Ferroxidase Activity Research Articles

Ferritin with methylglyoxal produces reactive oxygen species but remains functional

Iron is necessary for life, but the simultaneous iron-catalyzed formation of reactive oxygen species (ROS) is involved in pathogenesis of many diseases. One of them is diabetes mellitus, a widespread disease with severe long-term complications, including neuropathy, retinopathy, and nephropathy. Much evidence points to methylglyoxal, a potent glycating agent, as the key mediator of diabetic complications. In diabetes, there is also a peculiar dysregulation of iron homeostasis, leading to an expansion of redox-active iron. This in vitro study focuses on the interaction of methylglyoxal with ferritin, which is the main cellular protein for iron storage. Methylglyoxal effectively liberates iron from horse spleen ferritin, as well as synthetic iron cores; in both cases, it is partially mediated by superoxide. The interaction of methylglyoxal with ferritin increases the production of hydrogen peroxide, much above the generation of peroxide by methylglyoxal alone, in an iron-dependent manner. Glycation with methylglyoxal results in structural changes in ferritin. All of these findings can be demonstrated with pathophysiologically relevant (submillimolar) methylglyoxal concentrations. However, the rate of iron release by ascorbate, the ferroxidase activity, or the diameter of gated pores even in intensely glycated ferritin is not altered. In conclusion, although the functional features of ferritin resist alterations due to glycation, the interaction of methylglyoxal with ferritin liberates iron and markedly increases ROS production, both of which could enhance oxidative stress in vivo. Our findings may have implications for the pathogenesis of long-term diabetic complications, as well as for the use of ferritin as a nanocarrier in chemotherapy.

Cage Architecture and Reactivity are Preserved in Cysteine‐mutated Ferritin

The role of solvent‐exposed cysteines as reactive sites for bioconjugation with a variety of metal drugs or fluorescent probes is emerging in the use of human H‐ferritin (HuHf) as a nanocarrier for targeting various tumor cell lines. Here, we have investigated the role of these residues in controlling the structural stability and ferroxidase activity by analyzing the properties of three variants: C90A, C90AC102A, and C130A. Protein folding and cage assembly were unchanged. Ferroxidase activity was also unaffected, indicating that these cysteines are not involved in either the catalytic activity or the overall iron(II) uptake process. For comparison, activity measurements were also performed on two derivatives containing the solvent‐exposed cysteine residues.

A new spectrophotometric method for measuring ceruloplasmin ferroxidase activity: an innovative approach.

Ferroxidases are enzymes that participate in the iron metabolism of different organisms. They catalyze the oxidation of ferrous iron, Fe2⁺, into ferric iron, Fe3⁺, which is essential in iron homeostasis and physiological functioning. The present study describes a novel spectrophotometric method of serum ceruloplasmin ferroxidase activity. This method is easy to perform; it is also sensitive, specific, and rapid. In this method, ferrous ions are used as a substrate for the enzyme, with either salicylic acid or sulfosalicylic acid being taken as a chromogenic compound. These chromogens easily form a colored complex with ferric ions but are not formed with ferrous ions. In the enzymatic reaction, the ceruloplasmin ferroxidase enzyme catalyzes the oxidation of ferrous to ferric ions. The resulting increase in ferric ion concentration is then measured spectrophotometrically, following the formation of the colored complex. The complex formed has maximum absorbance at 540 nm in the case of salicylic acid and 490 nm in the case of sulfosalicylic acid. Comparatively, it was tested against the standard method to ascertain the new method's effectuality and reliability for assaying ferroxidase activity. The determined correlation coefficient amounted to 0.99, showing a strong correlation between the results obtained by the two methods. This new spectrophotometric technique offers a simplified, sensitive, specific, and fast means of estimating ferroxidase activity. It avoids using concentrated strong acids in the procedure and correlates excellently with the standard technique. This sets up a potential alternative for accurately determining ferroxidase activity in biological samples.

High-Yield Expressed Human Ferritin Heavy-Chain Nanoparticles in K. marxianus for Functional Food Development.

