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Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

Iron Mineralizing Bacterioferritin A from Mycobacterium tuberculosis Exhibits Unique Catalase-Dps-like Dual Activities Abhinav Mohanty,† Biswamaitree Subhadarshanee,†,‡ Pallavi Barman,†,§ Chinmayee Mahapatra,†,§ B. Aishwarya,†,§ and Rabindra K. Behera*,† †

Department of Chemistry, National Institute of Technology, Rourkela 769008, Odisha, India School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT), Bhubaneswar 751024, Odisha, India

‡

Inorg. Chem. Downloaded from pubs.acs.org by IDAHO STATE UNIV on 03/28/19. For personal use only.

S Supporting Information *

ABSTRACT: Mycobacterium tuberculosis (Mtb) expresses heme binding protein nanocages, bacterioferritin A (BfrA), along with nonheme bacterioferritin B (BfrB). BfrA is unique to bacteria and, like BfrB, carries out ferroxidase activity to synthesize iron oxide biominerals. The expression of BfrA, in the presence of BfrB, indicates that Mtb may utilize it for some additional purpose apart from its natural iron storage activity. However, the mechanism of ferroxidase activity (iron biomineralization) in Mtb BfrA still remains unexplored. H2O2 is secreted by the host during host−pathogen interaction. In some bacteria, heme containing Bfr and/or Dps (DNA binding protein during starvation) detoxify H2O2 by utilizing it during their ferroxidase activity. Interestingly, Mtb lacks the gene for Dps which protects DNA from H2O2-induced oxidative cleavage. Therefore, the current work investigates the kinetics of O2/H2O2-dependent ferroxidase activity, DNA protection, and catalase-like activity of recombinant Mtb BfrA. Ferroxidase activity by Mtb BfrA was found to proceed via the formation of a transient intermediate and its initial rate exhibited sigmoidal behavior, with increasing Fe2+ concentration. Moreover, Mtb BfrA exhibited catalase-like activity by evolving O2 upon reaction with H2O2, which gets inhibited in the presence of catalase inhibitors (NaN3 and NaCN). In addition, Mtb BfrA protected plasmid DNA from Fenton reagents (Fe2+ and H2O2), similar to Dps, by forming BfrA-DNA complexes. Thereby, Mtb BfrA executes multiple functions (ferroxidase, catalase, and Dps-like activities) in order to cope with the host generated oxidative stress and to promote pathogenesis.

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nanocavity.2,11−14 These ∼445 kDa natural iron repositories are composed of 24 identical subunits (in bacteria) that roughly form a spherical shaped, hollow protein nanocage. Each subunit is comprised of a four-helix bundle carrying the di-iron ferroxidase (Fox) center.15−19 Fe2+ sequestered by ferritin hydrophilic pores gets ferried to the Fox center, where its oxidation takes place.12,17,18,20−24 Oxidized Fe3+ at the Fox center either moves toward the central nanocavity25 or oxidizes the incoming Fe2+ for mineralization.22,26−28 As iron is vital to both the host and the pathogens, it takes the center stage during the host−pathogen battle, where the outcome of the battle decides whether infection will occur or not.4,5 During the course of this clash, H2O2 is secreted by the host to kill pathogens.29−32 Some bacteria have developed a counter mechanism against such activity, and as a result, instead of being exterminated by this hazardous oxidant, they actually utilize it for their survival.33−36 In bacteria, usually three types of ferritin exist: nonheme bacterial ferritin (Ftn), heme binding bacterioferritin (Bfr), and mini-ferritin (Dps).27,37,38 Dps or the mini-ferritin, during starvation,

INTRODUCTION Iron is an essential element for the survival of almost all forms of life,1−3 including pathogens for their virulence.4−8 Apart from playing a crucial role in physiological processes like respiration, gene regulation, DNA synthesis, oxygen transport, this transition element even participates in biocatalytic transformations.3,9 However, the bioavailability of free iron is extremely limited due to the low solubility of Fe3+ (∼10−18 M). On the other hand, Fe2+ can prove to be immensely toxic, if present in excess, particularly in the presence of H2O2, generating the highly deleterious reactive oxygen species (ROS) such as hydroxyl radicals (via Fenton chemistry).10 This reaction causes two potential problems: a drastic decrease in the bioavailability of the essential Fe2+ nutrient; and the oxidation of DNA, lipids and other cellular biomolecules, indiscriminately by the hydroxyl radicals. To fight against the detrimental effects of Fe2+/H2O2, organisms have evolved a multitude of protection mechanisms such as detoxification of H2O2 or inhibition of the Fenton reaction.10 To achieve these, eukaryotic and prokaryotic organisms utilize a family of intracellular iron storage proteins called ferritins, which sequester an excess of free Fe2+ and store it in the form of nontoxic hydrated ferric oxyhydroxide biomineral in its central © XXXX American Chemical Society

Received: September 27, 2018

A

DOI: 10.1021/acs.inorgchem.8b02758 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 1. UV−vis absorption spectra and iron loading ability of heme binding Mtb BfrA. (A) Schematic representation of a 24-mer Mtb BfrA protein, visualized along one of its 12 two-fold (C2) symmetry axis, highlighting the heme binding site. Cartoon representation in the enlarged section indicates the two monomeric polypeptide subunits, while the stick model represents the two methionine residues (Met52; one from each subunit) forming a bis-methionine heme complex at the dimer interface. This image is prepared from PDB ID: 3UOI using PyMOL. (B) Absorption spectra of oxidized (black) and reduced (red; in the presence of Na2S2O4) forms of Mtb BfrA (1.0 μM cage concentration; ∼4 heme/ cage). Inset: Characteristic 623 and 735 nm absorption bands indicative of high-spin Fe3+ heme signature and bis-methionine coordination to heme iron in Mtb BfrA, respectively. (C) Mtb BfrA protein samples were run in a 6% (w/v) nondenaturing gel, at 100 V in ice for 3 h. The gel was further treated with acidified K4[Fe(CN)6] (Prussian blue staining for checking its iron loading ability; top) and Coomassie brilliant blue (for checking intact protein cage; bottom). The ‘+’ and ‘−’ signs indicate Mtb BfrA in its mineralized and apo-form, respectively. The strong intense bands at the bottom of each gel indicates the 24-mer ferritin nanocage, while the weak faint bands at the top correspond to the cage multimers/aggregates of Mtb BfrA. (D) TEM image of iron loaded Mtb BfrA nanocages (1 mg/mL), negatively stained with 4% (w/v) uranyl acetate (Inset: SAED pattern).

additional role of BfrA, apart from functioning as cellular iron repository.44 Moreover, the kinetics/mechanisms of iron uptake/oxidation and mineralization (its natural function) inside the Mtb BfrA nanocavity are not explored and are different for different types of ferritin, even within the heme binding Bfr family (E. coli vs Pseudomonas aeruginosa).26,27,37 Therefore, the current work is targeted toward unravelling the new role of Mtb BfrA. Apart from the rapid iron mineralization, recombinant Mtb BfrA exhibited the dual catalase and Dps-like DNA binding cum protection activities. The current finding, in vitro, indicates how Mtb utilizes BfrA, in vivo, for its defense mechanism (exhibiting Dps and/or catalase-like activities) against Fe2+/H2O2-induced oxidative damage. Further, this study would not only help to understand the mechanism of iron mineralization in BfrA but would also work as a potential principle for developing newer antibacterial agents to prevent the growth of these life-threatening pathogens.45

primarily utilizes H2O2 for ferroxidase activity, synthesizing iron biomineral, instead of generating hydroxyl radical.29,34 Furthermore, some of the Dps (Escherichia coli, Brevibacillus brevis, and Mycobacterium smegmatis) provides resistance to the peroxide-induced oxidative stress by inhibiting DNA cleavage via the rapid formation of highly organized Dps-DNA complexes.29,34 Both the iron mineralizing property of Dps and its complex formation ability with DNA contribute toward DNA protection during Fe2+/H2O2-induced oxidative cleavage. Heme binding Bfr from E. coli and Desulfovibrio vulgaris carries out the oxidoreduction reaction by using either O2 and/or H2O2 as iron(II) oxidant.33,35 Additionally, organisms also make use of catalase, one of the fastest antioxidant enzymes, which disproportionates H2O2, nullifying their role in the generation of hydroxyl radicals, the most reactive among ROS.9,39 Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, encodes both a heme and a nonheme binding bacterioferritin, Mtb BfrA and BfrB, respectively, but it lacks the gene for the structural homologues of Dps.40−42 The overall tertiary/quaternary structure of BfrA is similar to that of BfrB and eukaryotic ferritins. However, the presence of 12 heme binding sites (forming bis-methionine heme complex by two conserved methionine (Met52) residues, one from each of the subunits) at the 12 dimer interfaces (C2 symmetry axes) differentiate BfrA from other ferritins (Figure 1).27,37,40−43 Expression of heme binding BfrA along with nonheme BfrB protein (similar to eukaryotic ferritins and a misnomer in case of Mtb) remains an unsolved puzzle, indicating some

