Nitroxides protect horseradish peroxidase from H2O2-induced inactivation and modulate its catalase-like activity

    Nitroxides protect horseradish peroxidase from H 2 O2 -induced inactivation and modulate its catalase-like activity Amram Samuni, Eric Maimon, Sara Goldstein PII: DOI: Reference:

S0304-4165(17)30112-5 doi:10.1016/j.bbagen.2017.03.021 BBAGEN 28812

To appear in:

BBA - General Subjects

Received date: Revised date: Accepted date:

5 January 2017 5 March 2017 20 March 2017

Please cite this article as: Amram Samuni, Eric Maimon, Sara Goldstein, Nitroxides protect horseradish peroxidase from H2 O2 -induced inactivation and modulate its catalaselike activity, BBA - General Subjects (2017), doi:10.1016/j.bbagen.2017.03.021

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Nitroxides protect horseradish peroxidase from H2O2-induced inactivation and

SC

RI P

T

modulate its catalase-like activity

a

MA NU

Amram Samunia, Eric Maimonb, Sara Goldsteinc*

Institute of Medical Research Israel-Canada, Medical School , The Hebrew University of

ED

Jerusalem, Jerusalem 91120, Israel, b Nuclear Research Centre Negev, Beer Sheva, Israel, c Institute of Chemistry, The Accelerator Laboratory, the Hebrew University of Jerusalem, Jerusalem 91904,

AC

CE

PT

Israel

* To whom all correspondence should be directed. Tel. 972-2-6586478; E-mail: [email protected]

1

ACCEPTED MANUSCRIPT Abstract Background: Horseradish peroxidase (HRP) catalyzes H2O2 dismutation while undergoing heme

T

inactivation. The mechanism underlying this process has not been fully elucidated. The effects of

RI P

nitroxides, which protect metmyoglobin and methemoglobin against H2O2-induced inactivation, have been investigated.

SC

Methods: HRP reaction with H2O2 was studied by following H2O2 depletion, O2 evolution and

MA NU

heme spectral changes. Nitroxide concentration was followed by EPR spectroscopy, and its reactions with the oxidized heme species were studied using stopped-flow. Results: Nitroxide protects HRP against H2O2-induced inactivation. The rate of H2O2 dismutation in the presence of nitroxide obeys zero-order kinetics and increases as [nitroxide] increases.

ED

Nitroxide acts catalytically since its oxidized form is readily reduced to the nitroxide mainly by H2O2. The nitroxide efficacy follows the order 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO) > 4-

PT

OH-TPO > 3-carbamoyl proxyl > 4-oxo-TPO, which correlates with the order of the rate constants

CE

of nitroxide reactions with compounds I, II, and III. Conclusions: Nitroxide catalytically protects HRP against inactivation induced by H2O2 while

AC

modulating its catalase-like activity. The protective role of nitroxide at µM concentration range is attributed to its efficient oxidation by P940, which is the precursor of the inactivated form P670. Modeling the dismutation kinetics in the presence of nitroxide adequately fits the experimental data. In the absence of nitroxide the simulation fits the observed kinetics only if it does not include the formation of a Michaelis-Menten complex. General Significance: Nitroxides catalytically protect heme proteins against inactivation induced by H2O2 revealing an additional important role played by nitroxides in vivo.

Key words: HRP, suicide inactivation, compound II, compound III, TPO, catalase-like activity, heme inactivation, EPR, kinetics

2

ACCEPTED MANUSCRIPT Abbreviations: 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid, ABTS2-; compound I, RPorFeIV=O; compound II, R-PorFeIV=O; compound III, R-PorFeIIIO2•–

+•

R-PorFeIIO2; 3-

CP, 3-carbamoyl proxyl; EPR, electron paramagnetic resonance; heme protein, R-PorFeIII; HRP,

T

horseradish peroxidase; hydroxylamine, R2NO-H; 4-OH-TPO, 4-hydroxy-2,2,6,6-

RI P

tetramethylpiperidine-N-oxyl; 4-oxo-TPO, 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl; nitroxide,

SC

R2NO•; oxoammonium cation, R2N+=O; PB, phosphate buffer; TON, turnover number; TPO

AC

CE

PT

ED

MA NU

,2,2,6,6-tetramethyl-piperidine-N-oxyl; TPO-H, 2,2,6,6-tetramethyl-1-hydroxypiperidine

3

ACCEPTED MANUSCRIPT 1. Introduction Peroxidases are ubiquitous enzymes that catalyze a variety of oxygen-transfer reactions and are thus potentially useful for industrial and biomedical applications [1-5]. However, peroxidases

T

including horseradish peroxidase (HRP) [6, 7], myeloperoxidase {Paumann-Page, 2013 #61},

RI P

ascorbate peroxidase [9], lactoperoxidase [10] and prostaglandin H synthase-1 [11] are progressively inactivated by their substrate H2O2. Hence, the commercial uses of peroxidases are

SC

limited, primarily by their low stability in the presence of high [H2O2]. The mechanism underlying

MA NU

this process, frequently denoted as “suicide inactivation”, has not been fully elucidated. The catalytic cycle of HRP (R-PorFeIII) is initiated by a rapid two-electron reduction of H2O2 involving donation of one electron by the metal to H2O2 and a second electron from the

ED

porphyrin ring forming a π-cation radical [12, 13].

k1 = 1.7 x 107 M-1s-1 [14]

(1)

PT

R-PorFeIII + H2O2  R-+•PorFeIV=O + H2O

CE

In the absence of reducing substrates excess of H2O2 can react with R-+•PorFeIV=O (compound I) as

AC

an electron donor. This reaction has been suggested to occur via one-electron reduction steps forming compound II (R-PorFeIV=O), compound III (R-PorFeIIIO2•–

R-PorFeIIO2), and O2•–

as intermediates [15, 16] or in a catalase-like two-electron process resulting in the formation of O2 [7, 17-19]. A competing pathway results in the progressive loss of the enzymatic activity due to the formation of an inactivated form absorbing at 670 nm named verdohemeperoxidase (P670) [20, 21], which has been presumably associated with H2O2 reaction with compound I [7, 15, 18, 19, 22], with compound II [21] or with compound III [16, 23, 24]. Because of the complexity of the system and the multiplicity of reactions, kinetic methods have been used in most studies for elucidating the catalytic and inactivation mechanisms. Recently, we have shown that stable nitroxide radicals catalytically protect metmyoglobin and methemoglobin against inactivation induced by H2O2 alone and in the presence of nitrite while 4

ACCEPTED MANUSCRIPT modulating their catalytic activities [25, 26]. In addition, nitroxides served as a powerful research tool for elucidating the mechanism underlying the catalysis and inactivation routes [25, 26]. In the present study we demonstrate that nitroxides catalytically protect HRP against inactivation induced

T

by H2O2 while modulating its catalase-like activity, and shed more light on the mechanisms

SC

RI P

underlying these processes.

