Chlorite reactivity with myoglobin: Analogy with peroxide and nitrite chemistry?

Accepted Manuscript Chlorite reactivity with myoglobin: Analogy with peroxide and nitrite chemistry?

Cristina Bischin, Augustin Mot, Andrei Stefancu, Nicolae Leopold, Denisa Hathazi, Grigore Damian, Radu SilaghiDumitrescu PII: DOI: Reference:

S0162-0134(16)30425-1 doi: 10.1016/j.jinorgbio.2017.04.017 JIB 10203

To appear in:

Journal of Inorganic Biochemistry

Received date: Revised date: Accepted date:

18 November 2016 19 April 2017 21 April 2017

Please cite this article as: Cristina Bischin, Augustin Mot, Andrei Stefancu, Nicolae Leopold, Denisa Hathazi, Grigore Damian, Radu Silaghi-Dumitrescu , Chlorite reactivity with myoglobin: Analogy with peroxide and nitrite chemistry?. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jib(2017), doi: 10.1016/j.jinorgbio.2017.04.017

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ACCEPTED MANUSCRIPT Chlorite reactivity with myoglobin: analogy with peroxide and nitrite chemistry? Cristina Bischina, Augustin Mota, Andrei Stefancub, Nicolae Leopoldb, Denisa Hathazia, Grigore Damian b, Radu Silaghi-Dumitrescua() Department of Chemistrya and Physicsb, Babes-Bolyai University, 11 Arany Janos street,

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() corresponding author: [email protected]

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Cluj-Napoca 400028, Romania

Abstract: Stopped-flow UV-vis data allow for the first time direct spectroscopic

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detection of a ferryl species during the reaction of met myoglobin (Mb) with chlorite,

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analogous to what is observed in the reaction with peroxides. Ferryl is also observed in the reaction of oxy Mb + chlorite. A pathway involving Fe-O-O-ClO2 is explored

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by analogy with the Fe-O-O-NO and Fe-O-O-NO2 previously proposed as

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intermediates in the reactions of oxy globins with nitric oxide and nitrite, respectively. However, Fe-O-O-ClO2 is not detectable in these stopped-flow

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experiments and is in fact, unlike its nitrogenous congeners, predicted by density functional theory (DFT) to be impossible for a heme complex. Deoxy Mb reacts with chlorite faster than met – suggesting that, unlike with hydrogen peroxide (with which deoxy Mb reacts slower than met), binding of chlorite to the heme is not a ratedetermining step (hence, most likely, an outer-sphere electron transfer mechanism); to correlate this, a Fe-O-Cl-O adduct was not observed experimentally for the met

ACCEPTED MANUSCRIPT or for the deoxy reactions – even though prior DFT calculations suggest it to be feasible and detectable.

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Keywords: ferryl, chlorite, myoglobin, iron-peroxochlorate, iron-peroxochlorite

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Introduction

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The reaction of hypochlorite with ferric heme proteins is reminiscent of those

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involving the related/isoelectronic hydrogen peroxide, involving high-valent ferryl species of the Compound I/II [1]–[11]. Similar reactivity is expected with chlorite,

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although this has been less extensively described. Chlorite dismutase (Cld) [12]

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catalyzes the disproportionation of chlorite to chloride and molecular oxygen in prokaryotes, protecting against oxidative stress [12], [13]. It features at the active

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site a pentacoordinated histidine-ligated heme [12]–[16], reminiscent of globins

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and of many peroxidases, where binding and heterolytic cleavage of chlorite would occur [12]–[14], yielding a high-valent ferryl center that would further interact

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with the emergent hypochlorite anion and eventually decompose to chloride and molecular oxygen [10]. Theoretical data by Hall et al. on porphyrin experimental models suggest that the mechanism involves intermediate spin state species and a homolytic cleavage of the Cl-O bond rather than a concerted mechanism [11]. More recently Obinger et al. reported high spin resting states for chlorite dismutases from several organisms [10]. Chloroperoxidase (CPO) and horseradish

ACCEPTED MANUSCRIPT peroxidase (HRP) are able to catalyze chlorination reactions with chlorite [1], [17]–[19]. Upon the disproportionation of chlorite by horseradish peroxidase a relatively stable intermediary assigned as Compound X [1] with similar spectral properties to that of HRP Compound I [20], was subsequently argued to in fact be

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Compound II [2], [3]. Radioisotope-labeling studies revealed the formation of an