The use of Generally Recognized as Safe (GRAS)-grade microbial cell factories to produce recombinant protein-based nutritional products is a promising trend in developing food and health supplements. In this study, GRAS-grade Kluyveromyces marxianus was employed to express recombinant human heavy-chain ferritin (rhFTH), achieving a yield of 11 g/L in a 5 L fermenter, marking the highest yield reported for ferritin nanoparticle proteins to our knowledge. The rhFTH formed 12 nm spherical nanocages capable of ferroxidase activity, which involves converting Fe2+ to Fe3+ for storage. The rhFTH-containing yeast cell lysates promoted cytokine secretion (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and -1β (IL-1β)) and enhanced locomotion, pharyngeal pumping frequency, egg-laying capacity, and lifespan under heat and oxidative stress in the RAW264.7 mouse cell line and the C. elegans model, respectively, whereas yeast cell lysate alone had no such effects. These findings suggest that rhFTH boosts immunity, holding promise for developing ferritin-based food and nutritional products and suggesting its adjuvant potential for clinical applications of ferritin-based nanomedicine. The high-yield production of ferritin nanoparticles in K. marxianus offers a valuable source of ferritin for the development of ferritin-based products.

Ceruloplasmin levels in β-thalassaemia major: therapeutic insights and implications for iron homeostasis

Ceruloplasmin (CP) is an enzyme that has ferroxidase activity and is important in maintaining iron homeostasis and serving as a copper-transporter in the bloodstream. Beta-thalassemia major (BTM), a common hereditary disorder in Iraq, can affect CP activity in patients with iron overload resulting from frequent blood transfusions in order to sustain haemoglobin levels. The current study is a single-center observational research. CP activity was measured in 304 patients (120 females and 184 males). Anthropometric indices were recorded and the iron load status was determined by measuring serum ferritin. Ninety-two healthy individuals were also included as a control group. Our findings re¬vealed no relationship between age, body mass index, or plasma ferritin and CP activity in BTM patients. No significant influence of sex on CP activity was observed. The outcomes provide insight into assumed pathways regulating CP, and add to the growing body of research on CP’s contribution to iron metabolism in BTM patients. To our knowledge, this work is the only study of its kind in Iraq, provides the groundwork for upcoming studies and potential therapeutic lines by generating insightful data on the multifaceted relationships between iron homeostasis, CP, and BTM.

Evidence for disrupted copper availability in human spinal cord supports CuII(atsm) as a treatment option for sporadic cases of ALS

The copper compound CuII(atsm) has progressed to phase 2/3 testing for treatment of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). CuII(atsm) is neuroprotective in mutant SOD1 mouse models of ALS where its activity is ascribed in part to improving availability of essential copper. However, SOD1 mutations cause only ~ 2% of ALS cases and therapeutic relevance of copper availability in sporadic ALS is unresolved. Herein we assessed spinal cord tissue from human cases of sporadic ALS for copper-related changes. We found that when compared to control cases the natural distribution of spinal cord copper was disrupted in sporadic ALS. A standout feature was decreased copper levels in the ventral grey matter, the primary anatomical site of neuronal loss in ALS. Altered expression of genes involved in copper handling indicated disrupted copper availability, and this was evident in decreased copper-dependent ferroxidase activity despite increased abundance of the ferroxidases ceruloplasmin and hephaestin. Mice expressing mutant SOD1 recapitulate salient features of ALS and the unsatiated requirement for copper in these mice is a biochemical target for CuII(atsm). Our results from human spinal cord indicate a therapeutic mechanism of action for CuII(atsm) involving copper availability may also be pertinent to sporadic cases of ALS.

Oxidant and antioxidant balance in children with bacteremia.

There is a crucial balance between oxidant and antioxidant defense mechanisms. We aimed to evaluate the role of the balance of these systems in children with bloodstream infection. We analyzed prospectively oxidant and antioxidant stress parameters from serum samples of children with BSI besides demographic and clinical data of children. Serum levels of the total antioxidant status (TAS), total oxidant status (TOS), albumin, plasma thiol, disulphide, catalase (CAT), myeloperoxidase (MPO), ischemia-modified albumin (IMA) levels, ferroxidase and arylesterase (ARES) activity were evaluated in both patients and healthy controls. A total of 113 children were evaluated, 50 of them had bacteremia and the remaining 63 were healthy subjects. The median TOS values were 18.5 µmol H<inf>2</inf>O<inf>2</inf>/L and 13.1 µmol H<inf>2</inf>O<inf>2</inf>/L in patient and control groups, respectively with a statistically significant difference between groups. The mean serum IMA levels were 0.8±0.1 absorbance unit (ABSU) in patients and 0.5±0.09 ABSU in control, the difference between groups was statistically significant. The native thiol, total thiol levels and the disulphide levels were significantly lower in the patient group as compared with the control group. The myeloperoxidase level was 136 U/L in patients and 107 in controls with a statistically significant difference between groups. TOS, IMA, MPO, and particularly plasma thiols seem good candidates for accurate diagnosis of bacteremia in children.

