Iron mobilization from intact ferritin: effect of differential redox activity of quinone derivatives with NADH/O2 and in situ-generated ROS.

Ferritins are multimeric nanocage proteins that sequester/concentrate excess of free iron and catalytically synthesize a hydrated ferric oxyhydroxide bio-mineral. Besides functioning as the primary intracellular iron storehouses, these supramolecular assemblies also oversee the controlled release of iron to meet physiologic demands. By virtue of the reducing nature of the cytosol, reductive dissolution of ferritin-iron bio-mineral by physiologic reducing agents might be a probable pathway operating in vivo. Herein, to explore this reductive iron-release pathway, a series of quinone analogs differing in size, position/nature of substituents and redox potentials were employed to relay electrons from physiologic reducing agent, NADH, to the ferritin core. Quinones are well known natural electron/proton mediators capable of facilitating both 1/2 electron transfer processes and have been implicated in iron/nutrient acquisition in plants and energy transduction. Our findings on the structure-reactivity of quinone mediators highlight that iron release from ferritin is dictated by electron-relay capability (dependent on E1/2 values) of quinones, their molecular structure (i.e., the presence of iron-chelation sites and the propensity for H-bonding) and the type/amount of reactive oxygen species (ROS) they generate in situ. Juglone/Plumbagin released maximum iron due to their intermediate E1/2 values, presence of iron chelation sites, the ability to inhibit in situ generation of H2O2 and form intramolecular H-bonding (possibly promotes semiquinone formation). This study may strengthen our understanding of the ferritin-iron-release process and their significance in bioenergetics/O2-based cellular metabolism/toxicity while providing insights on microbial/plant iron acquisition and the dynamic host-pathogen interactions.

Alteration of Coaxial Heme Ligands Reveals the Role of Heme in Bacterioferritin from Mycobacterium tuberculosis.

The uptake and utilization of iron remains critical for the survival/virulence of the host/pathogens in spite of the limitations (low bioavailability/high toxicity) associated with this nutrient. Both the host and pathogens manage to overcome these problems by utilizing the iron repository protein nanocages, ferritins, which not only sequester and detoxify the free Fe(II) ions but also decrease the iron solubility gap by synthesizing/encapsulating the Fe(III)-oxyhydroxide biomineral in its central hollow nanocavity. Bacterial pathogens including Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, encode a distinct subclass of ferritins called bacterioferritin (BfrA), which binds heme, the versatile redox cofactor, via coaxial, conserved methionine (M52) residues at its subunit-dimer interfaces. However, the exact role of heme in Mtb BfrA remains yet to be established. Therefore, its coaxial ligands were altered via site-directed mutagenesis, which resulted in both heme-bound (M52C; ∼1 heme per cage) and heme-free (M52H and M52L) variants, indicating the importance of M52 residues as preferential heme binding axial ligands in Mtb BfrA. All these variants formed intact nanocages of similar size and iron-loading ability as that of wild-type (WT) Mtb BfrA. However, the as-isolated heme-bound variants (WT and M52C) exhibited enhanced protein stability and reductive iron mobilization as compared to their heme-free analogues (M52H and M52L). Further, increasing the heme content in BfrA variants by reconstitution not only enhanced the cage stability but also facilitated the iron mobilization, suggesting the role of heme. In contrary, heme altered the ferroxidase activity to a lesser extent despite facilitating the accumulation of the reactive intermediates formed during the course of the reaction. The current study suggests that heme in Mtb BfrA enhances the overall stability of the protein and possibly acts as an intrinsic electron relay station to influence the iron mineral dissolution and thus may be associated with Mtb's pathogenicity.

Flavin-mediated reductive iron mobilization from frog M and Mycobacterial ferritins: impact of their size, charge and reactivities with NADH/O2.

In vitro, reductive mobilization of ferritin iron using suitable electron transfer mediators has emerged as a possible mechanism to mimic the iron release process, in vivo. Nature uses flavins as electron relay molecules for important biological oxidation and oxygenation reactions. Therefore, the current work utilizes three flavin analogues: riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which differ in size and charge but have similar redox potentials, to relay electron from nicotinamide adenine dinucleotide (NADH) to ferritin mineral core. Of these, the smallest/neutral analogue, RF, released more iron (~ three fold) in comparison to the larger and negatively charged FMN and FAD. Although iron mobilization got marred during the initial stages under aerobic conditions, but increased with a greater slope at the later stages of the reaction kinetics, which gets inhibited by superoxide dismutase, consistent with the generation of O2∙- in situ. The initial step, i.e., interaction of flavins with NADH played critical role in the iron release process. Overall, the flavin-mediated reductive iron mobilization from ferritins occurred via two competitive pathways, involving the reduced form of flavins either alone (anaerobic condition) or in combination with O2∙- intermediate (aerobic condition). Moreover, faster iron release was observed for ferritins from Mycobacterium tuberculosis than from bullfrog, indicating the importance of protein nanocage and the advantages they provide to the respective organisms. Therefore, these structure-reactivity studies of flavins with NADH/O2 holds significance in ferritin iron release, bioenergetics, O2-based cellular toxicity and may be potentially exploited in the treatment of methemoglobinemia. Smaller sized/neutral flavin analogue, riboflavin (RF) exhibits faster reactivity towards both NADH and O2 generating more amount of O2∙- and releases higher amount of iron from different ferritins, compared to its larger sized/negatively charged derivatives such as FMN and FAD.