Steady-state and transient kinetics of Escherichia coli nitric-oxide dioxygenase (flavohemoglobin) -: The B10 tyrosine hydroxyl is essential for dioxygen binding and catalysis

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