Structural evidence of the oxidation of iodide ion into hyper‐reactive hypoiodite ion by mammalian heme lactoperoxidase

Abstract

Lactoperoxidase (1.11.1.7, LPO) is a mammalian heme peroxidase found in the extracellular fluids of mammals including plasma, saliva, airway epithelial lining fluids, nasal lining fluid, milk, tears, gastric juices, and intestinal mucosa. To perform its innate immune action against invading microbes, LPO utilizes hydrogen peroxide (H₂O₂) to convert thiocyanate (SCN⁻) and iodide (I⁻) ions into the oxidizing compounds hypothiocyanite (OSCN⁻) and hypoiodite (IO⁻). Previously determined structures of the complexes of LPO with SCN⁻, OSCN⁻, and I⁻ show that SCN⁻ and I⁻ occupy appropriate positions in the distal heme cavity as substrates while OSCN⁻ binds in the distal heme cavity as a product inhibitor. We report here the structure of the complex of LPO with IO⁻ as the first structural evidence of the conversion of iodide into hypoiodite by LPO. To obtain this complex, a solution of LPO was first incubated with H₂O₂, then mixed with ammonium iodide solution and the complex crystallized by the addition of PEG‐3350, 20% (wt/vol). These crystals were used for X‐ray intensity data collection and structure analysis. The structure determination revealed the presence of four hypoiodite ions in the substrate binding channel of LPO. In addition to these, six other hypoiodite ions were observed at different exterior sites. We surmise that the presence of hypoiodite ions in the distal heme cavity blocks the substrate binding site and inhibits catalysis. This was confirmed by activity experiments with the colorimetric substrate, ABTS (2,2′‐azino‐bis(3‐ethylbenzthiazoline‐sulfonic acid)), in the presence of hypoiodite and iodide ions.

Short abstract

PDB Code(s): 7VE3;

1. INTRODUCTION

Lactoperoxidase (1.11.1.7, LPO) is a mammalian heme peroxidase observed in most mammals except some rodents. It is secreted from mammary, salivary, and other mucosal glands including the lungs, bronchi, and nasal passages. ¹ LPO is a member of the innate immune system and acts as a natural defense against bacterial and viral agents. ² It utilizes hydrogen peroxide (H₂O₂) to convert thiocyanate (SCN⁻) and iodide (I⁻) ions into the harsh antimicrobial compounds, hypothiocyanite (OSCN⁻) ³ , ⁴ and hypoiodite (IO⁻). ⁵ , ⁶ The substrate SCN⁻ is found in body fluids over a wide range of concentrations ⁷ , ⁸ , ⁹ and we mention here that a fraction of mammalian SCN⁻ may be synthesized from cyanide by sulfur transferase enzymes including mitochondrial rhodanese and gastric mercaptopyruvate sulfur transferase. ¹⁰ , ¹¹ Much SCN⁻ is ingested directly from plants with the cruciferous vegetables being a rich source of SCN⁻. ¹² On the other hand, the availability of substrate iodide is entirely dependent on the dietary intake. ¹³

The overall reaction with iodide ion can be summarized as

$${\mathrm{Fe}}^{+3}\left(\mathrm{LPO}\right)+{\mathrm{H}}_2{\mathrm{O}}_2\to \left(\mathrm{LPO}\right)\mathrm{Fe}=\mathrm{O}+{\mathrm{H}}_2\mathrm{O}$$ (1)

$$\left(\mathrm{LPO}\right)\mathrm{Fe}=\mathrm{O}+{\mathrm{I}}^{-}\to \left(\mathrm{LPO}\right){\mathrm{Fe}}^{+3}+{\mathrm{IO}}^{-}$$ (2)

The exact nature of the intermediate iron‐oxygen compound, termed compound I, is generally thought to be Fe⁺⁴, sometimes termed an oxyferryl heme, rather than Fe⁺⁵, with significant positive charge delocalized on to the porphyrin. ¹⁴ This intermediate is stable for several minutes in the absence of a reducing substrate, such as SCN⁻, I⁻, or ABTS. The IO⁻ produced is in equilibrium between this ion and HOI, hypoiodous acid, around neutrality since HOI is a weak acid.

The substrate H₂O₂ is produced by a range of oxidase enzymes including glucose oxidase for which simple glucose is the substrate and this combination is often used for in vitro experiments. ¹⁵ However, the most important source of lung and nasal H₂O₂ is the NADPH oxidase and dual oxidase (duox). ¹⁶ This is a flavin‐NADPH‐dependent enzyme, which appears to synthesize H₂O₂ directly without a superoxide intermediate, though a little H₂O₂ is enzymatically synthesized by superoxide dismutase enzymes through other pathways. ¹⁷ Along with LPO the two enzymes are termed the lactoperoxidase system.

