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Steady-state and Transient Kinetics of Escherichia coli Nitric-oxide Dioxygenase (Flavohemoglobin)

Escherichia coli expresses an inducible flavohemoglobin possessing robust NO dioxygenase activity. At 37 °C, the enzyme shows a maximal turnover number (V max) of 670 s−1 andK m values for NADH, NO, and O2 equal to 4.8, 0.28, and ∼100 μm, respectively. Individual reduction, ligand binding, and NO dioxygenation reactions were examined at 20 °C, where V max is ∼94 s−1. Reduction by NADH occurs in two steps. NADH reduces bound FAD with a rate constant of ∼15 μm −1s−1, and heme iron is reduced by FADH2 with a rate constant of 150 s−1. Dioxygen binds tightly to reduced flavohemoglobin, with association and dissociation rate constants equal to 38 μm −1 s−1and 0.44 s−1, respectively, and the oxygenated flavohemoglobin dioxygenates NO to form nitrate. NO also binds reversibly to reduced flavohemoglobin in competition with O2, dissociates slowly, and inhibits NO dioxygenase activity at [NO]/[O2] ratios of 1:100. Replacement of the heme pocket B10 tyrosine with phenylalanine increases the O2 dissociation rate constant ∼80-fold and reduces NO dioxygenase activity ∼30-fold, demonstrating the importance of the tyrosine hydroxyl for O2 affinity and NO scavenging activity. At 37 °C,V max/K m (NO) is 2,400 μm −1 s−1, demonstrating that the enzyme is extremely efficient at converting toxic NO into nitrate under physiological conditions.