Publications

• Nitric oxide dioxygenase: an enzymic function for flavohemoglobin.

  Gardner P.R., Gardner A.M., Martin L.A., Salzman A.L.

  Nitric oxide (NO*) is a toxin, and various life forms appear to have evolved strategies for its detoxification. NO*-resistant mutants of Escherichia coli were isolated that rapidly consumed NO*. An NO*-converting activity was reconstituted in extracts that required NADPH, FAD, and O2, was cyanide- ... >> More

  Nitric oxide (NO*) is a toxin, and various life forms appear to have evolved strategies for its detoxification. NO*-resistant mutants of Escherichia coli were isolated that rapidly consumed NO*. An NO*-converting activity was reconstituted in extracts that required NADPH, FAD, and O2, was cyanide-sensitive, and produced NO3-. This nitric oxide dioxygenase (NOD) contained 19 of 20 N-terminal amino acids identical to those of the E. coli flavohemoglobin. Furthermore, NOD activity was produced by the flavohemoglobin gene and was inducible by NO*. Flavohemoglobin/NOD-deficient mutants were also sensitive to growth inhibition by gaseous NO*. The results identify a function for the evolutionarily conserved flavohemoglobins and, moreover, suggest that NO* detoxification may be a more ancient function for the widely distributed hemoglobins, and associated methemoglobin reductases, than dioxygen transport and storage. << Less

  Proc. Natl. Acad. Sci. U.S.A. 95:10378-10383(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Flavohemoglobin detoxifies nitric oxide in aerobic, but not anaerobic, Escherichia coli. Evidence for a novel inducible anaerobic nitric oxide-scavenging activity.

  Gardner A.M., Gardner P.R.

  Nitric-oxide dioxygenase (NOD) and reductase (NOR) activities of flavohemoglobin (flavoHb) have been suggested as mechanisms for NO metabolism and detoxification in a variety of microbes. Mechanisms of NO detoxification were tested in Escherichia coli using flavoHb-deficient mutants and overexpres ... >> More

  Nitric-oxide dioxygenase (NOD) and reductase (NOR) activities of flavohemoglobin (flavoHb) have been suggested as mechanisms for NO metabolism and detoxification in a variety of microbes. Mechanisms of NO detoxification were tested in Escherichia coli using flavoHb-deficient mutants and overexpressors. flavoHb showed negligible anaerobic NOR activity and afforded no protection to the NO-sensitive aconitase or the growth of anoxic E. coli, whereas the NOD activity and the protection afforded with O(2) were substantial. A NO-inducible, O(2)-sensitive, and cyanide-resistant NOR activity efficiently metabolized NO and protected anaerobic cells from NO toxicity independent of the NOR activity of flavoHb. flavoHb possesses nitrosoglutathione and nitrite reductase activities that may account for the protection it affords against these agents. NO detoxification by flavoHb occurs most effectively via O(2)-dependent NO dioxygenation. << Less

  J. Biol. Chem. 277:8166-8171(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Flavohemoglobin and nitric oxide detoxification in the human protozoan parasite Giardia intestinalis.

  Mastronicola D., Testa F., Forte E., Bordi E., Pucillo L.P., Sarti P., Giuffre A.

  Flavohemoglobins (flavoHbs), commonly found in bacteria and fungi, afford protection from nitrosative stress by degrading nitric oxide (NO) to nitrate. Giardia intestinalis, a microaerophilic parasite causing one of the most common intestinal human infectious diseases worldwide, is the only pathog ... >> More

  Flavohemoglobins (flavoHbs), commonly found in bacteria and fungi, afford protection from nitrosative stress by degrading nitric oxide (NO) to nitrate. Giardia intestinalis, a microaerophilic parasite causing one of the most common intestinal human infectious diseases worldwide, is the only pathogenic protozoon as yet identified coding for a flavoHb. By NO amperometry we show that, in the presence of NADH, the recombinant Giardia flavoHb metabolizes NO with high efficacy under aerobic conditions (TN=116+/-10s(-1) at 1microM NO, T=37 degrees C). The activity is [O(2)]-dependent and characterized by an apparent K(M,O2)=22+/-7microM. Immunoblotting analysis shows that the protein is expressed at low levels in the vegetative trophozoites of Giardia; accordingly, these cells aerobically metabolize NO with low efficacy. Interestingly, in response to nitrosative stress (24-h incubation with 5mM nitrite) flavoHb expression is enhanced and the trophozoites thereby become able to metabolize NO efficiently, the activity being sensitive to both cyanide and carbon monoxide. The NO-donors S-nitrosoglutathione (GSNO) and DETA-NONOate mimicked the effect of nitrite on flavoHb expression. We propose that physiologically flavoHb contributes to NO detoxification in G. intestinalis. << Less

  Biochem. Biophys. Res. Commun. 399:654-658(2010) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Nitric-oxide dioxygenase activity and function of flavohemoglobins. Sensitivity to nitric oxide and carbon monoxide inhibition.

  Gardner P.R., Gardner A.M., Martin L.A., Dou Y., Li T., Olson J.S., Zhu H., Riggs A.F.

  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 dioxygenat ... >> More

  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. << Less

  J. Biol. Chem. 275:31581-31587(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Constitutive and adaptive detoxification of nitric oxide in Escherichia coli. Role of nitric-oxide dioxygenase in the protection of aconitase.

  Gardner P.R., Costantino G., Salzman A.L.

  Nitric oxide (NO.) is a naturally occurring toxin that some organisms adaptively resist. In aerobic or anaerobic Escherichia coli, low levels of NO. exposure inactivated the NO.-sensitive citric acid cycle enzyme aconitase, and inactivation was more effective when the adaptive synthesis of NO.-def ... >> More

  Nitric oxide (NO.) is a naturally occurring toxin that some organisms adaptively resist. In aerobic or anaerobic Escherichia coli, low levels of NO. exposure inactivated the NO.-sensitive citric acid cycle enzyme aconitase, and inactivation was more effective when the adaptive synthesis of NO.-defensive proteins was blocked with chloramphenicol. Protection of aconitase in aerobically grown E. coli was dependent upon O2, was potently inhibited by cyanide, and was correlated with an induced rate of cellular NO. consumption. Constitutive and adaptive cellular NO. consumption in aerobic cells was also dependent upon O2 and inhibited by cyanide. Exposure of aerobic cells to NO. accordingly elevated the activity of the O2-dependent and cyanide-sensitive NO. dioxygenase (NOD). Anaerobic E. coli exposed to NO. or nitrate induced a modest O2-independent and cyanide-resistant NO.-metabolizing activity and a more robust O2-stimulated cyanide-sensitive activity. The latter activity was attributed to NOD. The results support a role for NOD in the aerobic detoxification of NO. and suggest functions for NOD and a cyanide-resistant NO. scavenging activity in anaerobic cells. << Less

  J. Biol. Chem. 273:26528-26533(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.