One would think the strong oxidizing agent H₂O₂ itself should be capable of destroying most invasive microbes, but nature has provided a strong defense against H₂O₂ destruction by the dismutase activity of the highly active enzyme catalase, found in almost all cellular life. The noxious ions OSCN⁻ and OI⁻ are therefore required to circumvent H₂O₂ inactivation and are produced as the first line of mammalian defense against airborne microbes including viral agents.

While duox is a transmembrane enzyme, accepting NADPH from the cell interior and producing H₂O₂ at the exterior, LPO is extruded. This localizes the harsh LPO system products to the mucous linings of the lungs, bronchi, and nasal passages and not in tissues. It has been suggested recently that the anti‐viral activity of the LPO system against the SARS‐CoV‐2 virus may be enhanced by additional dietary iodine. ¹⁸ Such a positive effect of iodine has already been shown against the RSV virus in a lamb model. ¹⁹ and against influenza under in vitro conditions. ²⁰

We report here the first structural evidence of the conversion of iodide ion into hypoiodite ion using a lactoperoxidase system, which includes lactoperoxidase, hydrogen peroxide, and iodide ion (LPO + H₂O₂ + I⁻) to generate LPO with bound hypoiodite. This is a continuation of our work on the structural and functional studies of mammalian heme peroxidases, which began in our laboratory in 1995. ²¹ Since then we have reported the structures of native LPO from several different animal species. ²² , ²³ , ²⁴ Some of our work includes the structures of the complexes of LPO with the substrate SCN⁻, ²⁵ the oxidized product OSCN⁻, ²⁶ the substrates I⁻ and H₂O₂ as well as a ternary complex of LPO with I⁻ and H₂O₂. ²⁷ We have also reported the structures of the complexes of LPO with some small organic molecules and halides other than iodine. ²⁸ , ²⁹ , ³⁰ We crystallized this complex from a solution of LPO in the presence of H₂O₂ that was then treated with ammonium iodide and dehydrated by the addition of PEG‐3350, 20% (wt/vol). The resulting crystals were subjected to X‐rays and the diffracted information collected for structure analysis. The structure determination revealed the presence of four hypoiodite ions in the substrate binding channel with another seven hypoiodite ions about the protein exterior.

After this discovery we then investigated the possibility for OI⁻ product inhibition. Our experiments showed that the product IO⁻ inhibits the turnover of the colorimetric substrate ABTS in a manner consistent with a catalytic site crowded with product, confirming our observations of the complex structure.

2. RESULTS

2.1. Analysis of the activity of LPO in the presence of hypoiodite

The results of the absorbance at 413 nm of the oxidized ABTS catalyzed by LPO with increasing concentrations of the substrates ABTS and H₂O₂ are presented in Figure 1A. The inhibitory effects of additional iodide and the product hypoiodite are shown in Figure 1B,C, respectively. The curves D and E (Figure 1) represent the controls without LPO and H₂O₂. Curve A (Figure 1) shows that the ABTS product quantity increases linearly at lower initial ABTS concentrations but becoming nearly independent of ABTS at higher concentrations. This is the typical result for a catalyzed reaction reaching maximum turnover. The curves B and C correspond to the activities of LPO in the presence of iodide and hypoiodite ions, respectively. Although these curves show an overall similar behavior to that of curve A, the lower absorbance at every ABTS concentration indicates that both iodide and hypoiodite ions reduce the ABTS turnover. This suggests that (1) a fraction of the I⁻ and IO⁻ ions remain in the substrate binding site, which are hindering the reaction with H₂O₂ and ABTS turnover, (2) the I⁻ and IO⁻ ions residing in the substrate binding site block the binding of the substrate ABTS, inhibiting oxidation by active LPO, and (3) LPO may suffer both substrate and product inhibition.

FIGURE 1.

