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 Saccharomy ces cerevisiae, Alcaligenes eutrophus, and Escherichia coli share similar s pectra, 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 mu M O-2. The K-M values for NO are low and range from 0.1 to 0.25 mu M. V-max/K-M(NO) ratios of 900-2900 mu M-1 s(-1) indicate an extremely efficient dioxygenation mechanism. Approximate K-M values for O- 2 range from 60 to 90 mu M. NO inhibits the dioxygenases at NO:O-2 ratios o f greater than or equal to 1:100 and makes true K-M(O-2) values difficult t o determine, High and roughly equal second order rate constants for O-2 and NO association with the reduced flavoHbs (17-50 mu M-1 s(-1)) and small NO dissociation rate constants suggest that NO inhibits the dioxygenase react ion by forming inactive flavoHbNO complexes. Carbon monoxide also binds red uced flavoHbs with high affinity and competitively inhibits NO dioxygenases with respect to O-2 (K-I(CO) = similar to 1 mu M). These results suggest t hat flavoHbs and related hemoglobins evolved as NO detoxifying components o f nitrogen metabolism capable of discriminating O-2 from inhibitory NO and CO.