MA NU

2. Material and methods 2.1. Materials

All chemicals were of analytical grade and were used as received. Water for preparation of the solutions was purified using a Milli-Q purification system. The following products were purchased

ED

from Sigma-Aldrich: HRP (EC 1.11.1.7, Type VI, P8375, A403/A275= 3.0), 2,2'-Azino-bis(3-

PT

ethylbenzthiazoline-6-sulfonic acid (ABTS2-), 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO), 4-OHTPO, 4-oxo-TPO and 3-carbamoyl proxyl (3-CP). 2,2,6,6-tetramethyl-1-hydroxypiperidine

CE

hydrochloride (TPO-H.HCl) was prepared by bubbling HCl gas through ethanolic solution of the nitroxide followed by drying. Catalase from bovine liver was purchased from Boehringer

2.2. Analysis

AC

Biochemicals.

HRP was dissolved in 40 mM phosphate buffer (PB) at pH 6.3 or 7.2, and its concentration was determined spectrophotometrically using 403 = 100 mM-1cm-1 [27]. Hydrogen peroxide concentration was determined spectrophotometrically (240 = 39.4 M-1cm-1 [28]) or using the molybdate-activated iodine assay (352 = 25.8 mM-1cm-1) [29]. Compound II was prepared by adding 5 M H2O2 to 5 M HRP followed by the addition of 5.1 M ferrocyanide in 1 - 40 mM PB (pH 7.2). Compound II formation was evident by its typical Soret band at 419 nm and two absorption maxima at 527 and 555 nm [30]. Under these experimental conditions compound II decayed back to the native enzyme via a first-order kinetics (k = (5.7 ± 0.1) x 10-4 s-1 at 25oC). 5

ACCEPTED MANUSCRIPT Compound III was prepared by adding 10 mM H2O2 to pre-formed compound II followed 20 s later by catalase addition (120 U mL-1) to destroy residual un-reacted H2O2. The formation of compound III was manifested by its typical Soret band at 417 nm and two absorption maxima at 545 and 577

T

nm [30]. Compound III decayed back to the native HRP via a first-order kinetics resulting in k =

RI P

(3.0 ± 0.2) x 10-3 s-1 at 25oC in agreement with earlier reported values [17, 31]. 2.3. Inactivation of HRP. The peroxidative activity of HRP was determined using ABTS2- as a

SC

reducing substrate. A 10 L aliquot was withdrawn from the reaction mixture and plunged into a

MA NU

cuvette containing 1 mM ABTS2-, 0.5 mM H2O2 and 40 mM PB at pH 6.0 in a final volume of 3 mL. Activity was measured as the initial rate of the increase in the absorbance of ABTS•- during the first minute at 420 nm ( =36 mM-1cm-1) or 730 nm ( =19 mM-1cm-1). 2.4. Kinetics

ED

(i) Kinetic measurements were carried out using an HP 8452A diode array spectrophotometer coupled with a thermostat (HP 89075C Programmable Multicell Transport). The reaction was

PT

started by the addition of 10 – 30 L of H2O2 solution to 1 – 3 mL solutions containing HRP

CE

without and with nitroxide. The optical path length was 1 cm; (ii) rate constants of nitroxide reactions with compounds I, II, and III were determined using the Bio SX-17MV Sequential

AC

Stopped-Flow from Applied Photophysics with a 1 cm optical path. Compound I was formed in situ by mixing at a 1:1 ratio a solution of 5.1 – 5.4 M HRP with a solution containing 4.5 - 5 M H2O2 and the nitroxide or upon mixing a solution of 5 M H2O2 with a solution containing 5.3 M HRP and TPO-H to avoid premature oxidation of TPO-H by H2O2 to TPO. HRP was added to H2O2 slightly over the stoichiometric amount to ensure that no recycling occurs due to any residual H2O2. The reduction of compound I to compound II by excess of nitroxide or TPO-H was followed at 411 nm, which is the isosbestic point for HRP and compound II. Thus, any competition of compound II for the excess of nitroxide is eliminated as a source of error. Compound II or compound III were transferred into the syringe of the stopped-flow immediately after their preparation and mixed with excess of nitroxide solution at a 1:1 ratio. The reactions were monitored at 422 nm where maximum 6

ACCEPTED MANUSCRIPT change occurs in the absorbance between HRP and compound II or compound III. At least 3 independent experiments were preformed for each nitroxide concentration or pH and for each experiment 3 - 5 determinations of kobs.

T

2.5. Oximetry

RI P

Evolution of O2 during H2O2 dismutation catalyzed by HRP was followed using a Clark electrode oximetry coupled to an YSI Model 5300 Biological Oxygen Monitor unit, interfaced online with a

SC

PC computer. The sample was continuously stirred during the experiments and the temperature of

MA NU

the oximeter cell (1.65 mL volume) was controlled at 25 ± 0.2°C using a Julabo F10 circulating water bath. Argon was bubbled through the solution for experiments requiring anoxic conditions. The oximeter was calibrated for each experiment using aerated and anoxic solutions. Experiments were carried out in 40 mM acetate, phosphate or borate buffers. After temperature equilibration, the

ED

reaction was started by injecting the enzyme solution through the capillary tube at the stopper of the oximeter cell.

PT

2.6. Electron paramagnetic resonance (EPR) spectrometry

CE

Electron paramagnetic resonance (EPR) spectra were recorded using a Varian E4 X-band spectrometer operating at 9.36 GHz with the center field set at 3342 G, 100 kHz modulation

AC

frequency, 1 G field modulation amplitude, and 16 mW incident microwave power. Samples of the reaction mixture were injected into a flexible capillary inserted into a quartz tube placed within the EPR spectrometer cavity. The nitroxide concentration was calculated from the EPR signal intensity using standard solutions of the nitroxides. The experiments were carried out at room temperature (22 oC). 2.7. Kinetic simulations Modeling of the experimental results was carried out using INTKIN, a non commercial program developed at Brookhaven National Laboratories by Dr. H. A. Schwarz.