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METHODS

Materials. The plasmid pET21c/BfrA encoding for Mtb BfrA is a kind gift from Dr. Anil K. Tyagi and Dr. Garima Khare, Department of Biochemistry, University of Delhi South Campus. H2O2 (30% (v/ v); ε = 43.6 M−1 cm−1 at 240 nm) and DNA samples (pBR322, 100 ng/μL of 4361 bp) were purchased from EMPLURA and SRL, respectively, whereas Na2S2O4, FeSO4·7H2O, NaN3, NaCN, Amplex Red, horseradish peroxidase (HRP), hemin chloride, and bovine liver catalase were purchased from Sigma-Aldrich. Expression, Purification and Quantification of Mtb BfrA Protein. Recombinant WT Mtb BfrA protein was overexpressed in E. B

DOI: 10.1021/acs.inorgchem.8b02758 Inorg. Chem. XXXX, XXX, XXX−XXX

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(0−4 mM) at a fixed (100 Fe) iron content per BfrA cage (0.5 μM). Iron oxidation activity of Mtb BfrA was monitored both in the absence (only O2) and presence (O2 + H2O2) of H2O2 (0.05−4.0 mM). The ferroxidase reactions were monitored by the external addition of H2O2 to Mtb BfrA (H2O2 was added to Mtb BfrA protein ∼1 min prior to stopped flow mixing with Fe2+) under aerobic conditions where both H2O2 and O2 were present. Kinetics of Dissolved O2 Consumption during Ferroxidase Reaction of Mtb BfrA. Clark-type oxygen microelectrode (Oxygraph from Hansatech Instruments) was utilized to monitor the kinetics of dissolved O2 consumption.6,49 The microelectrode was calibrated with sodium dithionite (Na2S2O4), prior to the measurement, in order to establish zero O2 concentration in the oxygen electrode chamber.49 It was then air-saturated with distilled water to generate O2 (∼250 μM) in the chamber at 25 °C. Reaction sample (1.0 mL) was prepared by mixing Mtb BfrA (1.0 μM cage) in 100 mM MOPS buffer (pH 7.0), followed by the addition of FeSO4 (500 μM single addition and 120 μM stepwise addition) both in the absence and presence of H2O2 (250 μM) into the reaction mixture after 2 min of data acquisition. The slope obtained within the first 2 min of data recording (after the addition of H2O2 and FeSO4 with a delay of about 10 s between them) was analyzed to determine the rate of dissolved O 2 consumption during ferroxidase activity of Mtb BfrA. Detection of H2O2 Generated during Ferroxidase Activity of Mtb BfrA. By Oximetry Analysis. The time courses of O 2 concentration were monitored using a Clark-type microelectrode at 25 °C by adding varying concentrations of FeSO4 (0−48 μM) to Mtb BfrA (1.0 μM cage). The reaction samples were prepared in 100 mM MOPS buffer (pH 7.0), and required amounts of FeSO4 and catalase were added, with a time delay of ∼1 min between them. By H2O2 Assay Using Amplex Red/HRP. The formation of resorufin (ε = 5.4 × 104 M−1cm−1 at 571 nm) during ferroxidase activity of Mtb BfrA was monitored using H2O2 assay reagents (substrate: Amplex Red, catalyst: HRP) in a SHIMADZU UV−vis spectrophotometer. The concentrations of HRP and Amplex Red used for this experiment were 0.2 μM and 50 μM, respectively. HRP and Amplex Red solutions were added to Mtb BfrA protein samples (1.0 μM cage) in 100 mM MOPS buffer (pH 7.0). The absorption spectrum was then recorded after ∼3 min following the addition of FeSO4 (0−48 μM) to the above solution. Catalase Activity Assay. The assay solution comprised of Mtb BfrA (1.0 μM cage) in 100 mM MOPS buffer (pH 7.0). The reaction was initiated by the addition of H2O2 (0−3 mM) to the above solution, maintaining 1.0 mL total volume. The catalase activity of Mtb BfrA was analyzed by a Clark-type oxygen microelectrode to monitor the kinetics of O2 evolution resulting from the disproportionation of H2O2, by varying both the substrate (H2O2) as well as the enzyme (Mtb BfrA) concentration. Apparent values of Km and vmax were determined from the double reciprocal Lineweaver−Burk plot along with the calculation of kcat and catalytic efficiency of Mtb BfrA. Further, the effectiveness of the enzyme (Mtb BfrA) in catalytically converting H2O2 into O2 was tested over multiple cycles by the successive addition of H2O2 (250 μM) to the reaction mixture, after the completion of each cycle (∼40 min). It was then subsequently compared with the one obtained in the case of bovine liver catalase. The inhibition of catalase activity was also monitored using 2 mM of sodium azide (NaN3) and sodium cyanide (NaCN) which were preincubated with the buffered solution of Mtb BfrA protein, for 5 min prior to the addition of H2O2. The kinetics of O2 evolution were also monitored by the addition of H2O2 (5 mM) to the heme free Mtb BfrA variant (M52L) and the nonheme binding Mtb BfrB along with the as purified Mtb BfrA samples doped with lower amounts (6 and 12 μM) of externally added FeSO4. DNA Protection Assay. In vitro, DNA cleavage activity by hydroxyl radical was analyzed using the plasmid DNA (pBR322, ∼4361 bp) in 100 mM MOPS buffer (pH 7.0). Plasmid DNA (pDNA) (10 ng/μL) was incubated individually for 30 min with Mtb BfrA (1.6 μM cage). Freshly prepared FeSO4 (100 μM) was added to the samples and incubated for 10 min, followed by the addition of H2O2 (4.5 mM) to the reaction mixture to initiate the Fenton

coli [BL21(λDE3)] using earlier reported methods.44,46 Briefly, E. coli BL21(λDE3) cells were transformed with pET21c constructs encoding WT BfrA, followed by its culture in lysogeny broth medium containing ampicillin (100 μg/mL). Cells were grown at 37 °C until a cell density of 0.6−0.8 (A600 nm) was reached when isopropyl 1-thio-βD-galactopyranoside was added (0.5 mM final concentration) and continued for 4 h. Cells were disrupted by sonication, and the cell-free extracts were obtained after centrifugation (45 min, 16000g, 4 °C). The supernatant collected was filtered using a 0.45 μm syringe filter and was applied to a Q-Sepharose fast flow column. After initial washing, Mtb BfrA was eluted with a linear NaCl gradient (0−1 M) in 20 mM Tris-HCl, pH 8.0. Fractions containing Mtb BfrA were identified by 5% (w/v) native polyacrylamide gel electrophoresis (PAGE) and UV−vis absorption spectra. Purified Mtb BfrA proteins were concentrated by ultrafiltration using a 30 kDa cutoff membrane (Millipore). Protein concentration was determined using the Bradford assay with bovine serum albumin (Thermo Scientific) as a standard. Further, the heme content in the Mtb BfrA protein was determined by pyridine hemochromagen assay47 using SHIMADZU UV−vis spectrophotometer. Synthesis of Heme Free Mtb BfrA Variant and Nonheme Binding Mtb BfrB. The heme free Mtb BfrA variant (M52L) was prepared from WT Mtb BfrA plasmid by the Quick-Change Site Directed Mutagenesis Kit (Stratagene-Agilent). Oligonucleotide primer listed in Table S1, supplied by Integrated DNA Technology (IDT), was used for site-specific mutagenesis. The point mutation, M52L, was confirmed by sequencing the plasmid DNA at the DNA sequencing facility of Institute of Life Sciences, Bhubaneswar. The expression, purification, and quantification of M52L BfrA variant protein followed the same procedure as mentioned above for WT BfrA, while WT Mtb BfrB was synthesized as per earlier reports.48 Cage Integrity and Iron Loading Ability of Mtb BfrA Studied by Native PAGE and TEM. The iron loading into Mtb BfrA protein nanocages was carried out by the addition of a freshly prepared solution of FeSO4 (in 1 mM HCl) to Mtb BfrA, in 100 mM 3-(Nmorpholino) propane sulfonic acid (MOPS) buffer (pH 7.0), as per the earlier reports.6,14 It was followed by 2 h incubation at room temperature and overnight incubation at 4 °C. The final ratio of Fe atoms per protein cage was maintained at 625:1. For electrophoresis, 6% (w/v) nondenaturing polyacrylamide gels were used.14 A freshly prepared mixture of 2% K4[Fe(CN)6] and 2% 11.6 M HCl (1:1, v/v) was used for iron staining (Prussian blue formation) of mineralized Mtb BfrA. In order to stain the protein cage of apo-ferritin, the gels were first destained with distilled water and then restained with Coomassie dye. Purified Mtb BfrA protein (5 μL, 1 mg/mL) solution was mineralized (480 Fe/cage) as mentioned above and was dropped onto the carbon-coated copper grid (Formvar/300 mesh). The grids were washed with Millipore water, after 1 min incubation, and the excess solution was soaked away using filter paper. The samples were stained twice with 20 μL of a 4% (w/v) uranyl acetate solution (for 30 s). The grid was then dried with filter paper and then air-dried in a vacuum desiccator. TEM images were obtained in TECNAI-S30 transmission electron microscope operating at an accelerating voltage of 300 kV. Kinetics of Ferroxidase Reaction of Mtb BfrA Using Stopped Flow Spectrophotometer. Iron loading inside bacterioferritin protein nanocages was carried out by mixing equal volumes of freshly prepared FeSO4 (in 1 mM HCl) and a solution of Mtb BfrA protein in 100 mM MOPS buffer (pH 7.0) using a Hi-Tech SF61MX rapid mixing stopped-flow spectrophotometer. Final concentration of protein nanocage was maintained at 2.08 μM containing varied amounts of ferritin-caged iron (∼24 to 480 Fe/cage) in the presence of O2 only. Initial rates of formation of DFP-like species at 650 nm and [Fe3+-O]x species at 350 nm were calculated from the initial data points (within the first 30 ms). The participation of H2O2 (in the presence of O2) as Fe2+ oxidant during the ferroxidase activity of Mtb BfrA (2.08 μM cage) was monitored both by varying iron loading (24−480 Fe/Cage) at a fixed H2O2 (250 μM) concentration and by varying H2O2 concentration C