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enzyme-bound activated halogen intermediate most probably with –OCl or a –Cl

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as ligand [1], [4]. Shahangian and Hager showed that Compound X is a ferryl species with a chlorine oxide ligand which acts as a true halogenating intermediate

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when the pH is lowered to neutral or acidic values [5]. Resonance Raman

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spectroscopy suggested that compound X and II have similar but not identical heme structure and that the chlorine atom may rather be bound to a heme-

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coordinated aminoacid residue [6]. Recently, Obinger et al. demonstrated that

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chlorite induces the two-electron oxidation of ferric HRP to Compound I with the release of hypochlorous acid, and can also reduce both Compound I and

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Compound II, generating chlorine dioxide. These reaction products (hypochlorus

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acid and chlorine dioxide), known to induce heme destruction, can also compete with chlorite for binding to the ferric HRP and mediate Compound I formation -

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but are not able to react with Compounds I and II [7]. Chlorite high spin complexes are generated either for the mammalian myeloperoxidase (MPO) and lactoperoxidase (LPO) in their reaction with sodium chlorite, but this system, contrary to HRP and CPO, is not able to chlorinate a molecule [8]. Beside peroxidases, chlorite can react with other heme proteins such as cytochrome P450 [21], myoglobin (Mb) [22] and hemoglobin (Hb)[23]. For the latter, formation of

ACCEPTED MANUSCRIPT met hemoglobin from oxy [23] as well as peroxide-like reactivity with met myoglobin [22] have been described [24]–[28]. The physiological effects of exogenous sodium chlorite on humans and animals include some reported cases of methemoglobinemia, hemolysis and

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glutathione depletion [29], [30]. These medical links to globin chemistry provide

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an additional point of interest for the present investigation, besides generally trying

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to investigate hemoprotein-hypochlorite interactions with relevance to chlorite

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myeloperoxidase, and others [8], [33], [34].

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dismutases [31], [32] and to some extent haloperoxidases (chloroperoxidase,

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Experimental details

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Sodium chlorite and horse heart myoglobin were purchased from Sigma-Aldrich (Germany). UV-vis spectra were recorded on a Cary 50 (Varian, Inc) instruments.

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Stopped-flow spectra were collected on a Biologic SFM-300 system equipped with 3

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syringes and capable of sequential mixing, with a high-speed diode array detector. Stopped-flow data were analyzed within the SPECFIT32 software package (BioLogic

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Science Instruments, France) using Singular Value Decomposition (SVD) and global multiexponential fitting of the SVD treated data, with the spectra fitted to simple kinetic models using Levenberg-Marquardt or Simplex algorithms. The anaerobic condition was maintained using protocatechuate dioxygenase and protocatechuic acid (Sorachim, Switzerland).

ACCEPTED MANUSCRIPT EPR spectra were collected with a Bruker EMX Micro spectrometer with a liquid nitrogen cooling system. The conditions were as follow: microwave frequency 9.4359 GHz, microwave power 10.02 mW, modulation amplitude 3 G, sweep rate 180.224 G/s; conversion time 44 ms, average of three sweeps for each spectrum, temperature 100 K.

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For EPR spectra, a Bruker EMX Micro spectrometer with a liquid nitrogen cooling

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system was employed. Instrument conditions: microwave frequency 9.43 GHz,

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microwave power 15.89 mW, modulation frequency 100 kHz, modulation amplitude 3 G, sweep rate 22.6 G/s; time constant 81.92 ms, average of three sweeps for each spectrum,

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temperature 100 K.

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Raman spectra were acquired on a Renishaw inVia Raman Microscope, by using 50% of the 80 mW HeCd laser, emitting at 442 nm. The spectra were recorded with 10

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seconds exposure time and 2 accumulations, at room temperature. Myoglobin was used in

were done in triplicates.

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a concentration of 100 µM, peroxide - 400 µM and chlorite - 500 µM. The experiments

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Density functional theory (DFT) calculations were performed using the B3LYP/6-

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31G** level of theory as implemented in the Spartan software package. For the selfconsistent field (SCF) calculations a fine grid was used, and the convergence criteria

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were set to10−6 (for the root mean square of electron density) and 10−8 (energy), respectively. For geometry optimization, convergence criteria were set to 0.001 a.u. (maximum gradient criterion) and 0.0003 (maximum displacement criterion). No geometrical constraints were used. Partial atomic charges and spin densities were obtained from natural population analyses (NPA) as implemented in Spartan [35].