7

ACCEPTED MANUSCRIPT 3. Results 3.1. H2O2 dismutation catalyzed by HRP HRP is available from a number of sources having various grades of purity and isoenzymic

T

combinations. It has been demonstrated that all HRP types are susceptible to inactivation by H2O2

RI P

but might differ by their resistance towards inactivation, and their kinetic parameters [32]. Therefore, we selected a single type of HRP (Sigma-Aldrich, Type VI, P8375, A403/A275= 3.0), and

SC

re-determined its enzymatic activity and susceptibility toward inactivation induced by H2O2 alone

MA NU

as a basis for comparison with its behavior in the presence of nitroxides. The inclusion of 100 µM DTPA in the reaction mixtures had no effect on the results. Unless otherwise stated, all solutions contained 40 mM PB and the reactions were conducted at 25 oC. At relatively high [H2O2]/[HRP] ratios the kinetics of H2O2 decomposition demonstrates a gradually

ED

decreasing rate due to a progressive heme inactivation and loss of catalytic activity. Fig. 1A demonstrates that compound III is the predominant heme species throughout the catalysis process.

PT

The progressive heme destruction as indicated by the disappearance of the Soret peak and the

(Fig. 1A).

0.5

5

X9

B 4

0.3

[H2O2] (mM)

Absorbance

0.4

A

AC

CE

absorption peaks at 545 and 577 nm is accompanied by an increase of an absorption peak at 670 nm

0.2

0.1

0.0 300

3

2

1

400

500

600

0

700

0

20

40

60

80

100

Time (min)

Wavelength (nm)

Figure 1. Dismutation of 4.8 mM H2O2 catalyzed by 4.8 M HRP at pH 7.2. (A) Spectral changes monitored prior to the addition of H2O2 (black line) and 2, 24 and 79 min after its addition; (B) Kinetics of H2O2 consumption. The red curve represents simulated data calculated using the proposed mechanism (Scheme 1) and the rate constants listed in Table 4 where kcat/KM = 325 M-1s-1, kin = 0.42 M-1s-1 and k9 = 2.5 M-1s-1. 8

ACCEPTED MANUSCRIPT Complete loss of the Soret peak is observed at higher [H2O2]/[HRP] ratios, yet eventually, under such experimental conditions the absorption appearing at 670 nm slowly decays (data not shown). There is a good correlation between the extent of the loss of the Soret peak of HRP and the loss of

T

its peroxidative activity determined using ABTS2- as a reducing substrate (see experimental

RI P

section). For example, the loss of the Soret peak of 1.9 µM HRP during the dismutation of 2 mM

SC

H2O2 at pH 7.2 was 80  3% whereas the residual peroxidative activity was 23  2% implying that inactivation of the enzyme involves heme deterioration and release of its iron.

MA NU

As demonstrated in Fig. 2A, there is an initial fast absorbance increase at 417 nm reflecting the conversion of compound I to compound III. The initial rate of the absorption loss at 417 nm, which reflects enzyme inactivation, has been determined in the presence of 0.5 – 100 mM H2O2 both at pH 6.3 and 7.2. This rate is proportional to [HRP]o and therefore is expressed as a turnover number

ED

(TON) of inactivation, i.e., M inactivated HRP per M HRP per min, which depends on [H2O2]o.

PT

The concentration of the inactivated HRP was calculated using 417 = 89.5  4.5 mM-1cm-1 determined from the difference between the maximum absorption and that measured when the Soret

CE

band completely disappeared at relatively high [H2O2] and various [HRP]o. The effect of [H2O2]o on the TON of inactivation is presented in Fig. 2B demonstrating the same dependence at pH 6.3 and

AC

7.2 and a plateau value of 0.054  0.003 min-1, which is in agreement with previously reported value of 0.054 min-1 at [H2O2] < 1 M [33]. 0.06

B -1

TON of inactivation (min )

A

A417

0.32

0.28

0.24

0.04

0.02 pH 7.2 pH 6.3 0.00

0

200

0

400

20

40

60

80

100

[H2O2] (mM)

Time (s)

Figure 2. (A) Kinetics monitored at 417 nm when 5 mM H2O2 was added to 3.5 M HRP at pH 7.2; (B) Effect of [H2O2]o on the TON of HRP inactivation at pH 6.3 and 7.2 determined from the initial rate of the 9

ACCEPTED MANUSCRIPT decrease of the absorption at 417 nm using 417 = 89.5  4.5 mM-1cm-1 for calculating the concentration of inactivated HRP. Data represent mean values from duplicate determinations from at least two separate experiments.

T

The kinetics of H2O2 dismutation catalyzed by HRP was studied by following both the time-

RI P

dependence of H2O2 depletion and O2 evolution (e.g., Fig. 3). The same results were obtained under anoxia or under aerobic conditions. The kinetics of H2O2 loss was mirrored by the kinetics of O2

SC

production (Fig. 3A) implying that two molecules of H2O2 give rise to 1 molecule of O2. Fig. 3B shows the evolution of 620  80 M O2 when 1 M HRP was added to 20 mM H2O2, i.e., 1 M

MA NU

HRP catalyzes the dismutation of 1.24  0.15 mM H2O2 till it is inactivated. In this case the kinetics of O2 release demonstrates an initial fast rate followed by a relatively slower and progressively decreasing rate reflecting progressive enzyme inactivation (Fig. 3B). Similar kinetics of O2

ED

evolution has been previously observed in the case of HRP [18], myeloperoxidase [34] and methemoglobin [35]. The initial rapid reaction phase is observable using oximetric assay, which

PT

monitors the appearance of a reaction product, but hardly using iodometric assay, which follows

CE

substrate decay and is therefore much less sensitive.