DOI: 10.1021/acs.inorgchem.8b02758 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 2. Time courses of ferroxidase activity as a function of iron loading in Mtb BfrA. The progress curves of ferroxidase activity of Mtb BfrA: (A) [Fe3+-O]x formation and formation/decay of diferric peroxo (DFP)-like catalytic intermediate at 350 and 650 nm, respectively (at 240 Fe/cage). (B) and (C) The time courses of the above ferroxidase reaction with increasing Fe/cage (24−480). (D) The corresponding initial rates of ferroxidase activity as a function of iron loading per Mtb BfrA cage were fitted to the Hill equation. Mtb BfrA protein (4.16 μM cage) solution in 100 mM MOPS buffer (pH 7.0) was mixed with an equal volume of freshly prepared FeSO4 solution, at room temperature in a rapid mixing stopped flow spectrophotometer. reaction and was further incubated for 5 min. In order to check the effect of catalase activity of Mtb BfrA during its course of DNA protection, one of the reaction mixtures was incubated with catalase inhibitor, NaN3 (2.5 mM), for 5 min (prior to Fenton reaction). The control reaction for the DNA cleavage activity was carried out in the absence of Mtb BfrA. 1% (w/v) agarose gel was prepared in tris-acetate-EDTA (TAE) buffer, followed by its staining with ethidium bromide (EtBr).35 The gel was run at 90 V for 50 min, and the resultant gels were visualized by Gel Doc XR+. Electrophoretic Mobility Shift Assay (EMSA). In order to test the DNA binding ability of Mtb BfrA, EMSA was carried out in 1% (w/v) agarose gel stained with EtBr. DNA samples (pBR322, 14.3 ng/μL of 4361 bp) were incubated with different concentrations (0.5−3.0 μM cage) of Mtb BfrA for 10 min. In the control reaction, no Mtb BfrA was added to the DNA samples. The gel electrophoresis was carried out in TAE buffer for 50 min at 90 V.34,35 The resulting gel images were visualized in Gel Doc XR+.

of Na2S2O4 (red trace of Figure 1B) leads to the reduction of heme iron(III) present in Mtb BfrA causing a bathochromic shift of the Soret band from 410 to 425 nm along with the appearance of sharper α and β bands at 525 and 556 nm, respectively, in accordance with earlier reports.40,42 On the basis of symmetry, a single Bfr protein cage can accommodate 12 heme groups, in each of its 12 subunit dimer interfaces. However, the overexpressed recombinant Mtb BfrA cage in the current report is found to be undersaturated with only three to four bound heme groups. The soluble Mtb BfrA protein self-assembles to form an intact nanocage, which retains its iron sequestering ability and synthesizes iron oxide biominerals in its central nanocavity, which was confirmed by Prussian blue/Coomassie staining of nondenaturing gel (Figure 1C). The representative TEM image (Figure 1D) of iron loaded recombinant Mtb BfrA protein nanocages indicate spherical shaped discrete electron dense cores corresponding to the formation of a mineral core inside a BfrA central nanocavity, while negative staining with uranyl acetate confirms the intact nature of the apo-Mtb BfrA protein cage. The mean size of the Mtb BfrA protein nanocages was determined to be 12.3 (±1.7) nm, while the selected-area electron diffraction (SAED) pattern indicated the crystalline nature of the BfrA iron mineral core. Mtb BfrA Exhibits Ferroxidase Activity (as a Function of Iron Loading) via the Formation of DFP-like Transient Intermediate. Buffered solution of Mtb BfrA protein was mixed with equal volumes of freshly prepared ferrous sulfate solution in order to monitor the process of iron uptake and its oxidation (biomineralization) utilizing dissolved O2 as the oxidant (Figure 2). In most of the ferritins, the sequestration of Fe2+ occurs rapidly (in milliseconds) through the symmetric hydrophilic pores/channels.17,18,30 Further, the

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RESULTS UV−vis Absorption Spectra and Iron Loading Properties of Heme Binding Mtb BfrA. The 24-mer, self-assembled Mtb BfrA protein nanocages differ from Mtb BfrB and other nonheme binding ferritins, in terms of having a heme group at each of their subunit dimer interface (Figure 1A). The presence of heme in recombinant Mtb BfrA protein is reflected in its absorption spectrum (Figure 1B). The oxidized form of Mtb BfrA (black trace of Figure 1B) contains the characteristic 410 nm Soret band (for heme) along with the α and β bands. Moreover, the presence of a characteristic weak band at ∼735 nm (bis-methionine heme coordination; inset in Figure 1B, Table S4) along with ∼623 nm band (high spin Fe3+ heme signature; inset in Figure 1B, Table S4) in Mtb BfrA possibly indicate the existence of a mixture of low- and high-spin Fe3+heme species in solution,28,36,38,40,42,43,47 respectively. Addition D

DOI: 10.1021/acs.inorgchem.8b02758 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 3. Comparison of kinetics of ferroxidase activity and dissolved O2 consumption by Mtb BfrA in the presence/absence of H2O2, under aerobic conditions. Progress curves of ferroxidase reaction: (A) Formation/decay of DFP-like catalytic intermediate at 650 nm and (B) [Fe3+-O]x formation at 350 nm, for Mtb BfrA (at 48 Fe/cage) both in the absence (only O2) and presence (O2 + H2O2) of H2O2 (250 μM). BfrA protein (4.16 μM cage) solution in 100 mM MOPS buffer (pH 7.0) was mixed with an equal volume of freshly prepared FeSO4 solution (200 μM), at room temperature in a rapid mixing stopped flow spectrophotometer. The plots are obtained by taking average of five independent time courses (ΔA650 nm vs time and ΔA350 nm vs time) collected using three different sets of Mtb BfrA protein preparation. The time courses of O2 concentration were monitored using a Clark-type microelectrode at 25 °C: (C) by varying the concentration of H2O2 (0−250 μM) and keeping FeSO4 and Mtb BfrA cage concentrations fixed at 500 μM and 1.0 μM, respectively. The reaction samples were prepared in 100 mM MOPS buffer (pH 7.0), and the required amounts of H2O2 and FeSO4 were added, with a time delay of about 10 s between them. Control experiments for iron oxidation were also performed in the absence of Mtb BfrA protein. (D) Oxygen consumption during catalytic turnover of ferroxidase reaction: by adding 120 μM of FeSO4 (for each addition) to 1.0 μM Mtb BfrA cage. In another reaction, 60 μM H2O2 was added (for each addition) to 1.0 μM Mtb BfrA, prior to addition of FeSO4. Detection of H2O2 generated during ferroxidase activity of Mtb BfrA: (E) by oximetry analysis and (F) by Amplex Red/HRPbased H2O2 assay (see details in Methods section). The time points of addition of FeSO4, H2O2, and catalase to Mtb BfrA solution are indicated by the arrows (in yellow).