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Results and discussions Reported here are UV-vis data allowing for the first time direct

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spectroscopic detection of a ferryl species during the reaction of met Mb with

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chlorite Lower amounts of ferryl also appear to accumulate early in the reaction of

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oxy myoglobin with chlorite, analogously to the related reaction of nitrite with oxy myoglobin [36].

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The reaction of ferric (met) Mb with chlorite is strongly pH-dependent as

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shown in Figure 1. The protein is more easily degraded at a lower pH. The ferryl states of Mb and Hb, obtained by the reaction with hydrogen peroxide, are more

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reactive at acidic pH due to the possibility of protonation of the ferryl group [37].

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In our chlorite experiments too, ferryl was accumulated only at pH 10. Even at high concentrations of chlorite (2 mM) the protein was not completely degraded.

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Figure 1.A. (left panel), illustrating the spectrum of the product obtained when

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mixing met Mb with chlorite, reveals a displacement of the Soret band from 410 nm to 420 nm, alongside a slight decreasing in absorbance. This, together with the

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547 and 582-nm features (Figure 1A, right panel), are similar to the UV-vis features of the Compound II ferryl species such as for instance generated in the reaction of met myoglobin with peroxide.

[Figure 1 here]

ACCEPTED MANUSCRIPT The spectrum resulted upon mixing myoglobin with chlorite is rapidly converted back to the starting form (met-Mb) by addition of ascorbate or by changes in pH, as expected from a high-valent form of protein (Figure 2)[38].

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[Figure 2 here]

Figure 3 shows that 90% of met-Mb treated with chlorite can be converted

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to sulfheme by treatment with sulfide. The generally accepted mechanism of

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sulfheme formation entails interaction of the highly reactive ferryl Compound II with H2S yielding a sulfide radical (HS•) which further attacks the porphyrin ring

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generating the sulfheme derivative [38], [39]. This sulfide treatment is a common

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diagnostic for the presence of ferryl globins [40]; the observed 90% yield of sulfmyoglobin in our chlorite experiments, supports the conclusion of ferryl formation

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during the myoglobin-chlorite interaction.

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[Figure 3 here]

In an attempt to further investigate the reaction intermediate, Resonance Raman (RR) spectroscopy was employed. Spectra of Mb (pH 10), compared to Mb treated with hydrogen peroxide and chlorite, respectively, are plotted in Figure 4. The typical spectral features of heme proteins [41], [42] are visible; additionally, specific bands are easily identified that distinctly appear when Mb is treated with

ACCEPTED MANUSCRIPT hydrogen peroxide as well as when treated with chlorite (Figure 4). These specific bands are clustered into two groups, 1000-1350 cm-1 and 550-750 cm-1 – i.e., regions that typically reflect structural and electronic change in the heme. Since ferryl-Mb generated with peroxide presents RR spectral features very similar to the

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Mb-chlorite product under the same experimental conditions, it can then be safely

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stated that the dominant reaction intermediate in the latter case is ferryl-Mb as

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well.

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[Figure 4]

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The EPR spectra of myoglobin treated with chlorite are shown in Figure 5. At pH 7 a decrease of the high spin signal at g=5.874 and an increase in the free

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radical signal (g=2.006) are observed with increasing the concentration of chlorite,

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similar to those observed in the reaction of globins with hydrogen peroxide where ferryl species is generated. Also evident is a slight increase in the g=4.286 signal

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which corresponds to heme degradation. While ferryl is a triplet species and as

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such cannot be directly detected using EPR spectroscopy, the decrease in the high spin (ferric) signal and the increase in radical signal (presumably arising via a

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ferryl mechanism) is an indirect evidence that ferryl is formed. At pH 10, the g=2.592 signal corresponding to the classical alkaline species (hydroxide ligation) is replaced in the presence of chlorite by a signal at g=2.439. According to Rifkind [43] the wide range of low-spin complexes with g above 3 and below 1, which have a defined EPR spectrum other than those corresponding to the hydroxide ligation (g=2.59, 2.17 and 1.83) [44], are indicative for the

ACCEPTED MANUSCRIPT hemichrome complexes. Therefore, the signal seen at g=2.439 and 2.224, together with the increase in absorbance around 630 nm (Fig.1), can be regarded as evidence for the formation of hemichrome under these conditions.