250

600

150

[O2] (M)

AC

M

200

B

A

100

20 min

300

400

2 min

200

200

100

50

0

0

0 0

5

10

15

20

Time (min)

HRP

Figure 3. (A) O2 evolution and H2O2 consumption when 5 M HRP was added to 250 M H2O2 at pH 7.2; (B) O2 evolution when 1 M HRP was added to 20 mM H2O2 at pH 6.3. The inset is the expansion of the plot at 0 – 10 min. The red curves represent simulated data obtained using the proposed mechanism (Scheme 1) and the rate constants listed in Table 4 where kcat/KM = 325 M-1s-1, kin = 0.52 M-1s-1 and k9 = 2.5 M-1s-1.

The initial rates of the catalytic dismutation were evaluated from the second slower process (Vo = 10

ACCEPTED MANUSCRIPT kcat[HRP]o[H2O2]o) (Fig. 3B, inset), which depends proportionally on [HRP]o and therefore is expressed as TON of O2 evolution, i.e., M O2 released per M HRP per min. The TON of O2 evolution was found to be hardly dependent on the pH between 6.0 and 9.2 as previously reported

RI P

T

[16, 19].

It should be noted that although the kinetics of the decomposition of H2O2 as demonstrated in Figs

SC

1B and 3A appear to be quite similar, this similarity is misleading. Under the experimental conditions specified for Fig. 3A ([H2O2]o << KM), the decay of H2O2 obeys first-order kinetics.

MA NU

Hence, the progressive decrease in the rate of H2O2 decomposition or O2 evolution is due to [substrate] decrease. On the other hand, under the experimental conditions specified for Fig. 1B ([H2O2]o ≥ KM), the dependence of the reaction rate on [substrate] disappears while progressively decreases due to enzyme inactivation.

ED

The stoichriometric ratio of inactivation (expressed as M O2 released/M heme

PT

inactivated) has been determined by measuring the accumulation of O2 until its evolution ceased upon complete loss of HRP activity. The results presented in Fig. 4A demonstrate that the total

CE

yield of O2 released is independent of [H2O2] and pH 5.5 – 7.2. The stoichiometric ratio of inactivation was calculated to be 690  80, i.e., each molecule of HRP catalyzes the dismutation of

AC

1380  160 H2O2 molecules till it is inactivated. The stoichiometric ratio of inactivation has also been determined by comparing the TON of inactivation with that of O2 evolution to be 40/0.06 = 667 at pH 6.3 and 7.2 (Fig. 4B), which is in agreement with that determined from the data presented in Fig. 4A.

11

ACCEPTED MANUSCRIPT 0.06

400

200

0.02

0.00

0 0

20

40

60

0

80

40

0 60

80

100

[H2O2] (mM)

MA NU

SC

[H2O2] (mM)

20

10

Figure 4. (A) Effect of [H2O2] and pH on the total yield of O2 evolved during the dismutation of H2O2 catalyzed by 1 µM HRP; (B) Comparison of [H2O2] effect on the TON of O2 evolution (O) with that of TON of inactivation () during the dismutation of H2O2 catalyzed by HRP at pH 7.2. The error is 5% resulting from duplicate determinations from at least two separate experiments.

ED

The stoichiometric ratio of inactivation has been previously reported to be 700  100 at pH > 6.3 [19, 36], but close to zero at pH 5.5 [19], which does not agree with our observations. The

PT

hyperbolic dependence of the TON of O2 evolution on [H2O2] revealed that saturation kinetics are

CE

observed under steady-state conditions and values of kcat = 0.60  0.05 s-1 and Km = 4  2 mM are obtained from the data in Figs 2B and 4B where kcat/Km is within the lower limit previously

AC

determined for this enzyme [18, 19]. Thus, under the experimental conditions of Fig. 3A ([H2O2] = 250 µM, [HRP] = 5 µM) the inactivation of the enzyme is insignificant, but the rate of H2O2 consumption deviates from zero-order kinetics because [H2O2] < KM. In this case accumulation of compound II is observed when [H2O2] becomes low, which slowly decomposes to HRP (1/2 ~ 0.5 h).

3.2. H2O2 dismutation catalyzed by HRP in the presence of nitroxides (R2NO•) At low [HRP]/H2O2] ratios the predominant heme species observed during the catalysis process in the presence of TPO is compound II, which is converted to the native HRP towards full consumption of H2O2 (Fig. 5A). The rate of this conversion obeys first order kinetics and increases 12

-1

pH 5.5 pH 6.3 pH 7.2

20

T

600

30 0.04

RI P

[O2] (M)

800

40

B

-1

A

TON O2 evolution (min ) (O)

TON inactivation (min ) ()

1000

ACCEPTED MANUSCRIPT as [TPO] increases. Figure 5B demonstrates that the decay of H2O2 and the formation of O2 obey nearly zero-order kinetics.

A 200

0.40

150

T

0.45

M

Absorbance

B

250

403 nm

RI P

0.50

100

SC

0.35

419 nm

50

0.30 4

8

12

Time (min)

0

MA NU

0

16

0

1

2

3

4

5

Time (min)

ED

Figure 5. Dismutation of 250 M H2O2 catalyzed by 5 M HRP in the presence of 20 M TPO at pH 7.2. (A) Spectral changes monitored at 403 nm (Soret peak of HRP) and at 419 nm; (B) Kinetic traces of O2 evolution and H2O2 consumption. The red curve represents simulated data calculated using the proposed mechanism (Scheme 2) and the rate constants listed in Table 4 where k2 = 2 x 104 M-1s-1 (Table 2) and k3a = 9 x 103 M-1s-1.

PT

At relatively high [H2O2]/[HRP] ratios, the predominant heme species observed during the

CE

catalysis process is compound III, and the spectrum of the native HRP re-appears towards full

AC

consumption of H2O2 (e.g., Fig. 6).

