Tiansheng Li

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 colishare similar spectra, O2, 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 °C with 200 μm O2. The K M values for NO are low and range from 0.1 to 0.25 μm.V max/K M (NO) ratios of 900–2900 μm −1 s−1 indicate an extremely efficient dioxygenation mechanism. ApproximateK M values for O2 range from 60 to 90 μm. NO inhibits the dioxygenases at NO:O2ratios of ≥1:100 and makes true K M (O2) values difficult to determine. High and roughly equal second order rate constants for O2 and NO association with the reduced flavoHbs (17–50 μm −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 O2(K I (CO) = ∼1 μm). These results suggest that flavoHbs and related hemoglobins evolved as NO detoxifying components of nitrogen metabolism capable of discriminating O2 from inhibitory NO and CO.

Identification of the iron-carbonyl stretch in distal histidine mutants of carbonmonoxymyoglobin.

Soret-excitation resonance Raman (RR) spectra are reported for six distal histidine mutants of carbonmonoxymyoglobin including H64A, H64V, H64L, H64I, H64W, and H64W/L29F. Based on 13CO isotope shifts, the iron-carbonyl stretching vibrations are unambiguously identified. The correct assignment of these modes eliminates the differences in the conformational substate occupations predicted by the RR versus IR data.