Low ceruloplasmin levels exacerbate retinal degeneration in a hereditary hemochromatosis model.

In a previous report, a 39-year-old patient with high serum iron levels from hereditary hemochromatosis (HH) was diagnosed with a form of retinal degeneration called bull's eye maculopathy. This is atypical for patients with HH, so it was theorized that the low serum levels of ferroxidase ceruloplasmin (CP) of this patient coupled with the high iron levels led to the retinal degeneration. CP, by oxidizing iron from its ferrous to ferric form, helps prevent the oxidative damage caused by ferrous iron. To test this, a hepcidin knockout (KO) mouse model of HH was combined with Cp KO to test whether the combination would lead to more severe retinal degeneration. Monthly in vivo retinal images were acquired and, after 11 months, mice were euthanized for further analyses. Both heterozygous and homozygous Cp KO increased the rate and severity of retinal degeneration. These results demonstrate the protective role of CP, which is most likely owing to its ferroxidase activity. The findings suggest that CP levels may influence the severity of retinal degeneration, especially in individuals with high serum iron.

Determination of Ceruloplasmin Ferroxidase Activity, Iron &amp; Transferrin in Patients with Brain Tumor

Serum from healthy volunteers (n=30) was found to contain little ceruloplasmin (Cp) ferroxidase activity. Higher level of activity was found in the sera that obtained from patients (n=64) with benign and malignant brain tumors. The highest activity was measured in sera of malignant tumors patients. The differences were significant between Cp activity in sera of both type of patients in comparison to that of the control healthy group (p&lt;0.05), and between benign patients with that with malignant brain tumors (p&lt;0.01). Also the results of the current work illustrated the presence of a significant increase in [iron] (p&lt;0.01), as well as total iron binding capacity, in sera of the patients groups in comparison to that of healthy group. The increase in iron concentration was found to be accompanied with a significant decrease in transferrin concentration. This lead to an increase in the concentration of free iron, which is one of the pro oxidants that cause production of more free radicals.

Revisit the E2 Domain of Amyloid Precursor Protein: Ferroxidase, Superoxide and Peroxynitrite Scavenging Activities.

Amyloid precursor protein (APP) is the biological precursor of β-amyloids, a known histopathological hallmark associated with Alzheimer's disease (AD). The function of APP is of great interest yet remains elusive. One of the extracellular domains of APP, the E2 domain, has been proposed to possess ferroxidase activity and affect neuronal iron homeostasis. However, contradicting evidence has been reported, and its precise role remains inconclusive. Here, we studied the Cu-binding site of the E2 domain using extended X-ray absorption fine structure (EXAFS), UV-vis, and electron paramagnetic resonance (EPR) and discovered that a new labile water ligand coordinates to the Cu(II) cofactor in addition to the four known histidines. We explored the proposed ferroxidase activity of the Cu(II)-E2 domain through reactions with ferrous iron and observed single-turnover ferrous oxidation activity with a rate up to 1.0 × 102 M-1 s-1. Cu(I)-E2 reacted with molecular oxygen at a rate of only 5.3 M-1 s-1, which would restrict any potential multiturnover ferroxidase activity to this slow rate and prevents observation of activity under multiturnover conditions. The positive electrostatic potential surface of the protein indicates possible reactivity with negatively charged small substrates such as superoxide radicals (O2•-) and peroxynitrite (ONOO-) that are major contributors to the oxidative stress prevalent in the extracellular environment. Our assays showed that Cu(I)-E2 can remove O2•- at a rate of 1.6 × 105 M-1 s-1, which is slower than the rates of native SODs. However, the reaction between Cu(I)-E2 and ONOO- achieved a rate of 1.1 × 105 M-1 s-1, comparable to native ONOO- scavenger peroxiredoxins (105-107 M-1 s-1). Therefore, the E2 domain of APP can serve as an enzymatic site that may function as a ferroxidase under substrate-limiting conditions, a supplemental O2•- scavenger, and an ONOO- remover in the vicinity of the cellular iron efflux channel and protect neuron cells from reactive oxygen species (ROS) and reactive nitrogen species (RNS) damage.

Functional Characterization of Salmonella Typhimurium Encoded YciF, a Domain of Unknown Function (DUF892) Family Protein, and Its Role in Protection during Bile and Oxidative Stress.