binding of 2 equiv of ferrous ion at the di-iron catalytic site (Fox center), followed by its rapid oxidation, leads to the formation of a transient μ-peroxo diferric species (DFP) in the 600−680 nm wavelength range.12,19,38 This rapid process of iron sequestration, leading to the formation of protein encapsulated soluble ferrihydrite mineral, prevents the precipitation of iron in buffer solution.1,10 Loading of Fe2+ inside Mtb BfrA nanocages leads to the formation of a DFPlike transient intermediate species in the wavelength range 620−700 nm (within 100 ms) (Figure 2A,B and Figure S1), which rapidly decays but at a relatively slower rate than its formation, similar to human and frog M ferritin.24 Apart from Mtb BfrA used in the current report, the detection of similar

transient species has not been reported for any heme binding Bfr protein. Further, the kinetics of Fe2+ oxidation was monitored by recording the increase in absorbance at 350 nm corresponding to the formation of [Fe3+-O]x (for all types of ferric oxy species including DFP) during the course of iron mineralization (Figure 2A,C). The effect of iron loading to Mtb BfrA was further analyzed by monitoring time courses of formation of the transient intermediate and [Fe3+-O]x, with increasing iron concentration. The iron uptake assays (Figure 2B−D) indicated that the initial rates of iron oxidation increase with the gradual increase in the number of Fe/cage, reaching saturation at ∼200 Fe/cage (Figure 2D). The Hill coefficient (n ∼ 2−4) obtained E

DOI: 10.1021/acs.inorgchem.8b02758 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 4. Monitoring the catalase-like activity during the reaction of H2O2 with Mtb BfrA. The time courses of O2 evolution were monitored using a Clark-type microelectrode at 25 °C by adding H2O2 to Mtb BfrA. The kinetics of O2 evolution was investigated: (A) by adding H2O2 (250 μM) both in the absence and presence of Mtb BfrA (1.0 μM cage); (B) by varying the H2O2 concentration (0−3 mM) at fixed BfrA concentration (1.0 μM cage); (C) for the catalytic cycle (turnover) of Mtb BfrA (1.0 μM cage) by the successive addition of H2O2 (250 μM) at regular time intervals; (D) for the reaction of H2O2 (5 mM) with BfrA (0.3 μM cage concentration) both in the absence and presence of catalase inhibitors such as NaN3 (2 mM) and NaCN (2 mM); (E) for the reaction of H2O2 (5 mM) to Mtb BfrA, its heme free variant (M52L) and Mtb BfrB (1.0 μM cage in each case); (F) by adding H2O2 (5 mM) to the externally iron loaded (∼6 and 12 μM Fe2+) and the as-purified Mtb BfrA (1.0 μM cage in each case). The contribution from the self-disproportionation of H2O2 toward the O2 evolution has been subtracted for the plots shown in (E) and (F). All the reaction samples were prepared in 100 mM MOPS buffer (pH 7.0). The time point of addition of H2O2 to Mtb BfrA solution is indicated by the arrows (in yellow).

true catalytic center to oxidize the incoming ferrous ions. The detailed mechanism of iron mineralization in Mtb BfrA is lacking at present and requires further investigation. Ferroxidase Activity of Mtb BfrA Increases while the O 2 Consumption Decreases with Increasing H 2 O2 Concentration. The rate of iron oxidation was found to be faster in the presence of Mtb BfrA as compared to its autooxidation in buffer solution (Figure 3A,B), indicating the catalytic nature of Mtb BfrA. In this section, the participation of H2O2 (in the presence of O2) as Fe2+ oxidant during the ferroxidase activity of Mtb BfrA was examined. As the dissolved O2 concentration in buffer solution at 25 °C is about 250 μM, for the comparison purpose, the O2 and H2O2 concentration was maintained at 1:1 by externally adding 250 μM H2O2 to the buffer solution. At low iron loading ( 1) for the formation of DFP-like species, during the ferroxidase reaction in Mtb BfrA (Table S2). Though the origin of cooperativity in any kind of ferritins (including Mtb BfrA) is not understood, this phenomenon is frequently observed in multimeric proteins/enzymes.17 Since ferritin nanocages are composed of 24 subunits with each subunit carrying a dinuclear iron site, a Hill coefficient of ∼2− 4 must reflect cooperative interactions among a set of subunits.17,50−52 Positive cooperativity (n > 1) indicates that binding of Fe2+ at the di-iron sites in one subunit possibly influences its binding at the di-iron center of adjacent subunits in BfrA via distant sites at three-fold channels,19 where iron distribution occurs to three different ferroxidase centers. Moreover, different BfrAs exhibit different mechanisms of iron biomineralization.27 After initial iron oxidation (in E. coli Bfr), Fe3+ at the ferroxidase center remains there to behave as a F

DOI: 10.1021/acs.inorgchem.8b02758 Inorg. Chem. XXXX, XXX, XXX−XXX

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from the disintegration of ∼672 μM H2O2 (within 10 min of the start of catalytic reaction), nearly obeying the stoichiometric ratio of 2:1 as per the well-known reaction of H2O2 disproportionation by catalase (Figure S4A). This Clark-type microelectrode-based oximetry data indicated that both the rate and amount of O2 evolution increase with the increase in concentration of substrate, H2O2 (0−3 mM) keeping a fixed concentration of Mtb BfrA (1.0 μM cage) (Figure 4B and Figure S4B). Similarly, the rate of O2 generation increased with the increase in Mtb BfrA concentration (0−2 μM cage) in the solution containing fixed amount of H2O2 (250 μM) (Figure S5A). Moreover, the addition of H2O2 to Mtb BfrA decreased the Soret peak intensity of heme group (Figure S6A,B). Like catalases, the catalytic reaction of Mtb BfrA does not follow the well-established Michaelis−Menten pathway, as the rate of O2 evolution gradually increases with increasing H2O2 concentration (Figure S4B−D) without attaining saturation. Therefore, the apparent values of the kinetic parameters were calculated from Lineweaver−Burk Plot (Figure S4B−D) and are listed in Table S3. The apparent values of vmax for the catalase reaction of Mtb BfrA were found to be comparable to that of Mtb KatG enzyme, whereas the relatively higher apparent values of Km and significantly lower apparent values of kcat reflect its lower affinity for H2O2 binding and slower rate of catalase activity, respectively.54 Control experiments were also performed with heme-free M52L BfrA variant (where Met residues at 52 position were mutated to Leu residues in each of the 24 subunits, which do not bind heme; Figure S7) and with a nonheme binding Mtb BfrB along with lower doped iron (∼6 and 12 μM Fe2+) mineralized heme binding Mtb BfrA samples. Despite containing a similar amount of nonheme iron (∼10−12 Fe/ Cage) as that of WT Mtb BfrA (as purified), the heme-free M52L variant showed insignificant evolution of O2 upon treatment with H2O2 (Figure 4E), whereas the low iron doped mineralized samples exhibited exactly same kinetic profile for catalase activity as that of Mtb BfrA (Figure 4F). The results clearly indicated the role of bound heme groups in catalase activity of Mtb BfrA. The presence of minimal catalase activity exhibited by nonheme binding Mtb BfrB, containing ∼3−4 Fe/ Cage, indicates that the role of nonheme iron toward catalase activity cannot be completely ruled out. Almost 125 μM of O2 was found to evolve at each successive addition of equal amounts of H2O2 (250 μM) to the same solution of Mtb BfrA (1.0 μM cage). These results indicated the catalytic turnover of Mtb BfrA as it gets regenerated after every catalytic cycle to disproportionate H2O2 and evolves O2 (Figure 4C) at a comparable rate (Figure S5C). However, the comparison of catalase activity of Mtb BfrA with that of bovine liver catalase (keeping the heme concentration same) clearly indicated the superiority of catalase in terms of exhibiting an enhanced rate of O2 evolution (at an initial rate almost 60 times faster as compared to Mtb BfrA) under the same reaction conditions (Figure S5B). Further, to check the inhibition of catalase-like activity of BfrA, potent catalase inhibitors such as NaN3 and NaCN were pre-incubated (for 5 min) with BfrA (0.3 μM cage), followed by the addition of H2O2 (5 mM) which resulted in a negligible amount of change in O2 concentration (Figure 4D). The extent of inhibition of catalase-like activity of Mtb BfrA was found to increase with the increasing concentration of both azide and cyanide (Figure S5E,F). Further, the spectral features of heme in the presence