Absorbance

0.20

2.0

A

B

1.5

[H2O2] (mM)

0.24

403 nm

0.16

[TPO] = 50 M 1.0

0.5

0.12

[TPO] = 0

417 nm 0

20

40

195

210

0.0

225

Time (min)

0

50

100

Time (min)

13

150

200

ACCEPTED MANUSCRIPT 0.24

C

1

RI P

[H2O2] (mM)

0.16

T

403 nm

0.20

Absorbance

D

2

417 nm 0.12 5

10

15

20

0

25

SC

0

Time (s)

0

4

8

12

Time (min)

ED

MA NU

Figure 6. Dismutation of 2 mM H2O2 catalyzed by 1.9 M HRP in the presence of 50 M (A, B) or 2 mM TPO (C, D) at pH 7.2. (A, C) Spectral changes monitored at 403 nm (Soret peak of HRP) and at 417 nm; (B) Kinetics of H2O2 consumption in the absence and presence of 50 M TPO. The red curve represents simulated data calculated using the proposed mechanism (Scheme 2) and the rate constants listed in Table 4 where k2 = 9.4 x 103 M-1s-1 (Table 2) and k3a = 3.2 x 103 M-1s-1. The blue curve represents simulated data using the rate constants listed in Table 4 where kcat/KM = 325 M-1s-1, kin = 0.46 M-1s-1 and k9 = 2.8 M-1s-1. (D) Kinetics of H2O2 consumption in the presence of 2 mM TPO. The red curve represents simulated data calculated using the rate constants listed in Table 4 where k2 = 1.15 x 103 M-1s-1 (Table 2) and k3a = 8 x 102 M-1s-1.

PT

The nitroxide protects HRP against inactivation induced by H2O2 and its protective effect is dose-

CE

dependent. For example, under the experimental conditions of Fig. 6 (1.9 M HRP, 2 mM H2O2, pH 7.2) the residual peroxidative activity was about 20%, which increased to 80%, 93% and 100%

AC

in the presence of 50, 100 and 250 M TPO, respectively. The decay of H2O2 obeys zero-order kinetics and the rate increases as [TPO] increases (Fig. 6 and 7A). Indeed, the TON of O2 evolution was hardly dependent on [H2O2] when varied between 0.1 and 2 mM both at pH 6.0 and 7.2 (Fig. 7), but higher concentrations of H2O2 demonstrate some inhibitory effect (results not shown). The rate of H2O2 dismutation decreases as the pH increases (Fig. 7B).

14

ACCEPTED MANUSCRIPT 60

A

B -1

TON of O2 evolution (min )

-1

0.1 mM 0.2 mM 0.5 mM 2 mM

0 1

30

20 0.0

2

SC

0

40

T

20

pH 6.0

50

RI P

TON of O2 evolution (min )

40

[TPO] (mM)

0.5

pH 7.2

1.0

1.5

2.0

[H2O2] (mM)

MA NU

Figure 7. (A) Effect of [TPO] on the TON of O2 evolution during the dismutation of various concentrations of H2O2 at pH 7.2; (B) Effect of [H2O2] on the TON of O2 evolution in the presence of 1 mM TPO at pH 6.0 and 7.2.

The effect of various nitroxides (R2NO•) on the TON of O2 formation follows the order TPO

PT

R2N+=O/R2NO• couple [37].

ED

> 4-OH-TPO > 3-CP > 4-oxo-TPO (Table 1), which correlates with the reduction potential of the

Table 1. TON of O2 evolution in the presence of various nitroxides.

TPO

3-CP

TON (min-1)a

TON (min-1)b

TON (min-1)c

750

64.4  5.5

46.1  4.5

29.5  3.0

840

17.3  1.4

11.1  1.2

8.6  0.9

880

6.3  0.5

4.4  0.5

930

3.0  0.3

AC

4-OH-TPO

EO (mV)

CE

Nitroxide

4-oxo-TPO

a - 0.5 mM H2O2, 2 mM nitroxide, pH 6.0; b - 2 mM H2O2, 1 mM nitroxide, pH 7.2; c - 1 mM H2O2, 1 mM nitroxide, pH 7.2

In the presence of nitroxide H2O2 depletion and O2 evolution follow zero-order kinetics implying that the nitroxide acts catalytically. This was confirmed by following TPO using EPR spectroscopy. The EPR signal of 10 M TPO included in the reaction mixture of 250 M H2O2 and 5 M HRP at pH 7.2 decreased towards complete consumption of [H2O2] by ca. 20% and was not restorable by 2 mM ferricyanide, thus excluding accumulation of the respective EPR-silent hydroxylamine. Eventually, however, the nitroxide EPR signal recovered slowly to its initial 15

ACCEPTED MANUSCRIPT intensity. Since the oxoammonium cation is readily reduced back to the nitroxide by H2O2 [38], it is assumed that it is accumulated when [H2O2] becomes low, and is reducible back to the nitroxide by certain amino residues on the globin chain. The EPR signal of 25 M TPO included in the reaction

RI P

T

mixture of 2.5 mM H2O2 and 2 M HRP at pH 7.2 remained practically constant throughout the entire longer experiment since the residual concentration of H2O2 is sufficient to reduce the

SC

oxoammonium cation.

MA NU

3.3. Stopped-flow experiments

When solutions of 4.5 - 5 M H2O2 were mixed with 5.1 - 5.4 M HRP, the formation of compound I monitored at 403 nm or 411 nm was completed within less than 0.2 s. The conversion of compound I to compound II, and the subsequent decomposition of compound II to HRP were

ED

monitored by following the absorption increase at 411 nm and the subsequent slower absorption

PT

decrease at 422 nm. The half-life of compound I increased substantially when an interference filter (420 nm, ASAHI SPECTRA CO) was used implying that the spontaneous decay of compound I is

CE

accelerated by the white light of the Xe lamp as previously reported [39, 40]. The addition of 100 µM DTPA to the reaction mixture or lowering the buffer concentration from 40 mM to 5 mM had

AC

no effect of the half-life of compound I. The rate of the self-decomposition of compound II was too slow for determination using this technique. These processes were accelerated in the presence of nitroxide most probably due to reactions 2 and 3.

R-+•PorFeIV=O + R2NO•  R-PorFeIV=O + R2N+=O

(2)

R-PorFeIV=O + R2NO• + 2H+  R-PorFeIII + R2N+=O + H2O

(3)

Typical kinetic traces are shown in Fig. 8. The kinetic traces at 411 nm demonstrate the formation of compound I (reaction 1), and its reduction by TPO to compound II (reaction 2). The kinetic 16

ACCEPTED MANUSCRIPT traces at 422 nm show the reduction of compound I to compound II by TPO (reaction 2), and the subsequent slower reduction of compound II to HRP by TPO (reaction 3). 0.28

0.18

B

0.26

A422

0.14

0.22

SC

0.12

RI P

0.16

0.24

A411

T

A

0.20

0.18 0.00

0.07

0.14

4

Time (s)

MA NU

0.10

8

0

2

4

100

200

Time (s)

ED

Figure 8. Kinetic traces monitored at 411 (A) and 422 nm (B) upon mixing a solution of 5.4 µM HRP and 0.2 mM DTPA in 1 mM PB (pH 7.2) with a solution of 5 µM H2O2 and 200 M TPO in 20 mM PB (pH 7.2) at a 1:1 ratio.