The measurements of absorbance at 413 nm against various concentrations of substrate ABTS under different conditions were made. (A) Lactoperoxidase and hydrogen peroxide were mixed and after 15 min, various concentrations of ABTS were added in different vials. The results are shown as curve (–▲–). (B) Lactoperoxidase was mixed with ammonium iodide and after 15 min hydrogen peroxide was added and then measurements were made for different concentrations of ABTS and the resulting curve is shown (–●–). (C) Lactoperoxidase and hydrogen peroxide were mixed after which ammonium iodide was added to it and then the measurements were made on adding hydrogen peroxide at different concentrations of ABTS (–♦–). The curve (D) corresponds to measurements without lactoperoxidase (–*–) while curve (E) represents the data without hydrogen peroxide (–—–)

2.2. Structure of LPO

The structure of the complex of LPO with hypoiodite ions consists of 4,765 protein atoms from 595 amino acid residues, 43 atoms of the heme moiety, 28 atoms of 2 N‐acetylglucosamine (NAG) residues, 1 calcium ion, 22 atoms of 11 of hypoiodite ions, 6 iodide ions, 5 atoms of one sulfate ion, and 157 water oxygen atoms. Considering the molecular weight of 67.6 kDa for the protein molecule and 3.7 kDa for other non‐protein components including the heme moiety, the hypoiodite ions, the iodide ions, and the sugar residues, the total molecular weight for non‐water components is estimated to be 71.3 kDa. This yields a value of 2.3 Å³/Da for the Matthews coefficient (V _m) corresponding to a solvent content of 46.5%.

The structure has been refined to values of 0.195 and 0.277 for R‐factor (R _cryst) and free R‐factor (R _free) factors, respectively. As calculated using PROCHECK, ³¹ 89.5% residues are present in the most favored regions of the Ramachandran's φ, ψ map. ³² There are 10.3% residues in the additionally allowed regions and 0.2% residues are present in the generously allowed regions. There are no residues in the disallowed regions of the map.

The overall structure of the complex of LPO with hypoiodite ions is shown in Figure 2. The polypeptide chain of LPO folds to form an inverted pear‐shaped structure and consists of 20 α‐helices, H1(Ala75‐Val83), H2(Val98‐Leu111), H2a(His124‐Tyr132), H3(Pro197‐Leu203), H4(Pro236‐Ile240), H5(Ile260‐Leu283), H6(Gly289‐Phe317), H7(Asn321‐His324), H8(Ala341‐Val353), H9(Lys383‐Ile387), H10(Val391‐Leu401), H11(Asn415‐Pro419), H12(Leu433‐Tyr444), H13(Gly449‐Pro456), H14(Gln463‐Leu471), H15(Leu475‐Asp483), H16(Asn492‐Glu498), H17(Leu509‐Trp525), H18(Ser538‐Gly546), H19(Ile549‐Asp556) and two short β‐strands, B1(Leu356 ‐Asn359), B2(Thr373‐Glu375). The central portion of the LPO structure around the heme moiety is well ordered having relatively low values of B‐factors. The heme moiety is situated between two well formed α‐helices, H2 and H8 and it is surrounded by the columns of four α‐helices, H2, H5, H8, and H12. Helix H8 (Ala341‐Val353) is located on the proximal side and contains His351 which forms a coordination linkage with heme iron through N^ε2 while helix H2 (Val98‐Leu111) is located on the distal heme side with three important residues, Gln105, Asp108, and His109, which are critical for the stability and catalytic action of LPO. The two other important residues on the distal heme side are Arg255 and Glu258, which are from a long loop (Asn241‐Gln259). These residues are also critical for the catalytic action and stability of the heme moiety. The two ends of this important loop (Asn241‐Gln259) are held by two relatively stable α‐helices, H4 and H5. Even though this loop is quite flexible residues Arg255 and Glu258 belong to a rather rigid segment, Arg255‐Ala256‐Ser257‐Glu258, forming a tight type I, β‐turn conformation. An overall rigid architecture of the distal heme cavity consists of the heme moiety on one side and the α‐helix H2 and a rigid part of the loop (Asn241‐Gln259) on the other side. Thus the substrate binding site in LPO represents a well‐formed structure, which is located deep inside the enzyme.

FIGURE 2.

Showing the structure of lactoperoxidase with four hypoiodite ions in the substrate binding channel. The secondary structure elements such as α‐helices (cyan) and β‐strands (red) are shown. The N and C terminal ends are indicated. The heme moiety is also shown (yellow)

This is the first structure of the LPO complex with the product hypoiodite (IO⁻). Hereafter, the IO⁻ ion will be written as HOI for the purpose of discussion. In this structure, the conserved water molecule W1 is again present in the distal heme cavity forming a coordinate linkage with the heme iron at a distance of 2.07 Å. It also forms two hydrogen bonds with His109 N^ε2 and Gln105 N^ε2 at distances of 3.10 Å and 3.31 Å, respectively. Two hypoiodite ions, HOI‐1 (occupancy 0.5) and HOI‐2 (occupancy 0.5) are present in the proximity of heme moiety in the distal heme cavity, which means that either HOI‐1 is present or HOI‐2 is present (but not both simultaneously) as both positions are equally filled (Figure 3a). The two other hypoiodite ions HOI‐3 and HOI‐4 are also observed at two separate sites at the entrance/exit positions of the of the substrate binding tunnel (Figure 3a). The other seven HOI ions are located on the protein at different sites in the structure (Figure 3b). Six iodide ions are also bound to protein at various other sites (Figure 3c).

