UBC Theses and Dissertations
Catalysis of iron core formation in Escherichia coli bacterioferritin Wong, Steve
Iron is an essential element for almost all life, so iron homeostasis is an important concern for most living organisms. The chemical properties of iron as represented by the low aqueous solubility of ferric iron and the toxicity of hydroxyl radicals it can produce by means of the Fenton reaction make achievement of iron homeostasis both challenging and crucially important. Bacterioferritin (BFR) is a bacterial member of the ferritin family of proteins that stores iron as a microcrystalline ferric hydroxide core of ~2700 iron atoms. This core is surrounded by 24 identical protein subunits, each possessing a dinuclear iron centre that catalyzes the oxidation of Fe²⁺ to Fe³⁺. This structure affords storage, solubility and bioavailability of iron. To improve our incomplete knowledge of the mechanism of iron core formation, the properties of an assembly variant (Glu128Arg/Glu135Arg) and the wild-type of Escherichia coli BFR have been characterized by X-ray crystallography, site-directed mutagenesis, and iron oxidation kinetics. The crystal structure of the variant included two ethylene glycol (EG) molecules adjacent to the dinuclear (ferroxidase) site that catalyzes iron oxidation. One EG resides in the ferroxidase pore that provides a route from the solvent to the ferroxidase site. The other EG resides at the inner surface of the protein where the iron core presumably binds and is surrounded by three acidic residues: Glu47, Asp50, and Asp126. Kinetics studies revealed that Glu47Gln, Asp50Asn and Asp126Asn substitutions in the assembly variant and the wild-type 24-mer retarded iron core formation and that Glu47 is important in iron oxidation at the ferroxidase site whereas Asp50 and Asp126 are important for iron core nucleation. The 3-fold channel, 4-fold channel, B-site channel and the ferroxidase pore of BFR are possible routes of iron entry for core formation, but disruption of each of these sites individually in the 24-mer did not alter the kinetics of iron core formation. The intermediate states of the dinuclear site during iron oxidation are not well defined, but fast formation and decay of a μ-1,2-peroxodiferric intermediate (ƛmax = 650 nm) has been proposed. This intermediate was detected by multi-wavelength stopped flow kinetic analysis of wild-type BFR.
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