and H2O2 are very similar to the case in the absence of H2O2. However, with the increment in iron loading, the initial rates of formation of DFP-like species decrease in the presence of H2O2 (Figure S2F), indicating that H2O2 competes with O2 during the ferroxidase activity of Mtb BfrA and thus decreases the formation of DFP-like species. The effect of H2O2 on the process of iron oxidation in recombinant Mtb BfrA protein was further investigated by varying H2O2 concentration at fixed iron loading per BfrA nanocage (Figure S2C). Rates of DFPlike species (Figure S2A,F) formation decreased, while that of [Fe3+-O]x formation (iron mineralization) (Figure S2B−E) increased upon increment in H2O2 concentration, at higher Fe loading. Moreover, the relatively faster ferroxidase activity ([Fe3+-O]x species formation) of Mtb BfrA under anaerobic conditions (manual mixing; Figure S2G) indicates the stronger oxidizing nature of H2O2. The O2 consumption (Figure 3C) profile, in the presence of Mtb BfrA protein, displayed faster rates as compared to Fe2+ in buffer (due to auto-oxidation), again confirming the catalytic nature of Mtb BfrA. O2 consumption during iron oxidation reaction in the absence of Mtb BfrA (control experiment) was observed to be negligible, irrespective of the presence/absence of H2O2. In order to investigate whether H2O2 competes with O2 for ferroxidase activity, the kinetics of O2 consumption by Mtb BfrA was examined. As described earlier, the rate of Fe2+ oxidation in Mtb BfrA gets enhanced in the presence of H2O2. However, the rate and amount of dissolved O2 consumption gradually decrease upon increasing the concentration of H2O2 (Figure 3C and Figure S3A). Moreover, the O2 consumption during ferroxidase activity of Mtb BfrA, in the presence of H2O2, became slower in each step of Fe2+ addition and took a greater number of such additions to consume the entire amount of dissolved O2 present in the solution (Figure 3D and Figure S3B). Therefore, the lower amount of O2 consumption in the presence of H2O2 can be attributed to the existence of competition between H2O2 and O2 as Fe2+ oxidant during ferroxidase activity of Mtb BfrA. All of these results clearly indicated that Mtb BfrA sequesters both Fe2+ and H2O2 rapidly to synthesize nontoxic ferric oxide biomineral, similar to heme binding Bfr from E. coli and D. vulgaris.35 On the contrary, when H2O2 was added to Mtb BfrA (without adding FeSO4), the amount of O2 in the reaction mixture was found to increase, indicating catalase-like activity of Mtb BfrA (Figure 4), which was further investigated in detail in the next section. Further, the generation of H2O2 during the ferroxidase reaction of Mtb BfrA was detected by performing an oximetry experiment at low iron doping (≤48 Fe/Cage), upon addition of bovine liver catalase ∼1 min after FeSO4 addition. The O2 evolution at 36 and 48 Fe/Cage was a clear indication of the generation of H2O2 during the ferroxidase reaction of Mtb BfrA (Figure 3E). This result was further confirmed by the oxidation of Amplex Red in the presence of horseradish peroxidase (HRP)53 (Figure 3F). Mtb BfrA Exhibits Catalase-like Activity. The evolution of O2 observed during the reaction of H2O2 with Mtb BfrA indicated that it exhibits catalase-like activity (Figure 4A). Upon adding H2O2 (250 μM) to the buffer solution (in the absence of Mtb BfrA), O2 concentration remained unaltered, indicating negligible disproportionation of H2O2. However, in the presence of Mtb BfrA (1.0 μM cage) solution, O2 concentration increases with the passage of time (Figure 4A). Further, the addition of 5 mM H2O2 to Mtb BfrA (0.3 μM cage) solution resulted in the evolution of ∼316 μM of O2 G

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its oxidative damage, as seen in L6 of Figure 5A. As discussed in the previous section, recombinant Mtb BfrA exhibited catalase-like activity via disproportionation of H2O2. Hence, the effect of catalase-like activity of Mtb BfrA on its DNA protection role was investigated by using a catalase inhibitor, NaN3, which was pre-incubated with the solutions containing BfrA and DNA prior to the addition of Fe2+ and H2O2. However, BfrA protected plasmid DNA even in the presence of catalase inhibitor (L7 of Figure 5A), which eliminated the contribution of catalase activity of Mtb BfrA as a defense mechanism against H2O2-induced oxidative DNA damage. Appearance of an additional faint band in between the intense DNA bands in L6 of Figure 5A indicated the formation of BfrA-DNA complex (also observed in L1 and L2), which may possibly contribute toward DNA protection activity. Further, EMSA indicated the formation of Mtb BfrA-DNA complex (Figure 5B). With the increase in concentration of Mtb BfrA, the intensity of middle bands (BfrA-DNA complex) gradually enhanced, concomitant with the gradual disappearance of the lower DNA bands (L2−L7 of Figure 5B). Therefore, the recombinant Mtb BfrA was able to protect plasmid DNA from oxidative cleavage by forming a BfrA-DNA complex and by sequestering free Fe2+ and H2O2. Moreover, the binding of Mtb BfrA to DNA was confirmed by incubating Mtb BfrA and pDNA in the presence of high salt (600 mM NaCl) concentration, where the intermediate band due to BfrA-DNA complex formation was absent (Figure S9A). This observation indicates that high concentration of salt screens the surface charge and thereby affects DNA-Mtb BfrA interaction. The conformational changes in DNA induced by Mtb BfrA may affect the migration rate as observed in Figure 5B. Circular dichroism (CD) spectra (Figure S10A,B) revealed the condensation of plasmid DNA upon interaction with Mtb BfrA,55 possibly forming a complex which exhibited a slower migration rate. These results further confirm the binding of Mtb BfrA to DNA making the DNA structure more condensed and compact, which is able to resist the Fe2+/H2O2 mediated oxidative stress. Further, only BfrA was able to protect plasmid DNA from oxidative cleavage and not BfrB, indicating the preferential role and importance of BfrA during oxidative stress and pathogenesis of Mtb (Figure S9B). The binding assay of Mtb BfrA with linearized plasmid DNA (NheI-digested pBR322) was also performed (Figure S11) and revealed that either Mtb BfrA does not bind to long linear DNA fragments or the complexes, which, if formed, are not stable and probably dissociate during electrophoresis. The results suggest the preferential binding of Mtb BfrA to supercoiled forms of plasmid DNA, which was also observed in several other cases such as heme containing Bfr from D. vulgaris, histone-like HU proteins, or linker histone H1 isolated from chicken erythrocytes, etc.35

of these anionic ligands were also investigated which resulted in a decrease of the Soret peak intensity immediately following the addition of CN−/N3−, possibly indicating the binding of these anions to heme iron (Figure S6C−F). Similar spectral changes were also observed during cyanide/azide binding studies (at lower concentrations up to 2 mM) in the case of bovine liver catalase and hemin chloride (Figure S8). Further, the addition of H2O2 to the same solution (Mtb BfrA + CN−/ N3−) resulted in insignificant spectral changes associated with the absence of catalase activity (i.e., no O2 evolution), probably due to the strong coordination of CN−/N3− (Figure S6G,H). Therefore, the above results indicate that heme containing Mtb BfrA behaves similar to that of catalase enzyme in terms of exhibiting catalase-like activity, which can be inhibited in the presence of catalase inhibitors. Moreover in the presence of NaN3 (i.e., absence of catalase activity), Mtb BfrA exhibited relatively faster O2 consumption kinetics during ferroxidase activity as compared to the one in the absence of NaN3 (Figure S5D). This observation indicated that the catalase activity of Mtb BfrA is contributing to the overall consumption of dissolved O2. Mtb BfrA Protects Plasmid DNA from Oxidative Cleavage by forming BfrA-DNA Complex. The toxic hydroxyl radicals, generated as a result of Fe2+ and H2O2-based Fenton reaction, are known to cleave DNA.34 Dps protein protects DNA by sequestering both the substrates of Fenton reaction for its ferroxidase activity and by forming a Dps-DNA complex. However, Mtb lacks the Dps gene. Therefore, in order to investigate whether Mtb BfrA exhibits similar Dps-like activity or not, the DNA cleavage assay was carried out in the presence of Fenton reagents. This Fenton-mediated oxidative damage of plasmid DNA (pBR322) could be well documented in lane 5 (L5) of Figure 5A. Two intense bands in L4 indicate the two primary forms (possibly supercoiled and circular) of plasmid DNA which disappeared upon incubation with the Fenton reagent (L5 of Figure 5A). Exposure of this plasmid DNA to Fenton reagent in the presence of Mtb BfrA prevented

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DISCUSSION Iron behaves as a double-edged sword for both host and pathogens, being both beneficial (during O2 transport and oxidative metabolism) as well as harmful (generates toxic ROS, upon reaction with H2O2). During the host−pathogen clash, the host secretes H2O2, but bacterial pathogens utilize Dps protein to detoxify it. However, even in the absence of structural homologues for Dps, Mtb must have evolved with some kind of defensive strategies to survive against the oxidative stress and to continuously proliferate.44,56,57 Out of the two iron repositories (heme BfrA and nonheme BfrB)

