STUDIES OF THE INTERACTION OF NADH OXIDASE FROM THERMUS-THERMOPHILUS HB8 WITH TRIAZINE DYES

NADH oxidase from Thermus thermophilus HB8 was selected to study the i nteraction of flavoproteins with structurally defined dye ligands. The fact that the enzyme binds NADH in addition to FAD favours the enzyme as a model for studying the interaction of enzymes with biomimetic li gands. In addition, the crystal structure of the holoenzyme is known. Applying affinity partitioning in aqueous two-phase systems, differenc e spectroscopy, affinity chromatography and kinetics, results about th e chemistry of binding and the binding site(s) for various triazine dy es were obtained. The binding of the dyes to the enzyme is stabilized by hydrophobic and electrostatic forces. The binding behaviour is infl uenced by small differences in the structure of the dye ligands. In mo st instances the dye ligands occupy both FAD binding sites of the enzy me dimer; this is particularly shown for Procion Red H-8BN by kinetic and difference spectroscopic studies. An ''optimum'' structural model of a biomimetic ligand of NADH oxidase from Thermus thermophilus HB8 i s proposed, requiring the presence of a disulphonated aminonaphthol ri ng combined with another aromatic negatively charged residue and a hyd rophobic arm at a distance between 7 and 13 Angstrom.