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[Figure 5 here]

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Exposure of ferric Mb even to low concentrations of chlorite at pH 7 (Fig. S1, Supporting information) leads to oxidative bleaching of the Soret band. On the

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stopped-flow time-scale, the reaction of ferric Mb with chlorite (Figure 1A)

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reveals a sequence of spectra starting from the characteristic Fe(III) Soret band at 410 nm, followed by a buildup of a transient intermediate, whose spectrum is

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characterized by a lower extinction coefficient and an absorption maximum at 420,

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548 and 580 nm, similar with those of Compound II Mb-ferryl species generated in the reaction with peroxide. At myoglobin concentrations amenable to observing

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the Soret band, the course of absorbance was reasonably fitted according to an

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ABC scheme, with species B corresponding to the ferryl intermediate (Fig. S2.A, Supporting Information); an ABC scheme was also followed at higher

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Mb concentrations (50 μM) (Fig. 6. A and Fig. S2.B, Supporting Information). The kinetics of the interaction between myoglobin and chlorite could be studied only in a narrow interval of concentrations at pH 7 (200-800 mM) (Fig. S3, Supporting Information) so it is difficult to precisely determine the rate constant and the reaction order with respect to chlorite. The estimates within the ABC model are n=1.6; k=16.8 M-1s-1 for the AB step, and n=0.8; k=4.9 s-1 for BC. This

ACCEPTED MANUSCRIPT model is imperfect, as illustrated in Figure S3; nevertheless, it does agree with a mechanism where chlorite participates in the formation as well as in the decay of the ferryl. The 0.8 reaction order with respect to the second step of the reaction, the one-electron reduction of ferryl to met, may be interpreted to suggest that chlorite

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acts as quasi-stoichiometric reducing agent for ferryl at this pH. The computed

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reaction order for the first step of the A->B->C fit (transition from met to ferryl),

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may suggest that more than one chlorite ions would be involved – such as in a concerted process involving oxidation of met to Compound I by chlorite and

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reduction of Compound I to Compound II/ferryl by a second chlorite ion

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(Compound I in globins is known to be particularly unstable towards autoreduction [45] so that a clear n=2 would not be expected).

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Beyond 800 mM, the AB process is too fast to be detected. At pH 10 the

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kinetic of this reaction can be determined on a somewhat larger range of concentrations (200 – 1600 mM), following the same model as at pH 7

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(ABC), with n=0.3, k=4.5 M-1s-1 for the AB step and n=0.1, k=1.5 for BC

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(Fig. S4, Supporting Information). Apparently then, at pH 10 the ferryl does not decay in a reaction dependent on chlorite. The lower reaction order for the

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transition from met-hydroxo to ferryl is difficult to interpret; one explanation would be that at this pH chlorite is less efficient as reducing agent, so that a second myoglobin molecule steps in as reducing agent for Compound I to ferryl – making for a 2 Mb : 1 chlorite stoichiometry similar to the stoichiometry that one would also observe for the globin: peroxide reaction (also a process where Compound I,

ACCEPTED MANUSCRIPT or an equivalent thereof, is proposed to be very rapidly formed and then reduced before one can detect it).

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[Figure 6]

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To our knowledge, this is the first report of direct detection of ferryl within

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the reaction of a globin with chlorite. The data may be interpreted to indicate a partial chlorite dismutase-like mechanism, generating ferryl but not completing to

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full isomerization to dioxygen and chloride. A ferric-chlorite intermediate may be

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proposed as intermediate prior to formation of ferryl; Hall and co-workers have described such species with DFT calculations [46] (and we have replicated these as

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well with a different functional – data not shown); however, we were unable to

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directly observe any intermediate between met and ferryl in the met Mb + chlorite system.

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Figure 7 shows the UV-vis spectra collected after mixing oxy Mb with a

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large excess of chlorite. The lack of a clear isosbestic point suggests more than two Mb states to be involved in this reaction. The set of spectra could be reasonably

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fitted to a AB kinetic model consisting of a simple transition from oxy Mb to a species with some spectral characteristics of ferryl – the latter species accumulating only in a small amount and assignable based on its diagnostic absorbance features at 540 and 580 nm as well as by the slight redshift of the Soret band (Figure 7), while also displaying two other small bands at 650 and 700 nm.