PT

The rates of nitroxide oxidation by compound I and compound II obeyed first-order kinetics and the observed first-order rate constants (kobs) increased with increasing [nitroxide] exhibiting a non-

CE

linear dependency (e.g., Fig. 9). The kobs-values for nitroxide oxidation by compound II are similar

AC

to those obtained by mixing pre-formed compound II with the nitroxide (Fig. 9B). Some typical kobs values determined for various nitroxides are tabulated in Table 2 demonstrating that k2 is about an order of magnitude higher than k3. Also, the rates of both reactions 2 and 3 follow the order TPO > 4-OH-TPO > 3-CP > 4-oxo-TPO, and increase as the pH decreases. Such a non-linear dependence of kobs on [reductant] has been reported for the reaction of ferryl myoglobin with nitroxides [25, 41] and with ascorbic acid [42].

17

ACCEPTED MANUSCRIPT 6

0.5

A

B

4

2

0.3

T

0.6

0 .0 6

0.2

0.3

0 .0 3

0.1 0.0 0.00

0.05

0.10

0 2

4

6

0.0

8

0 .0 0 0 .0

0

SC

0

RI P

-1

kobs (s )

-1

kobs (s )

0.4

[TPO] (mM)

4

8

0 .1

12

0 .2

16

[TPO] (mM)

MA NU

Figure 9. Dependence of kobs on [TPO] when compound I (A) and compound II (B) react with excess TPO at pH 7.2. The open circles represent the values obtained by mixing pre-formed compound II with excess TPO. The insets are the expansion of the plots obtained at 0 – 0.1 mM TPO. The error is 5% resulting from duplicate determinations from at least two separate experiments.

pH

[nitroxide], mM

kobs(2), s-1

kobs(3), s-1

TPO

7.2

0.0125

0.28  0.01

0.012  0.001

0.025

0.38  0.03

0.019  0.002

0.05

0.48  0.03

0.032  0.001

7.2

0.1

0.60  0.04

0.049  0.002

7.2

1

1.53  0.05

0.107  0.003

6.4

1

1.86  0.04

0.113  0.004

5.5

1

2.16  0.07

0.122  0.004

5.1

1

5.50  0.08

0.235  0.005

7.2

2

2.30  0.05

0.135  0.005

7.2

0.1

0.28  0.02

0.017  0.001

7.2

1

0.74  0.05

0.069  0.003

6.4

1

1.30  0.07

0.084  0.003

3-CP

7.2

1

0.60  0.03

0.039  0.002

4-oxo-TPO

7.2

1

0.24  0.01

0.017  0.001

7.2

AC

CE

7.2

PT

nitroxide

ED

Table 2. Values of kobs measured for nitroxide reactions with compound I and compound II.

4-OH-TPO

18

ACCEPTED MANUSCRIPT Previously, k2 = (1.2  0.2) x 103 M-1s-1 and k3 = (5.7  0.5) x 102 M-1s-1 have been reported for 4OH-TPO both at pH 6.4 and 7.4 using sequential mixing and varying the nitroxide concentration between 20 and 100 M where nitroxide concentration was calculated using 242 = 1440 M-1cm-1

RI P

T

rather than 2050 M-1cm-1 [43].

The conversion of compound III to HRP in the presence of nitroxide was monitored at 422

SC

nm demonstrating a fast decrease in the absorption followed by a slower absorption decrease. Typical kinetic traces are shown in Fig. 10A. Both processes obeyed first-order kinetics where kobs

process was independent of [nitroxide]. 0.35

MA NU

of the first process increased linearly upon increasing [nitroxide] (Fig. 10B) and that of the slower

B

0.4

A

TPO, pH 6

0.3

0.20 0

CE

10

500

TPO, pH 7.2

-1

PT

0.25

kobs (s )

A422

ED

0.30

0.2 4-OH-TPO, pH 7.2 0.1

3-CP, pH 6

0.0 0

1000

5

10

15

[nitroxide] (mM)

AC

Time (s)

Figure 10. (A) Kinetic traces monitored at 422 nm upon mixing a solution of 4.5 µM compound III in 40 mM PB (pH 7.2) with 20 mM TPO in 40 mM PB (pH 7.2) at a 1:1 ratio; (B) Dependence of kobs of the reaction of compound III with excess TPO at pH 6.0 (), TPO at pH 7.2 (O), 4-OH-TPO at pH 7.2 () and 3-CP at pH 6.0 ().The error is 5% resulting from duplicate determinations from at least two separate experiments.

The reaction of compound III with nitroxide cannot be attributed to compound I formation via reaction 4 since this reaction must be followed by reactions 2 and 3. The slower process, which is independent of [nitroxide], was not observed when compound I reacted with excess nitroxide (Fig. 8).

R-PorFeIIIO2•– + R2NO• + 2H+  R-+•PorFeIV=O + R2N+=O + H2O 19

(4)

ACCEPTED MANUSCRIPT

Therefore, we assume that the nitroxide is reduced by compound III via an inner-sphere electrontransfer mechanism (reactions 5 and 6) where k5 >> k6 and the adduct absorption at 422 nm is higher

RI P

T

than that of HRP.