YciF (STM14_2092) is a member of the domain of unknown function (DUF892) family. It is an uncharacterized protein involved in stress responses in Salmonella Typhimurium. In this study, we investigated the significance of YciF and its DUF892 domain during bile and oxidative stress responses of S. Typhimurium. Purified wild-type YciF forms higher order oligomers, binds to iron, and displays ferroxidase activity. Studies on the site-specific mutants revealed that the ferroxidase activity of YciF is dependent on the two metal binding sites present within the DUF892 domain. Transcriptional analysis displayed that the ΔcspE strain, which has compromised expression of YciF, encounters iron toxicity due to dysregulation of iron homeostasis in the presence of bile. Utilizing this observation, we demonstrate that the bile mediated iron toxicity in ΔcspE causes lethality, primarily through the generation of reactive oxygen species (ROS). Expression of wild-type YciF, but not the three mutants of the DUF892 domain, in ΔcspE alleviate ROS in the presence of bile. Our results establish the role of YciF as a ferroxidase that can sequester excess iron in the cellular milieu to counter ROS-associated cell death. This is the first report of biochemical and functional characterization of a member of the DUF892 family. IMPORTANCE The DUF892 domain has a wide taxonomic distribution encompassing several bacterial pathogens. This domain belongs to the ferritin-like superfamily; however, it has not been biochemically and functionally characterized. This is the first report of characterization of a member of this family. In this study, we demonstrate that S. Typhimurium YciF is an iron binding protein with ferroxidase activity, which is dependent on the metal binding sites present within the DUF892 domain. YciF combats iron toxicity and oxidative damage caused due to exposure to bile. The functional characterization of YciF delineates the significance of the DUF892 domain in bacteria. In addition, our studies on S. Typhimurium bile stress response divulged the importance of comprehensive iron homeostasis and ROS in bacteria.

Functional characterization of the DNA-binding protein from starved cells (DPS) as a molecular chaperone under heat stress

The DNA-binding protein from starved cells, known as DPS, has been characterized as a crucial factor in protecting E. coli from external stresses. The DPS functions in various cellular processes, including protein-DNA binding, ferroxidase activity, compaction of chromosome and regulation of stress resistance gene expression. DPS proteins exist as oligomeric complexes; however, the specific biochemical activity of oligomeric DPS in conferring heat shock tolerance has not been fully understood. Therefore, we investigated the novel functional role of DPS under heat shock. To elucidate the functional role of DPS under heat shock conditions, we purified recombinant GST-DPS protein and demonstrated its thermostability and existence in its highly oligomeric form. Furthermore, we discovered that the hydrophobic region of GST-DPS influenced the formation of oligomers, which exhibited molecular chaperone activity, thereby preventing the aggregation of substrate proteins. Collectively, our findings highlight the novel functional role of DPS, as a molecular chaperone and may confer thermotolerance to E. coli.

OsLPR5 Encoding Ferroxidase Positively Regulates the Tolerance to Salt Stress in Rice.

Salinity is a major abiotic stress that harms rice growth and productivity. Low phosphate roots (LPRs) play a central role in Pi deficiency-mediated inhibition of primary root growth and have ferroxidase activity. However, the function of LPRs in salt stress response and tolerance in plants remains largely unknown. Here, we reported that the OsLPR5 was induced by NaCl stress and positively regulates the tolerance to salt stress in rice. Under NaCl stress, overexpression of OsLPR5 led to increased ferroxidase activity, more green leaves, higher levels of chlorophyll and lower MDA contents compared with the WT. In addition, OsLPR5 could promote the accumulation of cell osmotic adjustment substances and promote ROS-scavenging enzyme activities. Conversely, the mutant lpr5 had a lower ferroxidase activity and suffered severe damage under salt stress. Moreover, knock out of OsLPR5 caused excessive Na+ levels and Na+/K+ ratios. Taken together, our results exemplify a new molecular link between ferroxidase and salt stress tolerance in rice.

Biochemical Characterization of Caenorhabditis elegans Ferritins.

The nematode Caenorhabditis elegans contains genes for two types of ferritin (ftn-1 and ftn-2) that express FTN-1 and FTN-2. We have expressed and purified both proteins and characterized them by X-ray crystallography, cryo-electron microscopy, transmission electron microscopy, dynamic light scattering, and kinetically by oxygen electrode and UV-vis spectroscopy. Both show ferroxidase activity, but although they have identical ferroxidase active sites, FTN-2 is shown to react approximately 10 times faster than FTN-1, with L-type ferritin character over longer time periods. We hypothesize that the large variation in rate may be due to differences in the three- and four-fold channels into the interior of the protein 24-mer. FTN-2 is shown to have a wider entrance into the three-fold channel than FTN-1. Additionally, the charge gradient through the channel of FTN-2 is more pronounced, with Asn and Gln residues in FTN-1 replaced by Asp and Glu residues in FTN-2. Both FTN-1 and FTN-2 have an Asn residue near the ferroxidase active site that is a Val in most other species, including human H ferritin. This Asn residue has been observed before in ferritin from the marine pennate diatom Pseudo-mitzchia multiseries. By replacing this Asn residue with a Val in FTN-2, we show that the reactivity decreases over long time scales. We therefore propose that Asn106 is involved in iron transport from the ferroxidase active site to the central cavity of the protein.