ACCEPTED MANUSCRIPT [Figure 7 here]

The A->B transition in the reaction of oxy Mb with chlorite is computed to be only very slightly dependent on the concentration of chlorite (n=0.1, k=1.8 M-1s-1

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cf. Figure S5), suggesting that initial binding/reaction of chlorite to oxy Mb, or

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reaction of chlorite to the ensuing species, are not rate-limiting steps. On the other

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hand, nor is dissociation of molecular oxygen from the heme a rate-limiting step, cf. Figure 8: the rate of such dissociation (measured separately in the presence of

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dithionite) is different from the rate of oxy + chlorite, and different from the rate of

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deoxy + chlorite (see also below, Figure 10).

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[Figure 8]

The oxy+chlorite experiment was also monitored in the presence of guanidine

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hydrochloride at relatively low concentrations (300 µM - 500 µM, where full

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denaturation of protein does not occur) - in order to see if changes in protein conformation and solvent accessibility to the active site facilitate the accumulation

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of an intermediate [47]. Only an influence on the reaction rate was noted – without any changes in the species detected (data not shown). The reaction sequence leading to this point, proposed in Figure 7, mirrors to some extent the interaction of oxy myoglobin with nitrite or with nitric oxide, where the iron-bound oxygen molecule reacts with the incoming oxyanion/oxide; nevertheless, so far in both cases the elusive Fe-O-O-X-O2 species (X=Cl, N)

ACCEPTED MANUSCRIPT remains undetected [36]. Moreover, our DFT calculations (BP86/6-31G**, cf. Supporting Information Tables S1 and S2) reveal that a heme Fe-O-O-Cl-O2 adduct would feature particularly long FeOO-ClO2 distances - at ~3 Å with variations depending on spin state, distinctly longer than the sum of covalent radii

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(1.8 Å) and approaching the sum of van der Waals radii (3.3 Å) – suggestive of a

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caged pair trapped within the distal pocket as opposed to a true covalently bonded

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species (Table S1, Supporting Information). The spin densities computed for the chlorite in these species are relatively high (~0.7-0.8 spin units, cf. Table S2),

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suggestive of a partially-oxidized ClO2 moiety and implicating a likely outer-

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sphere mechanism for electron transfer without formation of an actual covalent FeOO-ClO2 bond.

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Figure 9 shows that oxy myoglobin catalyzes chlorite reduction by ascorbate

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(more efficiently at pH 5.5 than at pH 7, as is also the case for other globincatalyzed processes) [24], [48], as well as by dithionite. The very high Km values

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estimated for chlorite (in fact, a linear dependence in the studied range of

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concentrations) are expected by comparison with the Km of another strong oxidant

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(yet distinctly less sterically demanding than chlorite), peroxide.[49]

[Figure 9 here]

The reaction of deoxy-Mb with chlorite leads to the formation of ferryl as illustrated in Figure 10, with n=0.9 and k=1.1 x 103 s-1 (Fig. S6, Supporting information). This is faster than the reaction of met globin with ferryl. Since

ACCEPTED MANUSCRIPT generally the opposite is true when comparing the reactivity of ferrous vs. ferric hemoproteins towards exogenous ligands [7], [32], [50], [51], chlorite’s different reactivity towards the deoxy (ferrous) vs. met (ferric) states can be best interpreted as evidence for outer-sphere redox transfer at least in the deoxy case if not in both.

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determining step has equimolar Mb: chlorite stoichiometry.

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The reaction order is consistent with a dominant mechanism where the rate-

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[Figure 10 here]

Conclusions

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Mb appears to offer amenable chemistry with chlorite, including so far detection of

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ferryl species in reactions with the met, oxy and deoxy forms. Direct observation of Fe-O-Cl-O or Fe-O-O-ClO2 species, as putative intermediates en route to ferryl,

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was not possible. In fact, DFT calculations suggest that a heme Fe-O-O-ClO2

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would not exist (no local minimum identified), while the relative reaction rates for ferrous vs. ferric Mb with chlorite suggest an outer-sphere mechanism at least for

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the former.

Acknowledgements: the work shown here has been supported by the Romanian Ministry for Education and Research (grants PN-II-ID-PCE-2012-4-0488).

ACCEPTED MANUSCRIPT References: [1]

P. F. Hollenberg, T. Rand-Meir, L. P. Hager, J. Biol. Chem., 249 (1974), pp. 5816–25. W. D. Hewson, L. P. Hager, J. Biol. Chem., 254 (1979), pp. 3175–3181.