R-PorFeIIIO2•– + R2NO•  adduct + O2

(6)

MA NU

SC

adduct  R-PorFeIII + RNO-H

(5)

The k5-values follow the order TPO > 4-OH-TPO > 3-CP > 4-oxo-TPO, and slightly increase as the pH decreases (Table 3).

pH

TPO

6.0 7.2

23.9  1.1 22.7  1.6

7.2

16.2  0.9

6.0

4.3  0.9

AC

3-CP

6.0

34.5  2.1

CE

4-OH-TPO

k5, M-1s-1

PT

nitroxide

ED

Table 3. Values of k5 for nitroxide reaction with compound III.

a - Determined at [nitroxide] > 1 mM

RNO-H is not accumulated in the HRP/H2O2/nitroxide reactions system as evident by EPR spectroscopy. One may argue that RNO-H is readily oxidized back to the nitroxide by compound I, by compound II and/or by RN+=O. However, the rate of the latter process is relatively slow, e.g., for TPO k = 2 and 19 M-1s-1 at pH 6.0 and 7.2, respectively [44]. Also, we have determined the rate constants for compound I and by compound II reduction by 0.25 – 10 mM TPO-H at pH 6.0 to be 250  25 M-1s-1 and 11.0  1.5 M-1s-1, respectively. These oxidation processes are relatively slow, and therefore we assume that in the HRP/H2O2/nitroxide reactions system H2O2 reduction by the

20

ACCEPTED MANUSCRIPT adduct (reaction 7) competes efficiently with its self-decomposition (reaction 6), which is relatively slow, e.g., (3.8  0.4) x 10-3 s-1 for TPO at pH 7.2.

(7)

RI P

T

adduct + H2O2 + 2H+  R-PorFeIII + R2N+=O + 2H2O

SC

Eventually, the reaction of compound III with nitroxide in the presence of H2O2 yields the native

MA NU

enzyme and R2N+=O where the rate-determining step is reaction 5.

4. Discussion

In the absence of reducing substrates, HRP catalyzes the dismutation of H2O2 and undergoes progressive inactivation. Because of the complexity of the system and the multiplicity of reactions

ED

mechanistic insights have been achieved by quantifying H2O2 consumption, O2 evolution, heme spectral changes and modeling the results by kinetic simulations taking into consideration that: (i)

PT

one molecule of HRP catalyzes the dismutation of 1380  160 molecules of H2O2 till it is

CE

inactivated ; (ii) the hyperbolic dependence of the TON of O2 evolution on [H2O2] results in kcat = 0.60  0.05 s-1 and Km = 4  2 mM; (iii) at relatively high [H2O2] the kinetics of O2 evolution

AC

demonstrates an initial fast rate followed by a relatively slower and progressively decreasing rate; (iv) nitroxide protects HRP against inactivation already at M concentration range; (v) in the presence of nitroxide the rate of H2O2 dismutation obeys zero-order kinetics and increases as [nitroxide] increases; (vi) at low [H2O2]/[HRP] ratios the predominant heme species both in the absence and presence of nitroxide is compound II whereas at high ratios it is compound III. Previously, two main mechanisms have been proposed for the catalase-like activity of HRP (Scheme 1). Mechanism I assumes that compound I decomposes via consecutive one-electron transfer reactions (Scheme 1, reactions 1, 8 - 12) [15, 16] where k10 = 20 - 25 M-1s-1 [17, 30] and k12 = 3 x 10-3 s-1. Mechanism II assumes a two-electron transfer reaction as the main route of catalysis (reaction 13) accompanied by a minor path proceeding via one-electron reduction steps [7, 21

ACCEPTED MANUSCRIPT 17-19] where K-8 = KM, k13 = kcat, kcat/kin ~ 700 and k9 ~ 2kin [7, 18, 19]. At relatively high [H2O2] the

H2O

H2O2

.

III

R-PorFe

T

contribution of the oxidation of compound III by H2O2 (reaction 11) may be significant [16].

R-+ PorFeIV=O

RI P

k1

H2O2

K-8 H2O + O2

k12

k13

SC

.

O2 -

com I-H2O2

H2O2

III

k9

.

O2 -

MA NU

k11

R-PorFe O2

H2O

.

+ 2H+

k14

P670

.

O2 IV

R-PorFe =O

k15

O2 + H2O2

ED

2O2

P940

H2O2

k10

.

kin

PT

Scheme 1. Previously proposed mechanisms underlying H2O2 dismutation catalyzed by HRP. A competing pathway causes heme inactivation and the extent of inactivation increases as [H2O2]

CE

increases (Fig. 4B) forming P940, which slowly decomposes to P670 (k14 = (7.3  0.3) x 10-3 s-1 pH

AC

7, 20oC) [20, 21]. P940 is a heme protein species since it is reducible by dithionite and ascorbate to the ferrous and ferric enzyme, respectively [45], whereas P670 is an inactivated form whose spectrum resembles that of free biliverdin, i.e., the porphyrin ring is probably impaired [21]. Mechanism I is ruled out since the rate-determining step of the catalysis process is reaction 12 implying far too slow rate of H2O2 dismutation compared with the experimental observations, e.g., Figs 1B and 3. This contradiction has been resolved by assuming that the two-electron transfer reaction 13 is the main route of the catalysis (Mechanism II) [7, 17-19]. Previously simulation of the kinetics using mechanism II has been carried out [18]. Contrary to the experimental observations, the simulation neither implies compound III as the predominant species at [H2O2]/[HRP] = 103 and 104, nor demonstrates the observable initial transient rapid phase of O2

22

ACCEPTED MANUSCRIPT evolution resulting from the fast conversion of compound I to compound III [18]. Hence, mechanism II cannot adequately describe all the experimental observations. In the presence of electron donors the peroxidative activity of HRP is generally described by

T

reactions 1 – 3 where k2 > k3. Hence, heme inactivation and compound III formation are

RI P

insignificant and compound II is the predominant species observed during the catalytic process. When the electron donor is nitroxide, the oxidized product, i.e., the oxoammonium cation, is readily

SC

reduced back to the nitroxide by H2O2 and by O2•– [38] resulting in catalytic dismutation of H2O2

MA NU

forming O2 rather than decomposition of H2O2 to water. However, simulation of the kinetics of H2O2 dismutation using the k3-values determined by the stopped-flow (Table 2) is far too slow compared with the experimental observations. Therefore, it is assumed that reaction 3 proceeds via an intra molecular electron transfer process (reactions 3a and 3b) as previously proposed for 4-OH-

PT

ED

TPO reaction with compound II of myeloperoxidase [46].

R-PorFeIV=O + R2NO•

X

(3a)

CE

X + 2H+  R-PorFeIII + R2N+=O + H2O

AC

(3b)

Hence, k3 = k3ak3b(k-3a + k3b) and k3 = k3a when X is efficiently scavenged by H2O2 (reaction 16).

