Active site dynamics in NADH oxidase from Thermus thermophilus studied by NMR spin relaxation.

We have characterized the backbone dynamics of NADH oxidase from Thermus thermophilus (NOX) using a recently-developed suite of NMR experiments designed to isolate exchange broadening, together with (15)N R (1), R (1ρ ), and {(1)H}-(15)N steady-state NOE relaxation measurements performed at 11.7 and 18.8 T. NOX is a 54 kDa homodimeric enzyme that belongs to a family of structurally homologous flavin reductases and nitroreductases with many potential biotechnology applications. Prior studies have suggested that flexibility is involved in the catalytic mechanism of the enzyme. The active site residue W47 was previously identified as being particularly important, as its level of solvent exposure correlates with enzyme activity, and it was observed to undergo "gating" motions in computer simulations. The NMR data are consistent with these findings. Signals from W47 are dynamically broadened beyond detection and several other residues in the active site have significant R ( ex ) contributions to transverse relaxation rates. In addition, the backbone of S193, whose side chain hydroxyl proton hydrogen bonds directly with the FMN cofactor, exhibits extensive mobility on the ns-ps timescale. We hypothesize that these motions may facilitate structural rearrangements of the active site that allow NOX to accept both FMN and FAD as cofactors.