[3]

W. D. Hewson, L. P. Hager, J. Biol. Chem., 254 (1979), pp. 3182–3186.

[4]

R. Chiang, T. Rand-Meir, R. Makino, L. P. Hager, J. Biol. Chem., 251 (1976), pp.

IP

T

[2]

CR

6340–6.

S. Shahangian, L. P. Hager, J. Biol. Chem., 256 (1981), pp. 6034–40.

[6]

A. J. Sitter, C. M. Reczek, J. Terner, J. Biol. Chem., 261 (1986), pp. 8638–42.

[7]

C. Jakopitsch, H. Spalteholz, P. G. Furtmüller, J. Arnhold, C. Obinger, J. Inorg.

AN

US

[5]

Biochem., 102 (2008), pp. 293–302.

C. Jakopitsch, K. F. Pirker, J. Flemmig, S. Hofbauer, D. Schlorke, P. G.

M

[8]

[9]

ED

Furtmüller, J. Arnhold, C. Obinger, J. Inorg. Biochem., 135 (2014), pp. 10–19. R. Silaghi-Dumitrescu, Eur. J. Inorg. Chem., 2008 (2008), pp. 5404–5407.

PT

[10] J. Kostan, B. Sjöblom, F. Maixner, G. Mlynek, P. G. Furtmüller, C. Obinger, M.

CE

Wagner, H. Daims, K. Djinović-Carugo, J. Struct. Biol., 172 (2010), pp. 331–42. [11] J. M. Keith, M. M. Abu-Omar, M. B. Hall, Inorg. Chem., 50 (2011) pp. 7928–

AC

7930.

[12] K. S. Bender, M. R. Rice, W. H. Fugate, J. D. Coates, L. A. Achenbach, Appl. Environ. Microbiol., 70 (2004), pp. 5651–8. [13] H. Ueno, K. Oishi, Y. Sayato, K. Nakamuro, 38 (2000), pp. 1-6. [14] F. Maixner, M. Wagner, S. Lücker, E. Pelletier, S. Schmitz-Esser, K. Hace, E. Spieck, R. Konrat, D. Le Paslier, H. Daims, Environ. Microbiol., 10 (2008), pp.

ACCEPTED MANUSCRIPT 3043–56. [15] B. R. Goblirsch, B. R. Streit, J. L. Dubois, C. M. Wilmot, J. Biol. Inorg. Chem., 15 (2010), pp. 879–88. [16] A. Ebihara, A. Okamoto, Y. Kousumi, H. Yamamoto, R. Masui, N. Ueyama, S.

T

Yokoyama, S. Kuramitsu, J. Struct. Funct. Genomics, 6 (2005), pp. 21–32.

IP

[17] A. Taurog, E. M. Howells, J. Biol. Chem., 241 (1996), pp. 1329–39.

CR

[18] G. S. Bayse and M. Morrison, Arch. Biochem. Biophys., 145 (1971) pp. 143–148.

Acad. Sci., 244 (1975), pp. 80–93.

US

[19] L. P. Hager, P. F. Hollenberg, T. Rand-Meir, R. Chiang, D. Doubek, Ann. N. Y.

AN

[20] J. N. Rodríguez-López, D. J. Lowe, J. Hernández-Ruiz, A. N. P. Hiner, F. GarcíaCánovas, R. N. F. Thorneley, J. Am. Chem. Soc.,123 (2001), pp. 11838–11847.

M

[21] E. G. Hrycay, J.-Å. Gustafsson, M. Ingelman-Sundberg, L. Ernster, FEBS Lett., 56

ED

(1975), pp. 161–165.

[22] P. George, D. H. Irvine, Biochem. J., 58 (1954), pp. 188–95.

PT

[23] C. L. French, S. S. Yaun, L. A. Baldwin, D. A. Leonard, X. Q. Zhao, E. J.

CE

Calabrese, J. Appl. Toxicol., 15 (1995), pp. 167–74. [24] C. E. Cooper, R. Silaghi-Dumitrescu, M. Rukengwa, A. I. Alayash, P. W. Buehler,

AC

Biochim. Biophys. Acta - Proteins Proteomics, 1784 (2008), pp. 1415–1420. [25] B. J. Reeder, D. A. Svistunenko, C. E. Cooper, and M. T. Wilson, Antioxid. Redox Signal., 6 (2004), pp. 954–966. [26] B. J. Reeder, M. Grey, R. L. Silaghi-Dumitrescu, D. A. Svistunenko, L. Bulow, C. E. Cooper, M. T. Wilson, J Biol Chem, 283 (2008), pp. 30780–30787. [27] R. Silaghi-Dumitrescu, B. J. Reeder, P. Nicholls, C. E. Cooper, and M. T. Wilson,

ACCEPTED MANUSCRIPT Biochem. J., 403 (2007), pp. 391-395. [28] N. B. J. Vollaard, B. J. Reeder, J. P. Shearman, P. Menu, M. T. Wilson, C. E. Cooper, Free Radic. Biol. Med., 39 (2005), pp. 1216–1228. [29] A. Romanovsky, D. Djogovic, D. Chin, J. Med. Toxicol., 9 (2013), pp. 67–70.