FIGURE 3.

(a) Showing the stereo view of the backbone tracing of lactoperoxidase with substrate binding channel in yellow for four hypoiodite ions in the substrate binding channel, (b) showing the stereo view of the backbone with positions of seven additional hypoiodite ions (5–11), and (c) showing the stereo view of the backbone with positions of six iodide ions

The nearest hypoiodite ion to the heme iron in the distal heme cavity is HOI‐1. It forms multiple van der Waals contacts with atoms of the heme moiety, W1, His109, Arg255, and Glu258 and also forms a hydrogen bond with another water molecule W17. The second hypoiodite ion HOI‐2 interacts with the side chains of Arg255, Glu258, and Phe381. The position of HOI‐2 is further stabilized by a hydrogen bond with water molecule W3 which in turn is hydrogen bonded to Gln423 O^ε1. The third hypoiodite ion HOI‐3 located near the surface and forms van der Waals contacts with Pro234, Pro236, Phe239, Phe422, and Pro424. It also forms a hydrogen bond at a distance of 2.78 Å with the backbone nitrogen atom of Thr425. The fourth hypoiodite anion HOI‐4 is located near the surface and forms van der Waals contacts with Asn230, Lys232, Ser235, Pro236, and Cys248. It also forms two hydrogen bond with Lys232 N^ς and water molecule W32.

The seven other hypoiodite ions are located at different sites which are held in place by weak interactions with the protein. The HOI‐5 is located at the surface between Glu196 and Ser198 and forms van der Waals contacts with Glu196, Pro197 and Ser198. The next hypoiodite ion, HOI‐6, fits in a broad pocket at the surface and forms van der Waals contacts with the side chains of Ilu24, Pro197, and Ser201. HOI‐7 forms van der Waals contacts with Phe309, Trp529, and Trp530. Hypoiodite ion HOI‐8 forms van der Waals interactions with residues Pro461, Lys462, and Gly466 and four hydrogen bonds with Lys462 N, Thr463 N, Thr463 O^γ1 and water molecule W20. HOI‐9 is located in a narrow cleft on the surface and forms van der Waals contacts with Glu363, Tyr365, Arg397, and Ile559. It also forms a hydrogen bond with the backbone nitrogen atom N of Thr560. The next hypoiodite ion, HOI‐10 is located at a distance of approximately 4.5 Å inside the molecule. The van der Waals contacts are formed with residues Ala44, Arg45, Leu47, Ser340, and Val342. Two hydrogen bonds are also formed with Arg45 NH1 and Asn341 N. The last position is HOI‐11 located in a shallow cavity on the surface making van der Waals contacts with Ser359, Pro367, Ala372, and Glu373. It also forms a hydrogen bond with Lys402 N^ς.

In addition to the 11 hypoiodite ions, six iodide ions are also observed in this structure. These are bound weakly to protein at various sites on the surface. The first I1 is located between α‐helix H6 and a C‐terminal loop Thr560‐Phe576. It forms van der Waals contacts with Asp311, His565, Ala566, and Phe567 and one hydrogen bond with a backbone N atom of Ala565. The second, I2, is situated in a shallow cleft on the surface, which is stabilized by van der Waals interactions from Gln217, Asn218, and Phe229 as well as by a hydrogen bond with Phe229 N. Another iodide ion, I3, is observed near the side chains of Asn95, Arg96, and Arg506 and is stabilized by ionic interactions with Arg96 and Arg506, hydrogen bonds with Asn95 N^δ2, W767 and also forms van der Waals contacts with Arg504. I4 is observed with occupancy of 0.6 making van der Waals contacts with residues Ser198, Leu199, and Arg202 and forming a hydrogen bond with Arg203 NH1. I5 is located at a distance of 2.75 Å from I4 with occupancy of 0.4 and stabilized by van der Waals interactions with residues Ser198 and Arg202. The last iodine ion, I6, is observed in a well‐formed cleft‐like structure on the surface making van der Waals contacts with Pro145, Glu77, and Lys81. It also forms two hydrogen bonds with Asn80 N^δ1 and water molecule W735 which in turn is hydrogen bonded to Asn147 N.

