions between the two proteins did not lead to overall conformational changes in protein structure, as indicated by the results from circular dichroism spectroscopy in the far-UV region. However, subtle changes in the flavin environment of FMN-bound SsuE that occur in the presence of SsuD were identified by circular dichroism spectroscopy in the visible region. These data are supported by the results from fluorescent spectroscopy experiments, where a dissociation constant of 0.002 ± 0.001 µM was obtained for the binding of SsuE to SsuD. A 1:1 stoichiometric ratio for monomeric binding between SsuE and SsuD supports a structural model involving four dimers of SsuE bound to a tetramer of SsuD. The results from cross-linking experiments using a zero length cross-linker suggest that protein-protein interactions might occur between a negatively charge amino acid residue such as aspartate or glutamate of SsuE with a positively charge amino group of SsuD (arginine, lysine, or histidine). However, MALDI-TOF MS failed to determine the exact residues involved in protein-protein interactions due to the low amount of cross-linking products. Rapid reaction kinetic analyses were performed to investigate the effect of protein-protein interactions between SsuE and SsuD on SsuE-catalyzed flavin reduction and charge transfer formation. The results showed that in the presence of SsuD the rate of the third phase of flavin reduction increased approximately 200-fold compared to the reaction by SsuE alone. Furthermore, in a coupled-enzyme system monitored at 550 nm, the rate for the second phase representing the charge transfer formation between FMNH2 and NADP+ showed 10-fold increase compared to FMN reduction by SsuE alone. Taken together, these results suggest that SsuD is essential for efficient reduced flavin transfer. In this case, the reduced flavin may be rapidly transferred from SsuE to SsuD through a channeling mechanism. In addition, the results also suggest that SsuE-catalyzed flavin reduction is affected by the SsuD-catalyzed reaction through allosteric communication.