T

[30] D. Couri, M. S. Abdel-Rahman, R. J. Bull, Environ. Health Perspect., 46 (1982),

IP

pp. 13–7.

CR

[31] S. Hofbauer, C. Gruber, K. F. Pirker, A. Sündermann, I. Schaffner, C. Jakopitsch, C. Oostenbrink, P. G. Furtmüller, C. Obinger, Biochemistry, 53 (2014), pp. 3145–

US

57.

AN

[32] I. Schaffner, S. Hofbauer, M. Krutzler, K. F. Pirker, P. G. Furtmüller, C. Obinger, Arch. Biochem. Biophys., 574 (2015), pp. 18–26.

M

[33] R. Floris, R. Wever, Eur. J. Biochem., 207 (1992), pp. 697–702.

ED

[34] S. J. Klebanoff, J. Leukoc. Biol., 77 (2005), pp. 598–625. [35] “SPARTAN’06 for Windows Wave function Inc, 18401 Von Karman Avenue,

PT

Suite 370 Irvine, CA 92612, 2006.”

CE

[36] D. Hathazi, S. D. Mahuţ, F.-V. Scurtu, C. Bischin, C. Stanciu, A. A. Attia, G. Damian, R. Silaghi-Dumitrescu, JBIC J. Biol. Inorg. Chem., 19 (2014), pp. 1233–

AC

1239.

[37] B. J. Reeder, M. T. Wilson, Free. Radic. Biol. Med., 30 (2001), pp. 1311–1318. [38] B. B. Ríos-González, E. M. Román-Morales, R. Pietri, J. López-Garriga, J. Inorg. Biochem., 133 (2014), pp. 78–86. [39] J. A. Berzofsky, J. Peisach, W. E. Blumberg, J. Biol. Chem., 246 (1971), pp. 3367–3377.

ACCEPTED MANUSCRIPT [40] L. L. Bondoc, M. H. Chau, M. A. Price, and R. Timkovich, Biochemistry, 25 (1986), pp. 8458–8466. [41] S. Hu, K. M. Smith,T. G. Spiro, J. Am. Chem. Soc., 118 (1996), pp. 12638–12646. [42] S. Hu, I. K. Morris, J. P. Singh, K. M. Smith, T. G. Spiro, J. Am. Chem. Soc., 115

T

(1993), pp. 12446–12458.

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[43] J. M. Rifkind, O. Abugo, A. Levy, J. Heim, Methods Enzymol., 231 (1994), pp.

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449–80.

[44] D. A. Svistunenko, M. A. Sharpe, P. Nicholls, C. Blenkinsop, N. A. Davies, J.

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Dunne, M. T. Wilson, C. E. Cooper, Biochem J, 351 (2000), no. 3, pp. 595–605.

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[45] C. E. Cooper, M. Jurd, P. Nicholls, M. M. Wankasi, D. A. Svistunenko, B. J. Reeder, M. T. Wilson, Dalt. Trans., 2005 (2005), pp. 3483-3488.

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[46] J. M. Keith, M. M. Abu-Omar, M. B. Hall, Inorg. Chem., 50 (2011), pp. 7928–

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7930.

[47] C. Bischin, F. Deac, R. Silaghi-Dumitrescu, J. A. Worrall, B. S. Rajagopal, G.

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Damian, C. E. Cooper, Free Radic Res, 45 (2011), pp. 439–444.

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[48] J. Dunne, A. Caron, P. Menu, A. I. Alayash, P. W. Buehler, M. T. Wilson, R. Silaghi-Dumitrescu, B. Faivre, C. E. Cooper, Biochem. J., 399 (2006), pp. 513–24.

