Abstract
Diflavin-linked disulfide oxidoreductase (DDOR) from the cyanobacterium Synechocystis sp. PCC6803 is an NADPH-thioredoxin reductase (NTR) superfamily enzyme that forms a homodimer containing two FAD molecules (FAD1 and FAD2) per monomer. FAD1 is conserved in the NTR superfamily, while FAD2 is characteristic of DDOR. Under aerobic conditions, thiol reduction of DDOR produces an apparently air-stable neutral blue semiquinone, but the respective roles of FAD1 and FAD2 in the reduction process remain unclear. To address this, spectroscopic, structural and mutational analyses were performed to investigate the contributions of the two FAD molecules. Recombinant apo- and holo-DDOR synthesised in Escherichia coli cells were separated and purified. Fluorescence titration of the apo-DDOR with FAD revealed rapid binding of two FADs. Stopped-flow analysis indicated biphasic binding of FAD to apo-DDOR. X-ray crystal structures of apo- and holo-DDORs exhibited pronounced conformational differences in the FAD-binding sites, suggesting that the first FAD may bind to the FAD1-binding site and subsequently induce formation of the FAD2-binding site. Systematic replacement of amino acid residues in the FAD2-binding site showed that mutations at the characteristic loop region bearing the GKKDSLY motif resulted in binding of three flavin species of FAD, flavin mononucleotide and riboflavin. Moreover, replacement of both Trp53 and Tyr143 at the binding site of the isoalloxazine ring of FAD2 with Ala residues yielded exclusively the apo-form. These results indicate that the loop region restricts the flavin species bound at the FAD2-binding site, and Trp53 and Tyr143 are important for FAD binding to both the FAD1- and FAD2-binding sites.