3. DISCUSSION

The substrate binding site in LPO consists of a long substrate diffusion channel. The innermost part of the substrate binding site is termed as the distal heme cavity while the outermost portion, reminding one of the two arms of a forceps, are the entrance and exit areas (Figure 3). We show that the product, HOI, accumulating in the substrate binding site during catalytic turnover, sterically hinders substrate oxidation which we also observe as product inhibition. Our results are evidence that the products occupy three subsites: (1) distal heme site, (2) central region of the channel, and (3) the outermost parts of the forceps (Figure 4). Seven additional hypoiodite ions were also observed occupying other sites within and on the surface of the structure. These ions fit well in the electron densities observed in the minor clefts formed at the surface of the protein structure but these interactions are probably of little importance to catalysis.

FIGURE 4.

(a) Showing the positions of four hypoiodite ions in the substrate binding channel where HOI‐1 and HOI‐2 are in the distal heme cavity while HOI‐3 and HOI‐4 are near the exit points of the binding channel. (b) A grasp view of the substrate binding channel in lactoperoxidase with four hypoiodite ions

It may be stated here that the structure of the complex of LPO with hypoiodite ions provides the first structural evidence that LPO converts iodide ions into antimicrobial hypoiodite ions. To gain further insight into the ligand bindings in the distal heme cavity, the structure of the complex of LPO with hypoiodite ions is compared by superimposing it on the structures of the native unbound protein (PDB ID 7DAO, Figure 5a) and its complexes with iodide ions (PDB ID 7DE5, Figure 5b, ²² ), SCN⁻ ions (PDB ID 3ERH, Figure 5c, ²⁴ ), and OSCN⁻ ions (PDB ID 3BXI, Figure 5d, ²⁵ ). The superimpositions of these structures clearly show that the binding positions in the substrate binding site are essentially conserved. A comparison of the other binding sites with the structure of the complex of LPO with iodide ions ²³ indicates that the binding sites on the surface of the protein molecule seem to have formed naturally to play important role in the overall function of LPO. Overall, the substrate binding site plays a role of converting the substrates into products while other binding sites on the surface may possibly be used for temporary storage of the substrates and the product molecules.

FIGURE 5.

The superimpositions of the distal heme cavities showing (a) W1, HOI‐1, and HOI‐2 from the present structure and W1, W2′, and W3′ from the native structure (PDB ID, 7DAO), (b) W1, HOI‐1, and HOI‐2 from the present structure and three iodide ions from the structure of the complex of lactoperoxidase with iodide ions (PDB ID, 7DE5), (c) W1, HOI‐1, and HOI‐2 of the present structure and W1 and SCN⁻ of the structure of the complex of lactoperoxidase with SCN⁻ (PDB ID, 3ERH), and (d) W1, HOI‐1, and HOI‐2 from the present structure and OSCN⁻ from the structure of the complex of lactoperoxidase with OSCN⁻ (PDB ID, 3BXI)

It was reported previously that in the absence of H₂O₂, iodide ions bound to LPO in the substrate binding site as well as on several other sites on the surface of the protein. ²³ However, when H₂O₂ was added afterwards to the mixture of LPO and iodide we observed that while H₂O₂ was bound in the distal heme cavity it failed to attain the required orientation for reaction with iron due to steric constraints with the iodide ion. As a result, the formation of the required compound I could not take place. ³³ This means that if the complex reaction of the lactoperoxidase system (LPO + H₂O₂ + iodide) is to proceed, the H₂O₂ must first interact with LPO to form compound I and then iodide ions will interact with compound I, instead of the resting LPO, to form the final product of hypoiodite ion. If the hypoiodite ion is not utilized by the system, it will remain weakly bound to LPO in the substrate binding site as well as other sites. Further reaction of LPO with a second substrate, such as ABTS in the present case, will be hampered.

It is clear from present observations that in the presence of H₂O₂, iodide is converted into hypoiodite and the protective effects of the oxidized iodide have already been enumerated. ³⁴ It was reported that hypoiodite ions very rapid oxidize thiol groups, irreversibly oxidize both NAD(P)H and β‐nicotinamide mononucleotide, react directly with thioether groups to produce sulfoxides and stimulate the oxidation of the primary amine moieties. ³⁴ , ³⁵ , ³⁶ , ³⁷ These quick, irreversible effects of hypoiodite and hypothiocyanite results in elimination of many microbial pathogens. ²⁰ , ³⁸ , ³⁹ , ⁴⁰