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[49] F. V Deac, A. Todea, A. M. Bolfa, P. Podea, P. Petrar, R. Silaghi-Dumitrescu, Rom. J. Biochem., 46 (2009), pp. 115–121. [50] D. Hathazi, S. D. Mahuţ, F.-V. Scurtu, C. Bischin, C. Stanciu, A. A. Attia, G. Damian, R. Silaghi-Dumitrescu, JBIC J. Biol. Inorg. Chem., 19 (2014), pp. 1233– 1239. [51] R. Silaghi-Dumitrescu, D. A. Svistunenko, D. Cioloboc, C. Bischin, F. Scurtu, C.

ACCEPTED MANUSCRIPT E. Cooper, Nitric Oxide - Biol. Chem., 42 (2014), pp. 32–39.

Figure 1. A. left: UV-vis spectra recorded in the Soret region for 10 µM met-Mb

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mixed with 2 mM chlorite at pH 10, borate buffer, 100 mM; right: the ɑ and β

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bands of the same reaction mixture, and the reference spectrum of ferryl-Mb

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obtained with a slight excess of hydrogen peroxide in the absence of chlorite. B.

chlorite at the pH shown in the legend.

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Time course for the reaction at 410 nm of 10 µM met-Mb mixed with 2 mM

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Figure 2. UV-vis spectra of ferryl Mb obtained by mixing met-Mb with chlorite (1 mM) (pH 10), and then rapidly transferred to a solution of 500 µM ascorbate, pH

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Figure 3. UV-vis spectra of species obtained after addition of 1 mM sulfide (metMb + chlorite + sulfide) to the mixture of met-Mb and 300 µM chlorite (met-Mb +

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chlorite).

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Figure 4. Resonance Raman spectra in the spectral region of A) low frequency (200-800 cm-1) and B) high frequency (900-1650) of met-Mb (100 µM) treated

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with chlorite (500 µM) and peroxide (400 µM), pH 10. Figure 5. EPR spectra of Mb (200 µM) treated with chlorite frozen at 30 s after mixing, at pH 7 (A) and at pH 10 (B). Figure 6. A) Left: UV-vis spectra of the Soret band collected upon mixing of metMb (10 µM) with chlorite (533 mM), in phosphate buffer (100 mM), pH 7, over a range of 2.4 seconds; right: computed spectra resulted from a fit of the

ACCEPTED MANUSCRIPT experimental data to an AB; BC model (global multiexponential fitting of the SVD treated data, using a Levenberg-Marquardt algorithm). B) Left: UV-vis spectra collected upon mixing of met-Mb (50 µM) with chlorite (400 mM). Right: computed spectra resulted from a fit of the experimental data to an AB; BC

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model (global multiexponential fitting of the SVD treated data, using a Levenberg-

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Marquardt algorithm) model, pH 7, over a range of 2.4 seconds. Lower panel:

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proposed reaction scheme.

Figure 7. A) UV-vis spectra collected in the first 2.4 s after mixing oxy-Mb (10

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µM) with chlorite (533 mM); right: UV-vis spectra collected in the first 2.4 s after

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mixing oxy-Mb (50 µM) with chlorite (880 mM), in phosphate buffer (100 mM), pH 7. B) Left: computed spectra for the species involved in the AB simulated

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reaction model. Conditions: oxy-Mb-50 µM; chlorite-880 mM, over a range of 1,6

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s; right: fitting at 580 nm trace for the AB kinetic model. Lower panel: proposed reaction scheme.

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Figure 8. Time course at 550 nm of the reaction between deoxy-Mb with 400 mM

pH 7.

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dithionite, deoxy-Mb with 533 mM chlorite, and oxy-Mb with 533 mM chlorite, at

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Figure 9. The initial reaction rate of the consumption of ascorbate (at 290 nm) and dithionite (at 315 nm) in the reaction of oxy-myoglobin (2 µM) with chlorite, at two different pH. Figure 10. A) UV-vis spectra of 50µM deoxy-Mb mixed with 1.3 mM chlorite, pH 7. B) The last spectrum from the A compared with the ferryl-Mb obtained in the

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Graphical Abstract

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UV-vis, RAMAN and EPR are used to investigate the reaction of chlorite with myoglobin (Mb);

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Ferryl species were detected by stopped-flow spectroscopy in the reactions with

Ferryl species were detected by stopped-flow spectroscopy in the reactions with

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met-Mb

oxy-Mb;

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Fe-O-Cl-O and Fe-O-O-ClO2 were explored as possible reaction intermediates.

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