Purification and characterization of Flavohemoglobin - A flavoheme enzyme

Abstract

Flavohemoglobin is a flavoheme enzyme that catalyzes dioxygenation of nitric oxide (NO) to nitrate NO3–. NO is diatomic radical gas produced by enzymatic and non-enzymatic oxidation of reduced nitrogenous compounds. NO acts as a membrane-permeable signal molecule in mammals, in addition to being a toxin at higher concentrations and being utilized by the immune system against invading pathogens. FHb is involved in protection against the cytotoxic effects of NO (termed nitrosative stress) and is found in a range of bacterial species and some fungi. FHbs are not found in higher eukaryotes, but is employed by a range of pathogens, and is a potential therapeutic target. In this thesis we sought to investigate the FHb from Bacillus cereus by determination of the X-ray crystal structure as only a few structures of FHb from different organisms have been obtained. The protein was successfully cloned and expressed and a purification protocol has been developed, with ammonium sulfate precipitation, anion exchange chromatography and gel filtration. A high yield with high purity was obtained, but with heme and FAD at substoichiometric levels. Crystallization screening resulted in some small needles grown from precipitate, although they were non-reproducible and no crystal structure was obtained. A heme assay was performed in order to identify the heme/protein ratio and protein assays was performed to calculate protein concentration. Testing of reconstitution with heme and FAD was performed with promising results.