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 approximately 100 microM, respectively. Individual reduction, ligand binding, and NO dioxygenation reactions were examined at 20 degrees C, where V(max) is approximately 94 s(-1). Reduction by NADH occurs in two steps. NADH reduces bound FAD with a rate constant of approximately 15 microM(-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 microM(-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 approximately 80-fold and reduces NO dioxygenase activity approximately 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 microM(-1) s(-1), demonstrating that the enzyme is extremely efficient at converting toxic NO into nitrate under physiological conditions.
Gardner
A M
AM
Division of Critical Care Medicine, Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA. gardp0@chmcc.org
Martin
L A
LA
Gardner
P R
PR
Dou
Y
Y
Olson
J S
JS
D000596:Amino Acids / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D016296:Mutagenesis; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry*; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism
D000596:Amino Acids; D008024:Ligands; D005182:Flavin-Adenine Dinucleotide; D009569:Nitric Oxide; D014443:Tyrosine; D009249:NADP; D010105:Oxygenases; C114415:nitric oxide dioxygenase; D010100:Oxygen