Figure 5. DNA protection exerted by Mtb BfrA and detection of DNA-protein complex formation by EMSA. DNA protection activity of Mtb BfrA and its interaction with DNA was studied by agarose gel (1%, w/v) electrophoresis. (A) DNA protection activity of Mtb BfrA and (B) DNA-BfrA interaction by EMSA. In (B), Mtb BfrA protein concentration was varied (0.5−3.0 μM), keeping the concentration of pDNA fixed (5 nM; with [Protein]:[DNA] ratio varied from 100 to 600). ‘+’ and ‘−’ signs for all lanes indicate the presence and absence of the concerned reactants, respectively. H

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recent report on SynFtn.61 These results suggested that some amount of generated H2O2 probably gets consumed by unreacted Fe2+, MOPS buffer, and catalase-like activity of Mtb BfrA within ∼1 min gap between the addition of FeSO4 and catalase in the reaction mixture. Therefore, the O2 consumption analysis indicates competition between O2 and H2O2 during ferroxidase activity of Mtb BfrA, but there are uncertainties existing around Fe(II):O2 ratios, which needs further investigation. The rate of O2 evolution by Mtb BfrA (catalase activity in the absence of Fe2+) increased with H2O2 (substrate) concentration, very similar to catalase, one of the fastest naturally occurring antioxidative enzymes. Catalase, and relatively slower KatG protein, protects the cells against the toxic effects of excess H2O2. The presence of four heme groups in the homotetrameric catalase enzyme catalyzes the disproportionation of two H2O2 molecules to one O2 and two H2O molecules within submicroseconds (which approaches a diffusion limited condition, kcat/Km = 4 × 107 M−1 s−1), preventing ROS influenced damage. The two-step catalytic reaction of catalase involves the generation/decay of oxy-ferryl porphyrin cation radical species (compound I) via the oxidation/reduction of heme by H2O2. This process requires the availability of an unsaturated heme coordination site for H2O2 binding. Though the mechanism of catalase activity of Mtb BfrA is not known, the presence of heme groups in it provides a clue to understand its observed catalase activity. The role of heme toward catalase-like activity of Mtb BfrA was further confirmed by the retardation in the O2 evolution with the introduction of catalase inhibitors such as sodium azide and sodium cyanide. Therefore, the observed catalase-like activity of Mtb BfrA can be proposed to arise via two possible mechanisms: (1) Binding of H2O2 to five-coordinated ferric heme, if already present, in the Mtb BfrA reaction mixture to generate compound I. The presence of both high-spin ferric heme (623 nm band) along with low-spin bis-methionine coordinated heme (735 nm weak band) signatures, in the UV−vis absorption spectrum of Mtb BfrA, indicates the possibility of a presence of both five and six-coordinated heme (for detail see Table S4). This high-spin feature not only explains the existence of proposed species but also can explain our observations on catalase-like activity in Mtb BfrA along with its inhibition and spectral changes associated with cyanide/azide binding. (2) In situ generation of a fivecoordinated iron(III)-heme species in Mtb BfrA (via H2O2 mediated oxidation of axial methionine ligands) to coordinate H2O2, giving rise to compound I.62 This compound I might further react with another molecule of H2O2, thereby continuing the catalytic cycle. The intensity of both 623 and 735 nm band decreased upon increasing the concentration of cyanide (Figure S14) but did not disappear completely, which cannot account for the complete loss of catalase activity in Mtb BfrA. Hence, the detailed mechanism of Mtb BfrA’s catalase-like activity requires further investigation to understand the structure−function relationship in detail. Moreover, the uncertainty existing about the involvement of the number of heme (all or partial) during the catalase-like activity of Mtb BfrA makes it difficult to compute the absolute value of catalytic constant (kcat) for Mtb BfrA to compare with that of catalase. Mtb BfrA also exhibits DNA protection activity, similar to Dps, by forming a BfrA-DNA complex. Dps protein either shields the DNA by forming a complex with it or brings about

identified in the Mtb gene, the role of BfrA has been proposed to be more than just iron biomineralization since it can be expressed under both low and high iron levels. Therefore, the current report investigated the kinetics of ferroxidase activity along with DNA protection and catalase-like activities of Mtb BfrA. Iron biomineralization in Bfr proceeds via uptake of Fe2+ ions through its hydrophilic pores followed by its binding at the di-iron Fox center (active site). In eukaryotic ferritins such as human and frog M, the electrostatics around three-fold pores drives Fe2+ inside,21 but in the case of Mtb BfrA, in addition to three-fold pores, iron entry through the four-fold and B pores cannot be ruled out (Figure S12A,B). Moreover, BfrA from E. coli employs B pores to sequester iron for its ferroxidase activity. After entering inside the nanocages of Mtb BfrA, Fe2+ ions gets oxidized forming the DFP-like transient species, which subsequently participates in further mineralization. Our current observations during the ferroxidase activity of Mtb BfrA indicate the rapid generation/decay of a DFP “like” transient intermediate, very similar to the one observed in case of eukaryotic ferritins, having a similar spectroscopic and kinetic profile in the wavelength range 620−700 nm. Interestingly, this DFP intermediate has not been detected in any other heme containing Bfr. But in frog M, human, and many other ferritins, formation of a blue colored transient DFP species is detected during ferroxidase activity, which also exhibits a characteristic absorption band in the wavelength range 600−680 nm. Therefore, the future perspectives of the current report involve challenges in terms of determining the exact Fe2+ entry pathway along with providing the detailed structural and spectral characterization of the DFP-like transient intermediate and its fate in Mtb BfrA. This preliminary study on ferroxidase activity would further help to establish the mechanism of its iron mineralization and would relate to heme containing BfrA from various sources (E. coli and P. aeruginosa). Mtb BfrA detoxifies H2O2 during ferroxidase activity by utilizing it as a co-substrate along with O2, for the synthesis of nontoxic protein encapsulated ferrihydrite biomineral. The kinetics of dissolved O2 consumption during the ferroxidase reaction of Mtb BfrA further revealed the participation of both H2O2 and O2 as Fe2+ oxidant. The variation obtained in the Fe2+/O2 stoichiometry (Figure S13) during the single and multiple additions of FeSO4 to Mtb BfrA can be attributed to several factors such as the effects of catalase activity (a new finding in the current report), Fe2+ loading per cage, MOPS buffer, etc., which significantly affects the Fe2+/O2 stoichiometry during the ferroxidase activity of ferritin.58−60 Similar to eukaryotic ferritin, high iron loading (∼500 Fe/Cage) leads to complete reduction of O2 to H2O with the stoichiometry being very close to the expected value of 4:1 (Figure 3C). However, with 120 Fe/Cage, the ratio (2.0 ± 0.1) was close to the value (∼2:1) since the H2O2 generated via the oxidation of 2 mol of Fe2+ by 1 mol of O2 further gets possibly consumed by MOPS buffer as well as by catalase-like activity of Mtb BfrA, thus preventing it from generating the desired stoichiometry of 4:1 (Figure 3D and Figure S13). When H2O2 is added externally, in addition to the internally generated H2O2 (via ferroxidase reaction), it resulted in the increase of Fe2+/O2 stoichiometry (Figure S13). Moreover, the amount of O2 generated (∼4 μM for 36 Fe/Cage), via the addition of bovine liver catalase during the ferroxidase activity of Mtb BfrA, was lower than expected (∼9 μM for 36 Fe/Cage) (Figure 3E), similar to a I

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ACKNOWLEDGMENTS This work was supported by DST-SERB India (EMR/2016/ 003894) to R.K.B., DST-INSPIRE Faculty Award (IFA-13 CH-111) to R.K.B. and B.S., and the intramural funding from National Institute of Technology (NIT) Rourkela, India, to R.K.B. We are thankful to Dr. S. Mazumdar and Mr. B. T. Kansara (TIFR, Mumbai) for the collection of stopped flow kinetics data. We are also thankful to Dr. Anil K. Tyagi and Dr. Garima Khare (University of Delhi South Campus) for their generous support in providing the Mtb Bfr clone and for their critical suggestions.

conformational changes in DNA into highly ordered chromatin structures, which resists oxidative cleavage by hydroxyl radicals. Therefore, in Mtb, BfrA in addition to histone and histone-like proteins (Lsr2) might safeguard DNA against peroxideinduced oxidative stress.57 Hence, the current work emphasizes on the multiple functions of BfrA, involving the newly identified catalase and Dps-like activities besides its natural physiological function as an iron repository, which might possibly play an important role during the survival and pathogenesis of Mtb.

