Kinetic parameters and standard redox potentials of FerBHis₆ for the wild-type and mutant variants.

The apparent Michaelis constant (Km) for NADH and the catalytic constant (kcat) were obtained by the initial velocity kinetic analysis at a constant UQ-0 concentration of 0.1 mM. The standard two-electron redox potential (E° ´) was determined spectrophotometrically by equilibrating the protein with a redox dye. The first-order rate constant for flavin cofactor reduction (kobs) was measured upon rapid mixing the enzyme with 5 mM NADH at 10 °C under anoxic conditions. Experimental details on these measurements are given under Experimental Procedures section.