Structural and Biochemical Characterization of Silver/Copper Binding by Dendrorhynchus zhejiangensis Ferritin.

Ferritin with a highly symmetrical cage-like structure is not only key in the reversible storage of iron in efficient ferroxidase activity; it also provides unique coordination environments for the conjugation of heavy metal ions other than those associated with iron. However, research regarding the effect of these bound heavy metal ions on ferritin is scarce. In the present study, we prepared a marine invertebrate ferritin from Dendrorhynchus zhejiangensis (DzFer) and found that it could withstand extreme pH fluctuation. We then demonstrated its capacity to interact with Ag+ or Cu2+ ions using various biochemical and spectroscopic methods and X-ray crystallography. Structural and biochemical analyses revealed that both Ag+ and Cu2+ were able to bind to the DzFer cage via metal-coordination bonds and that their binding sites were mainly located inside the three-fold channel of DzFer. Furthermore, Ag+ was shown to have a higher selectivity for sulfur-containing amino acid residues and appeared to bind preferentially at the ferroxidase site of DzFer as compared with Cu2+. Thus, it is far more likely to inhibit the ferroxidase activity of DzFer. The results provide new insights into the effect of heavy metal ions on the iron-binding capacity of a marine invertebrate ferritin.

Tartrate resistant acid phosphatases and osteodystrophy in chronic renal failure as related to oxidative stress: - tartrate resistant acid phosphatase is a proxidant

Objective: Oxidative stress has emerged as a constant feature of Chronic Renal Failure (CRF), this possibly helps to promote the progression and complications of CRF. This study aims to evaluate the relation of tartrate resistant acid phosphatase (TRACP) to oxidative stress in patients with End Stage Renal Disease (ESRD). Methods: Forty Iraqi patients with ESRD underwent hemodialysis and thirty seven healthy individuals were included in the present study. Several biochemical parameters such as lipid peroxidation through the concentration of malondialdehyde (MDA), [copper], [iron], [transferrin], [uric acid] and ceruloplasmin ferroxidase activity were measured in sera of control and ESRD patients. Results: The results revealed presence of a significant increase (P&lt; 0.001) in both [MDA] and uric acid level in sera of ESRD patients in comparison with that of the control group. Meanwhile a non-significant increase (P&gt;0.05) was found in copper concentration with a significant decrease (P&lt;0.001) in serum [iron], total iron binding capacity (TIBC),unsaturated iron binding capacity (UIBC), and [transferrin] of the patients group in comparison with that of the control group .Moreover no difference was observed in the saturation percentage of transferrin with iron between both groups and non-significant decrease in both the activity and the specific activity of ceruloplasmin ferroxidase (P &gt; 0.05) in sera of the patients group. Conclusion: we concluded that the increased of TRACP activity takes part in the measured oxidative stress (as indicated by the increased MDA level) in Iraqi patients with ESRD.

Asbestos fibers promote iron oxidation and compete with apoferritin enzymatic activity

ABSTRACT Asbestos fibers interact with many different proteins and may affect either their structure or functions. The aim of this study was to determine whether ferritin absorbed onto fibers might modify its ferroxidase activity. By measuring apo-ferritin ferroxidase activity, data demonstrated that ferritin in the presence of fibers did not significantly modify this enzymatic activity. However, fibers in the absence of ferritin promoted ferrous iron oxidation. Evidence suggests that asbestos fibers may promote iron oxidation and subsequently affect cellular iron homeostatic mechanisms.

Multicopper oxidases with laccase-ferroxidase activity: Classification and study of ferroxidase activity determinants in a member from Heterobasidion annosum s. l.