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CONCLUSIONS The present study elucidates the antioxidative properties of heme binding Mtb BfrA protein nanocages by enlisting three different modes of action. Mtb BfrA sequesters Fenton substrate(s) and carries out ferroxidase activity rapidly to synthesize the iron biomineral inside its central nanocavity. This rapid iron oxidation and mineralization process by Mtb BfrA proceeds via the formation of a DFP-like transient intermediate. Mtb BfrA protected the plasmid DNA from H2O2-induced oxidative stress by forming BfrA-DNA complexes. Further, H2O2 gets detoxified by its catalase-like activity, but it requires further investigation to understand its structure−function relationships in detail. The above in vitro studies suggest new functions of BfrA, which may possibly be exploited by Mtb during its pathogenesis. Thus, the current findings can be further utilized as a future platform for designing target-specific inhibitors against heme binding Mtb BfrA, to curb the pathogenesis of Mtb.44,56

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REFERENCES

(1) Theil, E. C.; Tosha, T.; Behera, R. K. Solving Biology’s Iron Chemistry Problem with Ferritin Protein Nanocages. Acc. Chem. Res. 2016, 49 (5), 784−791. (2) Theil, E. C.; Behera, R. K.; Tosha, T. Ferritins for chemistry and for life. Coord. Chem. Rev. 2013, 257, 579−586. (3) Crichton, R. In Iron Metabolism; John Wiley & Sons, Ltd: Chichester, 2009; pp 17−58. (4) Cassat, J. E.; Skaar, E. P. Iron in infection and immunity. Cell Host Microbe 2013, 13 (5), 509−19. (5) Weinberg, E. D. Iron availability and infection. Biochim. Biophys. Acta, Gen. Subj. 2009, 1790 (7), 600−5. (6) Koochana, P. K.; Mohanty, A.; Das, S.; Subhadarshanee, B.; Satpati, S.; Dixit, A.; Sabat, S. C.; Behera, R. K. Releasing iron from ferritin protein nanocage by reductive method: The role of electron transfer mediator. Biochim. Biophys. Acta, Gen. Subj. 2018, 1862 (5), 1190−1198. (7) Dehner, C.; Morales-Soto, N.; Behera, R. K.; Shrout, J.; Theil, E. C.; Maurice, P. A.; Dubois, J. L. Ferritin and ferrihydrite nanoparticles as iron sources for Pseudomonas aeruginosa. JBIC, J. Biol. Inorg. Chem. 2013, 18 (3), 371−81. (8) Pandey, R.; Rodriguez, G. M. IdeR is required for iron homeostasis and virulence in Mycobacterium tuberculosis. Mol. Microbiol. 2014, 91 (1), 98−109. (9) Poulos, T. L. Heme enzyme structure and function. Chem. Rev. 2014, 114 (7), 3919−62. (10) Theil, E. C. Ferritin: The Protein Nanocage and Iron Biomineral in Health and in Disease. Inorg. Chem. 2013, 52 (21), 12223. (11) Liu, X.; Theil, E. C. Ferritins: dynamic management of biological iron and oxygen chemistry. Acc. Chem. Res. 2005, 38 (3), 167−75. (12) Bou-Abdallah, F. The iron redox and hydrolysis chemistry of the ferritins. Biochim. Biophys. Acta, Gen. Subj. 2010, 1800, 719−731. (13) Watt, R. K.; Hilton, R. J.; Graff, D. M. Oxido-reduction is not the only mechanism allowing ions to traverse the ferritin protein shell. Biochim. Biophys. Acta, Gen. Subj. 2010, 1800, 745−759. (14) Subhadarshanee, B.; Mohanty, A.; Jagdev, M. K.; Vasudevan, D.; Behera, R. K. Surface charge dependent separation of modified and hybrid ferritin in native PAGE: Impact of lysine 104. Biochim. Biophys. Acta, Proteins Proteomics 2017, 1865 (10), 1267−1273. (15) Le Brun, N. E.; Crow, A.; Murphy, M. E.; Mauk, A. G.; Moore, G. R. Iron core mineralisation in prokaryotic ferritins. Biochim. Biophys. Acta, Gen. Subj. 2010, 1800 (8), 732−44. (16) Honarmand Ebrahimi, K.; Hagedoorn, P.-L.; Hagen, W. R. Unity in the Biochemistry of the Iron-Storage Proteins Ferritin and Bacterioferritin. Chem. Rev. 2015, 115 (1), 295−326. (17) Behera, R. K.; Theil, E. C. Moving Fe(2+) from ferritin ion channels to catalytic OH centers depends on conserved protein cage carboxylates. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (22), 7925− 7930. (18) Behera, R. K.; Torres, R.; Tosha, T.; Bradley, J. M.; Goulding, C. W.; Theil, E. C. Fe(2+) substrate transport through ferritin protein cage ion channels influences enzyme activity and biomineralization. JBIC, J. Biol. Inorg. Chem. 2015, 20 (6), 957−69.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b02758. Details of apparent kinetic parameters of ferroxidase activity and catalase-like activity of Mtb BfrA, dissolved O2 consumption rate plots, effect of salt on protein (BfrA)-DNA interaction, CD spectrum for DNA-BfrA interaction, UV−vis spectral features of heme group of Mtb BfrA, bovine liver catalase and hemin chloride in the presence of H2O2 and CN−/N3− and catalase-like activity of Mtb BfrA both in the presence and absence of H2O2/catalase inhibitors/heme/iron loading, spectral changes in methionine-heme coordination band in Mtb BfrA, detection of H2O2 during ferroxidase activity of Mtb BfrA (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +91-661-2462980. Fax: +91-661-2462651. ORCID

Abhinav Mohanty: 0000-0002-3843-1960 Rabindra K. Behera: 0000-0003-2849-3292 Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest. J

DOI: 10.1021/acs.inorgchem.8b02758 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry (19) Tosha, T.; Behera, R. K.; Theil, E. C. Ferritin ion channel disorder inhibits Fe(II)/O2 reactivity at distant sites. Inorg. Chem. 2012, 51 (21), 11406−11. (20) Bradley, J. M.; Le Brun, N. E.; Moore, G. R. Ferritins: furnishing proteins with iron. JBIC, J. Biol. Inorg. Chem. 2016, 21 (1), 13−28. (21) Chandramouli, B.; Bernacchioni, C.; Di Maio, D.; Turano, P.; Brancato, G. Electrostatic and Structural Bases of Fe2+ Translocation through Ferritin Channels. J. Biol. Chem. 2016, 291 (49), 25617− 25628. (22) Chasteen, N. D.; Harrison, P. M. Mineralization in ferritin: an efficient means of iron storage. J. Struct. Biol. 1999, 126 (3), 182−94. (23) Bernacchioni, C.; Ghini, V.; Theil, E. C.; Turano, P. Modulating the permeability of ferritin channels. RSC Adv. 2016, 6, 21219−21227. (24) Pozzi, C.; Di Pisa, F.; Bernacchioni, C.; Ciambellotti, S.; Turano, P.; Mangani, S. Iron binding to human heavy-chain ferritin. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2015, 71 (9), 1909−1920. (25) Turano, P.; Lalli, D.; Felli, I. C.; Theil, E. C.; Bertini, I. NMR reveals pathway for ferric mineral precursors to the central cavity of ferritin. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (2), 545−50. (26) Bradley, J. M.; Moore, G. R.; Le Brun, N. E. Diversity of Fe2+ entry and oxidation in ferritins. Curr. Opin. Chem. Biol. 2017, 37, 122−128. (27) Bradley, J. M.; Moore, G. R.; Le Brun, N. E. Mechanisms of iron mineralization in ferritins: one size does not fit all. JBIC, J. Biol. Inorg. Chem. 2014, 19 (6), 775−785. (28) Rivera, M. Bacterioferritin: Structure Function and Protein− Protein Interactions, 30 ed.; World Scientific Publishing Co. Pte. Ltd.: Singapore, 2013; p 135−178. (29) Chiancone, E.; Ceci, P. The multifaceted capacity of Dps proteins to combat bacterial stress conditions: Detoxification of iron and hydrogen peroxide and DNA binding. Biochim. Biophys. Acta, Gen. Subj. 2010, 1800 (8), 798−805. (30) Bellapadrona, G.; Stefanini, S.; Zamparelli, C.; Theil, E. C.; Chiancone, E. Iron translocation into and out of Listeria innocua Dps and size distribution of the protein-enclosed nanomineral are modulated by the electrostatic gradient at the 3-fold ″ferritin-like″ pores. J. Biol. Chem. 2009, 284 (28), 19101−9. (31) Gupta, S.; Chatterji, D. Bimodal Protection of DNA by Mycobacterium smegmatis DNA-binding Protein from Stationary Phase Cells. J. Biol. Chem. 2003, 278 (7), 5235−5241. (32) Ng, V. H.; et al. Role of KatG catalase-peroxidase in mycobacterial pathogenesis: countering the phagocyte oxidative burst. Mol. Microbiol. 2004, 52 (5), 1291−1302. (33) Bunker, J.; Lowry, T.; Davis, G.; Zhang, B.; Brosnahan, D.; Lindsay, S.; Costen, R.; Choi, S.; Arosio, P.; Watt, G. D. Kinetic studies of iron deposition catalyzed by recombinant human liver heavy, and light ferritins and Azotobacter vinelandii bacterioferritin using O2 and H2O2 as oxidants. Biophys. Chem. 2005, 114 (2), 235− 244. (34) Liu, X.; Kim, K.; Leighton, T.; Theil, E. C. Paired Bacillus anthracis Dps (Mini-ferritin) Have Different Reactivities with Peroxide. J. Biol. Chem. 2006, 281 (38), 27827−27835. (35) Timoteo, C. G.; Guilherme, M.; Penas, D.; Folgosa, F.; Tavares, P.; Pereira, A. S. Desulfovibrio vulgaris bacterioferritin uses H(2)O(2) as a co-substrate for iron oxidation and reveals DPS-like DNA protection and binding activities. Biochem. J. 2012, 446 (1), 125−33. (36) Wong, S. G.; Abdulqadir, R.; Le Brun, N. E.; Moore, G. R.; Mauk, A. G. Fe-haem bound to Escherichia coli bacterioferritin accelerates iron core formation by an electron transfer mechanism. Biochem. J. 2012, 444 (3), 553−60. (37) Rivera, M. Bacterioferritin: Structure, Dynamics, and Protein− Protein Interactions at Play in Iron Storage and Mobilization. Acc. Chem. Res. 2017, 50 (2), 331−340. (38) Theil, E. C.; Behera, R. K. In Coordination Chemistry in Protein Cages; John Wiley & Sons, Inc.: Hoboken, NJ, 2013; Chapter 1, pp 3−24.

