Nitric-oxide dioxygenase activity and function of flavohemoglobins. sensitivity to nitric oxide and carbon monoxide inhibition.
Widely distributed flavohemoglobins (flavoHbs) function as NO dioxygenases and confer upon cells a resistance to NO toxicity. FlavoHbs from Saccharomyces cerevisiae, Alcaligenes eutrophus, and Escherichia coli share similar spectra, O(2), NO, and CO binding kinetics, and steady-state NO dioxygenation kinetics. Turnover numbers (V(max)) for S. cerevisiae, A. eutrophus, and E. coli flavoHbs are 112, 290, and 365 NO heme(-1) s(-1), respectively, at 37 degrees C with 200 microm O(2). The K(M) values for NO are low and range from 0.1 to 0.25 microm. V(max)/K(M)(NO) ratios of 900-2900 microm(-1) s(-1) indicate an extremely efficient dioxygenation mechanism. Approximate K(M) values for O(2) range from 60 to 90 microm. NO inhibits the dioxygenases at NO:O(2) ratios of > or =1:100 and makes true K(M)(O(2)) values difficult to determine. High and roughly equal second order rate constants for O(2) and NO association with the reduced flavoHbs (17-50 microm(-1) s(-1)) and small NO dissociation rate constants suggest that NO inhibits the dioxygenase reaction by forming inactive flavoHbNO complexes. Carbon monoxide also binds reduced flavoHbs with high affinity and competitively inhibits NO dioxygenases with respect to O(2) (K(I)(CO) = approximately 1 microm). These results suggest that flavoHbs and related hemoglobins evolved as NO detoxifying components of nitrogen metabolism capable of discriminating O(2) from inhibitory NO and CO.
Division of Critical Care Medicine, Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA. gardp0@chmcc.org
P R
PR
Gardner
A M
AM
Gardner
L A
LA
Martin
Y
Y
Dou
T
T
Li
J S
JS
Olson
H
H
Zhu
A F
AF
Riggs
D000421:Alcaligenes / Q000201:enzymology / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002248:Carbon Monoxide / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D006420:Hemeproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D013053:Spectrophotometry
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D006420:Hemeproteins; D008024:Ligands; C086193:flavohemoprotein, Bacteria; D005182:Flavin-Adenine Dinucleotide; D009569:Nitric Oxide; D002248:Carbon Monoxide; D010105:Oxygenases; C114415:nitric oxide dioxygenase; D010100:Oxygen