Mechanisms of Iron Oxidation in the Ferroxidase Centre of Escherichia coli Bacterioferritin: A Comparison to Other Ferritins

Ferritins are iron storage proteins that are both widespread and abundant in nature, being an essential means of solubilising and storing iron in a non-toxic manner for most organisms. While ferritins have been known and studied since the mid-20th century, it has become increasingly clear that despite their outward spherical appearances and ferroxidase activity, ferritins have intimate differences in their structures which lead to differing mechanisms of iron storage and an additional role in oxidative stress prevention. Ferritins have been studied rigorously since 1937 (Wang et al., 2010), yet even the mechanisms of the most extensively studied mammalian ferritins, are the subject of debate within the academic community; it is hotly debated whether ferritins operate via a single ‘unifying’ mechanism or through a diverse range of different mechanisms (Hagen, Hagedoorn and Honarmand Ebrahimi, 2017). New ferritins are still discovered; the unprecedented observation of a mixed-valent ferroxidase centre forming in the cyanobacterial ferritin SynFtn directly upon reaction with O2 was incompatible with any reaction scheme currently published in the literature. This unprecedented reactivity was detected again in human mitochondrial ferritin and another newly discovered ferritin, n ferr, in quick succession. The detection of H2O2 as the product of O2 reduction suggested that ferroxidase centres operate in pairs, transferring electrons over long distances. Thus, the experimental results in Chapter 3 may be the early identifiers of a new class of oxidative-stress-protectant ferritins that has as of now gone undescribed. SynFtn (a recently discovered cyanobacterial ferritin) variant proteins where residues in the three-fold channels have been substituted are also discussed here, showing subtle differences in the way iron enters and exits the diiron sites. Unlike many ferritin studies, this thesis has primarily focused on the reactivity of the ferroxidase centre, rather than the ferritin’s mineralisation activity. Characterization of the ferroxidase centres provides information that gives major clues about the functional role of the ferritin in vivo. The kinetics of Fe2+ oxidation at the diiron centres in E. coli bacterioferritin have expanded upon the evidence that the protein exists to prevent oxidative stress. This was performed in novel anaerobic setups by directly measuring the protein’s reactivity with H2O2. UV/ vis spectroscopy highlighted that H2O2 is a preferential oxidant for bacterioferritin, and that there are several optically distinct phases of iron oxidation. Through a collaboration with the Blondin Group, Mössbauer spectroscopy allowed for the assignment of these phases. Chapter 4 details these findings and proposes an updated mechanism that expands upon the existing models. Electron paramagnetic resonance spectroscopy revealed that at supra-stoichiometric concentrations of H2O2, large protein-based free radical signals are observed. Each of the ferritins studied in this thesis form free radicals upon the aerobic addition of Fe2+, and these have been simulated and assigned to a universally conserved tyrosine at the ferroxidase centre. Chapter 5 focuses on electron paramagnetic resonance, depicting microwave power saturation and temperature dependence studies which reveal several free radicals that form in the H2O2-driven Fe2+ oxidation by E. coli bacterioferritin. This is given consideration with respect to the protein’s native role, which may be to prevent uncontrolled Fenton chemistry occurring elsewhere within the bacterium. The experiments and results described in this thesis should be of broad interest to those who study ferritins and iron biochemistry in general.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of Essex

Year dc:date.issued

2020

Author dc:creator

• Pullin, Jacob

• Q Science (General)
• QD Chemistry

Language dc:language

en