It was reported years ago that povidone‐iodine application to the nasal region was highly beneficial against an influenza pandemic in India. ⁴¹ A recent clinical trial has also shown that application of povidone‐iodine reduces the SARS‐CoV‐2 infection rate. ⁴² Interestingly, the infection rate and deaths from COVID‐19 has been and remains comparatively very low in Japan. ⁴³ Despite being densely populated, where many people live crowded lives, Japan has not enforced a nationwide lock‐down. As of June 2021, about 40,000 Japanese people were infected of 125 M citizens, with a cumulative death totaling about 15,000. With less than 5% of the Japanese fully vaccinated, deaths are running about 1/12 of the United States and Europe. ⁴⁴ Presumably this remarkable resistance to the COVID‐19 pandemic may be due to the high level of dietary iodine in Japan. ⁴⁵ The high level of iodine content may stimulate the lactoperoxidase system leading to the resistance to the SARS‐CoV‐2 viral agent. More independent evidence is the fact that smokers in general are suffering terribly from this virus because carbon monoxide binds to and inactivates lactoperoxidase. ⁴⁶ , ⁴⁷

4. CONCLUSIONS

The present studies clearly show that LPO with H₂O₂ converts iodide into hypoiodite. We emphasize here that the production of strong oxidants by the lactoperoxidase system (LPO + H₂O₂ + SCN⁻/I⁻) plays a powerful role in the innate immune defense against pathogenic bacteria, fungi, viruses, and parasites. This means that LPO is an indispensable enzyme in the fight against microbial infections and the iodine supplementation is a practical means of supporting this protection. We also mention that the enzymatic reactions involving mammalian heme peroxidases are complex with multiple sensitive steps as various small molecules can promote or reduce dramatically the antimicrobial activity of the LPO system. Several points should be taken into account to favor the enzymatic reaction for in vitro and in vivo antimicrobial applications, (1) avoid the presence of entities in the distal heme cavity which do not allow the binding and proper orientation of H₂O₂ thus preventing the formation of compound I, (2) avoid the presence of iodide competitors at the distal heme cavity of LPO‐compound I, (3) avoid an excess of H₂O₂ which can destroy the active enzyme, and (4) avoid an excess of iodide and hypoiodite which can also inhibit the formation of LPO‐compound I. While our work presented here does not directly address the current pandemic we strongly suggest the correlation between the very low infection and death rates in Japan from the SARS‐CoV‐2 virus is due to the high level of dietary iodine in the typical Japanese diet. The lactoperoxidase system in human nasal passages, bronchi and lungs utilizes iodine to prevent influenza infections including SARS‐CoV‐2.

5. MATERIALS AND METHODS

5.1. Purification of lactoperoxidase

LPO was isolated from the colostrum of sheep (Ovis aries) maintained at the Indian Veterinary Research Institute, Izzat Nagar, India. It was purified using a procedure reported earlier, ²¹ , ²² , ²³ , ²⁴ , ²⁵ , ²⁶ , ²⁷ in which a simple modification was made to the buffer added to the skimmed colostrum which is now 50 mM Tris–HCl, pH 8.0 and 2.5 mM CaCl_2. The buffered colostrum was mixed with CM‐Sephadex C‐50 (GE Healthcare, Uppsala, Sweden) suspended in 50 mM Tris–HCl, pH 8.0 at a concentration of 8 g L⁻¹ and the unbound proteins were removed by washing with excess buffer. The washed gel was loaded into a column (10 × 2.5 cm) then equilibrated with more buffer and the bound proteins were eluted using a linear gradient of 0.0–0.6 M NaCl in the same buffer. The brown‐colored fractions which eluted around 0.25 M NaCl were pooled, desalted and concentrated using an Amicon Ultrafiltration Cell (Millipore Corporation, Billerica, USA). The concentrate was loaded on a Sephadex G‐100 column (100 × 2 cm) equilibrated with 50 mM Tris–HCl buffer, pH 8.0 and purified LPO eluted using the same buffer at a flow rate of 5.8 ml/h. Tiny portions of the eluted fractions were examined using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). ⁴⁸ The fractions that developed bands corresponding to a molecular weight of about 68 kDa were pooled and lyophilized; these were typically judged to be >98% LPO.

5.2. Lactoperoxidase activity measurements

Three separate experiments were conducted to compare the effects of bound I⁻ and IO⁻ ions on the activity of LPO with the substrate ABTS (2,2′‐azino‐bis(3‐ethylbenzthiazoline‐sulfonic acid), which was procured from Sigma‐Aldrich (St. Louis, Missouri, USA). It may be mentioned here that ABTS is commonly used to measure peroxidase activity. Because neither HOI nor an alkaline salt of IO⁻ are commercially available we created IO⁻ in situ for some experiments. LPO was dissolved in 25 mM phosphate buffer, pH 6.0 (buffer) at a concentration of 5.0 μM then divided into small portions. A stock solution of H₂O₂ (Thermo Fisher Scientific India Pvt. Ltd., Mumbai, India) was prepared at a concentration of 1.0 M. Solutions of ABTS at 0.0, 5.0, 10.0, 20.0, 50.0, 100.0, 200.0, 400.0, 600.0, 800.0, and 1,000.0 μM were prepared in the same buffer.