Multi-copper oxidases (MCO) share a common molecular architecture and the use of copper ions as cofactors to reduce O2 to H2O, but show high sequence heterogeneity and functional diversity. Many new emerging MCO genes are wrongly annotated as laccases, the largest group of MCOs, with the widest range of biotechnological applications (particularly those from basidiomycete fungi) due to their ability to oxidise aromatic compounds and lignin. Thus, comprehensive studies for a better classification and structure-function characterisation of MCO families are required. Laccase-ferroxidases (LAC-FOXs) constitute a separate and unexplored group of MCOs with proposed dual features between laccases and ferroxidases. We aim to better define this cluster and the structural determinants underlying putative hybrid activity. We performed a phylogenetic analysis of the LAC-FOXs from basidiomycete fungi, that resulted in two subgroups. This division seemed to correlate with the presence or absence of some of the three acidic residues responsible for ferroxidase activity in Fet3p from Saccharomyces cerevisiae. One of these LAC-FOXs (with only one of these residues) from the fungus Heterobasidion annosum s. l. (HaLF) was synthesised, heterologously expressed and characterised to evaluate its catalytic activity. HaLF oxidised typical laccase substrates (phenols, aryl amines and N-heterocycles), but no Fe (II). The enzyme was subjected to site-directed mutagenesis to determine the key residues that confer ferroxidase activity. The mutated HaLF variant with full restoration of the three acidic residues exhibited efficient ferroxidase activity, while it partially retained the wide-range oxidative activity of the native enzyme associated to laccases sensu stricto.

Complementary Roles of Two DNA Protection Proteins from Deinococcus geothermalis.

The roles of two interrelated DNA protection protein in starved cells (Dps)-putative Dps Dgeo_0257 and Dgeo_0281-as orthologous proteins to DrDps1 for DNA binding, protection, and metal ion sensing were characterised in a Deinococcus geothermalis strain. Dgeo_0257 exhibited high DNA-binding affinity and formed a multimeric structure but lacked the conserved amino acid sequence for ferroxidase activity. In contrast, the Dgeo_0281 (DgDps1) protein was abundant in the early exponential phase, had a lower DNA-binding activity than Dgeo_0257, and was mainly observed in its monomeric or dimeric forms. Electrophoretic mobility shift assays demonstrated that both purified proteins bound nonspecifically to DNA, and their binding ability was affected by certain metal ions. For example, in the presence of ferrous and ferric ions, neither Dgeo_0257 nor Dgeo_0281 could readily bind to DNA. In contrast, both proteins exhibited more stable DNA binding in the presence of zinc and manganese ions. Mutants in which the dps gene was disrupted exhibited higher sensitivity to oxidative stress than the wild-type strain. Furthermore, the expression levels of each gene showed an opposite correlation under H2O2 treatment conditions. Collectively, these findings indicate that the putative Dps Dgeo_0257 and DgDps1 from D. geothermalis are involved in DNA binding and protection in complementary interplay ways compared to known Dps.

Modification of 4-Fold and B-Pores in Bacterioferritin from Mycobacterium tuberculosis Reveals Their Role in Fe2+ Entry and Oxidoreductase Activity

The self-assembled ferritin nanocages, nature's solution to iron toxicity and its low solubility, scavenge free iron to synthesize hydrated ferric oxyhydroxide mineral inside their central cavity by protein-mediated ferroxidase and hydrolytic/nucleation reactions. These complex processes in ferritin commence with the rapid influx of Fe2+ ions via the inter-subunit contact points (i.e., pores/channels). Investigation of these pores as Fe2+ uptake routes in ferritins remains a subject of intense research, in iron metabolism, toxicity, and bacterial pathogenesis, which are yet to be established in the bacterioferritin (BfrA) from Mycobacterium tuberculosis (Mtb). The electrostatic properties of this protein indicate that the 4-fold and B-pores might serve as potential Fe2+ entry routes. Therefore, in the current work, electrostatics at/along these pores was altered by site-directed mutagenesis to establish their role in Fe2+ uptake/oxidation (ferroxidase activity) in Mtb BfrA. Despite forming self-assembled protein nanocompartment, these 4-fold and B-pore variants exhibited partial loss of ferroxidase activity and lower accumulation of transient species, which not only indicated their role in Fe2+ entry but also suggested the existence of multiple pathways. Although the B-pore variants inhibited the rapid ferroxidase activity to a larger extent, they had minimal impact on their cage stability. The current work revealed the relative contribution of these pores toward rapid Fe2+ uptake/oxidation and cage stability, possibly as consequences of their differential symmetry, number of modified residues (at each pore), and heme content. Therefore, these findings may help to understand the role of these pores in iron acquisition and Mtb proliferation under iron-limiting conditions to control its pathogenesis.