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

• Published in: Biochemical and biophysical research communications Vol. 399; no. 4; pp. 654 - 658
• Main Authors: Mastronicola, Daniela, Testa, Fabrizio, Forte, Elena, Bordi, Eugenio, Pucillo, Leopoldo Paolo, Sarti, Paolo, Giuffrè, Alessandro
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.09.2010
• Subjects: Aerobiosis; Anaerobic protozoa; Detoxification; Flavin; Giardia; Giardia lamblia - drug effects; Giardia lamblia - metabolism; Hemeprotein; Hemoglobins - genetics; Hemoglobins - metabolism; Humans; NAD - metabolism; Nitric Oxide - metabolism; Nitric Oxide Donors - pharmacology; Nitrosative stress; Nitroso Compounds - pharmacology; NO amperometry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; S-Nitrosoglutathione - pharmacology; PBS; NO; Anaerobic protozoa; EDTA; GSNO; Nitrosative stress; NO amperometry; FDP; NOS; Flavin; Hemeprotein; DETA-NONOate; flavoHb
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2010.07.137

► NO degradation by Flavohemoglobin (FlavoHb) was measured by NO amperometry. ► FlavoHb was detected in Giardia intestinalis cells by immunoblotting. ► G. intestinalis cells were exposed to nitrosative stress using nitrite and NO-donors. ► G. intestinalis overexpresses FlavoHb under nitrosative stress conditions. ► The FlavoHb-overexpressing cells degrade NO more efficiently (∼×10) than controls. 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 ± 10 s −1 at 1 μM NO, T = 37 °C). The activity is [O 2]-dependent and characterized by an apparent K M,O2 = 22 ± 7 μM. 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 ⩾5 mM 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.