(39) Singh, R.; Switala, J.; Loewen, P. C.; Ivancich, A. Two [Fe(IV)O Trp*] intermediates in M. tuberculosis catalaseperoxidase discriminated by multifrequency (9−285 GHz) EPR spectroscopy: reactivity toward isoniazid. J. Am. Chem. Soc. 2007, 129 (51), 15954−63. (40) McMath, L. M.; Contreras, H.; Owens, C. P.; Goulding, C. W. The structural characterization of bacterioferritin, BfrA, from Mycobacterium tuberculosis. J. Porphyrins Phthalocyanines 2013, 17 (03), 229−239. (41) Khare, G.; Gupta, V.; Nangpal, P.; Gupta, R. K.; Sauter, N. K.; Tyagi, A. K. Ferritin Structure from Mycobacterium tuberculosis: Comparative Study with Homologues Identifies Extended CTerminus Involved in Ferroxidase Activity. PLoS One 2011, 6 (4), e18570. (42) Gupta, V.; Gupta, R. K.; Khare, G.; Salunke, D. M.; Tyagi, A. K. Crystal Structure of Bfr A from Mycobacterium tuberculosis: Incorporation of Selenomethionine Results in Cleavage and Demetallation of Haem. PLoS One 2009, 4 (11), e8028. (43) Yasmin, S.; Andrews, S. C.; Moore, G. R.; Le Brun, N. E. A New Role for Heme, Facilitating Release of Iron from the Bacterioferritin Iron Biomineral. J. Biol. Chem. 2011, 286 (5), 3473−3483. (44) Khare, G.; Nangpal, P.; Tyagi, A. K. Differential Roles of Iron Storage Proteins in Maintaining the Iron Homeostasis in Mycobacterium tuberculosis. PLoS One 2017, 12 (1), e0169545. (45) McLean, K. J.; Munro, A. W. Drug targeting of heme proteins in Mycobacterium tuberculosis. Drug Discovery Today 2017, 22 (3), 566−575. (46) Gupta, V.; Gupta, R. K.; Khare, G.; Salunke, D. M.; Tyagi, A. K. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of bacterioferritin A from Mycobacterium tuberculosis. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2008, 64 (5), 398−401. (47) Behera, R. K.; Nakajima, H.; Rajbongshi, J.; Watanabe, Y.; Mazumdar, S. Thermodynamic effects of the alteration of the axial ligand on the unfolding of thermostable cytochrome C. Biochemistry 2013, 52 (8), 1373−84. (48) Khare, G.; Nangpal, P.; Tyagi, A. K. Unique Residues at the 3Fold and 4-Fold Axis of Mycobacterial Ferritin Are Involved in Oligomer Switching. Biochemistry 2013, 52 (10), 1694−1704. (49) Koochana, P. K.; Mohanty, A.; Subhadarshanee, B.; Satpati, S.; Naskar, R.; Dixit, A.; Behera, R. K. Phenothiazines and phenoxazines: as electron transfer mediators for ferritin iron release. Dalton Transactions 2019, 48, 3314. (50) Tosha, T.; Hasan, M. R.; Theil, E. C. The ferritin Fe2 site at the di-iron catalytic center controls the reaction with O2 in the rapid mineralization pathway. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (47), 18182. (51) Liu, X.; Theil, E. C. Ferritin reactions: Direct identification of the site for the diferric peroxide reaction intermediate. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (23), 8557. (52) Schwartz, J. K.; Liu, X. S.; Tosha, T.; Theil, E. C.; Solomon, E. I. Spectroscopic Definition of the Ferroxidase Site in M Ferritin: Comparison of Binuclear Substrate vs Cofactor Active Sites. J. Am. Chem. Soc. 2008, 130 (29), 9441−9450. (53) Behera, R. K.; Goyal, S.; Mazumdar, S. Modification of the heme active site to increase the peroxidase activity of thermophilic cytochrome P450: A rational approach. J. Inorg. Biochem. 2010, 104 (11), 1185−1194. (54) Singh, R.; Wiseman, B.; Deemagarn, T.; Jha, V.; Switala, J.; Loewen, P. C. Comparative study of catalase-peroxidases (KatGs). Arch. Biochem. Biophys. 2008, 471 (2), 207−214. (55) Bhanjadeo, M. M.; Nayak, A. K.; Subudhi, U. Cerium chloride stimulated controlled conversion of B-to-Z DNA in self-assembled nanostructures. Biochem. Biophys. Res. Commun. 2017, 482 (4), 916− 921. (56) Sharma, D.; Bisht, D. Role of Bacterioferritin & Ferritin in M. tuberculosis Pathogenesis and Drug Resistance: A Future Perspective by Interactomic Approach. Front. Cell. Infect. Microbiol. 2017, 7, 240. K

DOI: 10.1021/acs.inorgchem.8b02758 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry (57) Colangeli, R.; Haq, A.; Arcus, V. L.; Summers, E.; Magliozzo, R. S.; McBride, A.; Mitra, A. K.; Radjainia, M.; Khajo, A.; Jacobs, W. R.; Salgame, P.; Alland, D. The multifunctional histone-like protein Lsr2 protects mycobacteria against reactive oxygen intermediates. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (11), 4414−4418. (58) Mehlenbacher, M.; Poli, M.; Arosio, P.; Santambrogio, P.; Levi, S.; Chasteen, N. D.; Bou-Abdallah, F. Iron Oxidation and Core Formation in Recombinant Heteropolymeric Human Ferritins. Biochemistry 2017, 56 (30), 3900−3912. (59) Zhao, G.; Bou-Abdallah, F.; Arosio, P.; Levi, S.; JanusChandler, C.; Chasteen, N. D. Multiple Pathways for Mineral Core Formation in Mammalian Apoferritin. The Role of Hydrogen Peroxide. Biochemistry 2003, 42 (10), 3142−3150. (60) Zhang, B.; Wilson, P. E.; Watt, G. D. Ferritin-catalyzed consumption of hydrogen peroxide by amine buffers causes the variable Fe2+ to O2 stoichiometry of iron deposition in horse spleen ferritin. JBIC, J. Biol. Inorg. Chem. 2006, 11 (8), 1075−86. (61) Bradley, J. M.; Svistunenko, D. A.; Pullin, J.; Hill, N.; Stuart, R. K.; Palenik, B.; Wilson, M. T.; Hemmings, A. M.; Moore, G. R.; Le Brun, N. E. Reaction of O2 with a di-iron protein generates a mixedvalent Fe2+/Fe3+ center and peroxide. Proc. Natl. Acad. Sci. U. S. A. 2019, 116 (6), 2058. (62) Nugraheni, A. D.; Ren, C.; Matsumoto, Y.; Nagao, S.; Yamanaka, M.; Hirota, S. Oxidative modification of methionine80 in cytochrome c by reaction with peroxides. J. Inorg. Biochem. 2018, 182, 200−207.

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DOI: 10.1021/acs.inorgchem.8b02758 Inorg. Chem. XXXX, XXX, XXX−XXX