In the first experiment, LPO and H₂O₂ were mixed in equal molar proportions for 15 min at 15°C after which different concentrations, 0.0–1,000.0 μM of ABTS were added, the mixtures incubated at 15°C for 15 min, then the absorptions of the oxidized ABTS measured at 413 nm (Figure 1, curve A) using a Lambda 25 (Perkin‐Elmer, Waltham, MA, USA) spectrophotometer. For the second experiment LPO at 5.0 μM and ammonium iodide at 20 mM concentrations were incubated for 15 min at 15°C then the H₂O₂ solution was added. Aliquotes of ABTS from 0.0 to 1,000.0 μM were then mixed and the mixtures incubated for another 15 min at 15°C and the absorptions measured (Figure 1, curve B). In the third experiment, LPO and H₂O₂ at equal molar ratios were added to 20 mM ammonium iodide solution which were first mixed and incubated at 15°C for 15 min. Then additional H₂O₂ and ABTS were added at concentrations from 0.0 to 1,000.0 μM, the mixtures incubated for 15 min at 15°C and the absorptions measured (Figure 1, curve C). All the experiments were performed in triplicate and mean errors were estimated. For display purposes only, the best fit curves were estimated empirically using the regression software Sigma Plot 14.0 (Systat Software Inc., San Jose, CA, USA).

5.3. Crystallization

Small portions of purified and lyophilized samples of LPO were dissolved in 0.01 M phosphate buffer, pH 7.0 to a concentration of 25 mg/ml. This was mixed in a 1:1 (vol:vol) ratio with 4 mM H₂O₂ and incubated for 15 min at 15°C, then an equal amount of 2 mM ammonium iodide was added to produce the LPO–HOI complex. Next 5 μl of this complex was mixed with 5 μl of a reservoir solution containing 20% (wt/vol) PEG‐3350 to prepare the 10 μl drops for the hanging drop vapor diffusion crystallization routine. After 1 month at 4°C, rectangular shaped, brown‐colored crystals measuring up to 0.3 × 0.2 × 0.2 mm³ were obtained and used for data collection.

5.4. X‐ray intensity data collection and structure determination

The LPO crystals were transferred to a small portion of reservoir solution and glycerol was added to a 22% concentration for data collection at low temperature. The X‐ray intensity data were collected at 100 K to 2.70 Å resolution with a MAR CCD‐225 detector (Marresearch, Norderstedt, Germany) using synchrotron beamline, BM14 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. The program, HKL 2000 ⁴⁹ was used for data processing. The crystals belong to monoclinic space group, P2₁ with cell dimensions, a = 53.9 Å, b = 80.2 Å, c = 76.1 Å, and β = 102.9°. As indicated by the unit cell dimensions and molecular weight of LPO, a single protein molecule was considered as the asymmetric unit. The structure was determined by the molecular replacement method using the program MOLREP. ⁵⁰ The coordinates of the structure of native LPO (PDB ID, 3GC1, Reference 21) were used as a search model. The refinement was carried out with the program REFMAC. ⁵¹ The manual model building was carried out using program COOT. ⁵² A difference Fourier (F _o − F _c) electron density map was calculated at R _cryst value of 0.261. The map showed electron densities above 3.0 σ cutoff at four positions in the distal heme cavity (Figure 6a) into which iodide ions were placed and refined. After 20 cycles of refinement, the electron density map with coefficients of (2F _o − F _c) was computed and clearly showed extra densities at all the four positions of iodide ions in the substrate binding channel (Figure 6b). Therefore, the hypoiodite ions were placed in the iodide ion positions thus taking into account the extra electron densities (Figure 6c). After further refinement for several cycles, an omit map was calculated after removing the four hypoiodite ions from the substrate binding channel, which clearly showed electron densities for hypoiodite ions into which hypoiodite ions were placed appropriately (Figure 6d). After further refinement, an electron density map with (2F _o − F _c) coefficients was calculated which showed perfect densities for the hypoiodite ions (Figure 6e). Seven more hypoiodite ions were identified at different sites on the protein exterior structure in a similar manner. In addition to seven exterior hypoiodite ions, six iodide ions were also observed loosely bound to the structure. The coordinates of all the extra entities were included in the subsequent refinement cycles which were performed using default restraints. The protein backbone was further adjusted using (2F _o − F _c) and (F _o − F _c) electron density maps.

