FLAVOHEMOGLOBIN: The sole heme enzyme of Giardia intestinalis is a flavohemoglobin, an enzyme found in other micoorganisms including E. coli. These are a fusion of an amino-terminal heme-binding globin domain and a C-terminal FAD-binding ferredoxin NADP reductase (FNR) domain. Such enzymes are nitric oxide dioxygenases and use oxygen and an electron to oxidize the reactive free radical nitric oxide to nitrate, which is less harmful. The Giardia flavohemoglobin (gFlHb) shares this activity, but it has unusual features of unknown significance. Owing to a pair of sequence inserts in its globin and FNR domains it is larger than all other known flavohemoglobins. These inserts are also predicted to contact each other and to increase the contact surface between domains. We are now studying the effect of mutations within these domains on the nitric oxide dioxygenase activity of gFlHb. We are also studying the role of gFlHb in combatting oxidative stress in Giardia by measure its NADH oxidase activity under different conditions.
We use a free radical analyzer equipped with an NO-sensitive electrode to measure the nitric oxide dioxygenase activity of flavohemoglobins. UV-visible spectrophotometers are used to measure their NADH oxidase activities.
We also use a uv-visible fluorescence microplate reader to develop a rapid screening assay to identify flavohemoglobin inhibitors.