FIGURE 6.

The electron density maps covering the substrate binding channel (a) the initial (F _o − F _c) map at 3σ showing extra electron densities into which iodide ions were placed and refined, (b) (2F _o − F _c) map at 1.5σ showing residual densities (green) indicating extra densities at 3σ, (c) placed hypoiodite ions in place of iodide ions, (d) omit electron density map at 3σ after removing hypoiodite ions from the substrate binding channel in which hypoiodite ions fit well, and (e) final (2F _o − F _c) electron density map at 1.5σ matching well for hypoiodite ions

A total of 157 water oxygen atoms were observed from the difference Fourier maps. The conserved water molecule W1 was also observed in the present structure at its usual position in the distal heme cavity. In the subsequent refinement cycles, the coordinates of all the water oxygen atoms were also included. The final values of R _cryst and R _free factors were 0.195 and 0.277, respectively. The quality of the final model was assessed with PROCHECK. ³¹ A validation report of the final model obtained using wwPDB validation pipeline showed the clash score and other indicators were in the optimum range. The final refined structure contained four hypoiodite ions in the substrate binding channel (Figure 3a) while seven hypoiodite ions were present at other positions (Figure 3b). The structure also contained six iodide ions (Figure 3c). The coordinates and other crystal data on this structure are deposited in the protein data bank with an accession code of 7VE3. The data collection and refinement statistics are given in Table 1. We note that it is not surprising that the average resolution of this structure is only 2.70 Å while many other LPO structural studies have yielded better data. ²³ , ²⁷ The presence of concentrated HOI ions in the present crystals may have affected the crystal quality.

TABLE 1.

Data collection and refinement statistics of lactoperoxidase at 2.70 Ǻ resolution with hypoiodite. Values in parentheses are for the highest resolution shell

Data collection
Beam line	ESRF BEAMLINE BM14
Wavelength (Å)	0.97
Resolution range (Å)	43.97–2.70 (2.75–2.70)
Space group	P 21
Unit‐cell parameters (Å, °)	a = 53.9, b = 80.2, c = 76.1 and β = 102.9
Number of molecules in the asymmetric unit	1
V m (Å3/Da)	2.30
Solvent content (%)	46.6
Number of unique reflections	17,167
Overall completeness (%)	98.1 (93.0)
R sym	0.07 (0.24)
I/σ(I)	46 (10)
Refinement statistics
R cryst	19.5
R free	27.7
Number of protein atoms	4,765
Heme moiety atoms	43
No of hypoiodite (11) atoms	22
No. of iodide (6) atoms	6
NAG (2) atoms	28
Sulphate (1) atom	5
Calcium (1) atom	1
Number of water oxygen atoms	157
R. M. S. deviations
Bond length (Å)	0.005
Bond angles (°)	1.4
Dihedral angles (°)	15.40
Mean B‐factor (Å2)
Main chain	69.3
Side chain and water oxygen atoms	71.4
Overall	70.0
Ramachandran plot statistics
Residues in the most favored region (%)	89.5
Residues in the additional allowed region (%)	10.3
Residues in the generously allowed regions (%)	0.2
PDB ID	7VE3

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest with the contents of this article.

AUTHOR CONTRIBUTIONS

Prashant Kumar Singh: Conceptualization (equal); data curation (equal); formal analysis (equal); investigation (equal); methodology (equal); software (equal). Nayeem Ahmad: Conceptualization (equal); data curation (equal); formal analysis (equal); investigation (equal); methodology (equal). Shavait Yamini: Data curation (equal); investigation (equal); methodology (equal). Rashmi Prabha Singh: Data curation (equal); investigation (equal); methodology (equal). Amit Kumar Singh: Data curation (equal); formal analysis (equal); methodology (equal). Pradeep Sharma: Data curation (equal); formal analysis (equal); methodology (equal). Michael L. Smith: Conceptualization (equal); formal analysis (equal); investigation (equal); validation (equal); writing – review and editing (equal). Sujata Sharma: Formal analysis (equal); investigation (equal); supervision (equal); validation (equal); writing – review and editing (equal). Tej P. Singh: Conceptualization (lead); investigation (lead); methodology (equal); resources (lead); supervision (lead); validation (lead); visualization (lead); writing – original draft (lead); writing – review and editing (lead).

Singh PK, Ahmad N, Yamini S, Singh RP, Singh AK, Sharma P, et al. Structural evidence of the oxidation of iodide ion into hyper‐reactive hypoiodite ion by mammalian heme lactoperoxidase. Protein Science. 2022;31:384–395. 10.1002/pro.4230