X + H2O2 + 2H+  R-+•PorFeIV=O + R2N+=O + 2H2O

(16)

The mechanism in the presence of nitroxide is described in Scheme 2. Irrespective whether the mechanism does or does not include a Michaelis-Menten complex formation between compound I and H2O2, the simulation adequately fits the decay of H2O2 under different experimental conditions: (i) decay of 250 M H2O2 in the presence of 5 M HRP and 20 M TPO (Fig. 5B) using the rate constants listed in Table 4, k2 = 2 x 104 M-1s-1 (Table 2) where k3a = 9 x 103 M-1s-1 and k16 > 1 x106 23

ACCEPTED MANUSCRIPT M-1s-1 are extracted as best fits from the simulation procedure; (ii) decay of 2 mM H2O2 in the presence of 1.9 M HRP and 2 mM TPO (Fig. 6D) using the rate constants listed in Table 4, k2 = 1.15 x 103 M-1s-1 (Table 2) where k3a = 8 x 102 M-1s-1 and k7 > 200 M-1s-1 are extracted as best fits

H2O2

H2O

.

IV

R-PorFe =O

SC

III

R-PorFe O2 -

k10

.

k12

.

H2O2

k5 O2

O2

k9

H2O2

k13/K-8

R2N+=O

H2O2

.

O2 -

H2O2

O2 + H2O

MA NU

R2NO

RI P

T

from the simulation procedure.

H2O

+.

III

R-PorFe

IV

R- PorFe =O

k1

R2N+=O

k3b

k17 O2

.

X

R2NO

k14

P940

P670

.

k2

k16

k3a

PT

R2NO

.-

kin

R2NO

H2O2

ED

R2N+=O

H2O2

H2O2

k19 R2N+=O

. IV

R-PorFe =O

III

R-PorFe

k-3a

CE

Scheme 2. Proposed mechanism underlying H2O2 dismutation catalyzed by HRP in the presence of nitroxide. The reactions involving the nitroxide are presented in red color.

AC

TPO at relatively low concentrations inhibits heme inactivation, e.g., Fig. 6A, where the residual peroxidative activity is ca. 20% in the presence of 50 M TPO. Hence, one has to assume that P940 reduction by TPO (reaction 19) competes with the conversion of P940 to P670 (reaction 14) and k19 = 40 M-1s-1 is extracted from the best fit to the experimentally observed residual activity (Fig. 6A).

P940 + TPO  HRP + TPO+

(19)

24

ACCEPTED MANUSCRIPT Table 4. Rate constants employed for simulations in the presence of TPO at pH 7.2 (Scheme 2). Reaction No. 1

Rate constant

comment

reference

1.7 x 107 M-1s-1

[14] [TPO] dependent

Table 2

3

[TPO] dependent

Table 2

T

2 23.9  1.1 M-1s-1

7

> 200 s-1

simulated value

8

K-8 = 4  2 mM

K-8 = KM

9

2.6  0.2 M-1s-1

simulated value

in

0.48  0.06 M-1s-1

(k13/KM)/kin = 690 ± 80

10

20 - 25 M-1s-1

11

0.11 M-1s-1

12

(3.0 ± 0.2) x 10-3 s-1

13

0.6  0.05 s-1

14

(7.3 ± 0.3) x 10-3 s-1

15

3 x 105 M-1s-1

16

> 1 x 106 M-1s-1 -1 -1

6 x 10 M s

18

3 x 109 M-1s-1

19

40 M-1s-1

SC

ED

k13 = kcat

pH-dependent

Table 3

This study

This study [17, 30] Fig. 3B This study, [17, 31] This study [20, 21] [47]

simulated value pH-dependent

[38] [38]

simulated value

AC

CE

17

MA NU

simulated at 20 mM H2O2

PT

3

RI P

5

The kinetic modeling of the reactions system in the absence of nitroxide fits adequately the experimentally kinetic curves in Figs 1B, 2B, 6B assuming that kcat/KM = k13/K-8 = 325 M-1s-1, (kcat/KM )/kin = 690 ± 80 and k9 = 2.5 ± 0.1 M-1s-1 (Scheme 2). In the presence of 20 mM H2O2 formation of compound I from compound III (reaction 11, k11 = 0.11 M-1s-1) takes place but its contribution under moderate concentrations of H2O2 is insignificant. This mechanism does not include the formation of a Michaelis-Menten complex in contrast with the experimental observations (Fig. 4B) implying that the catalatic mechanism is still not fully elucidated.

25

ACCEPTED MANUSCRIPT 5. Conclusions Nitroxide catalytically protects HRP against inactivation induced by H2O2 and modulates its catalase-like activity. The results in the presence of nitroxides support the catalatic mechanism

T

proceeding mainly via a two-electron transfer route. Under in vivo conditions bioreduction of

RI P

nitroxides should not pose a problem since the respective hydroxylamines can be oxidized back to the nitroxide by both compounds I and II and probably by biological reducing substrates such as

SC

thiols, ascorbic acid and uric acid when [H2O2] and [O2•] are too low to effectively reduce the

MA NU

oxoammonium cation back to the nitroxide. The protective effect of nitroxides against heme inactivation induced by H2O2 appears to be general and points towards an additional role played by stable cyclic nitroxide radicals in vivo.

ED

Acknowledgements

AC

CE

PT

This work has been supported by the Pazy Foundation Grant number 276/17.

26

ACCEPTED MANUSCRIPT References

J. Everse, K.E. Everse, Peroxidases in Chemistry and Biology, CRC Press, Boca Raton,

T

[1]

[2]

RI P

1991.

N. Duran, E. Esposito, Potential applications of oxidative enzymes and phenoloxidase-like

SC

compounds in wastewater and soil treatment: a review, Appl. Catalysis B: Environmental 28

[3]

MA NU

(2000) 83-89.

A.M. Azevedo, V.C. Martins, D.M.F. Prazeres, V. Vojinovie, J.M.S. Cabral, L.P. Fonseca, Horseradish peroxidase: a valuable tool in biotechnology, Biotecnol. Ann. Rev. 9 (2003) 1387-2656.

T.L. Poulos, Heme enzyme structure and function, Chem. Rev. 114 (2014) 3919-3962.

[5]

F.W. Krainer, A. Glieder, An updated view on horseradish peroxidases: recombinant

ED

[4]

PT

production and biotechnological applications, Appl. Microbiol. Biotechnol. 99 (2015) 1611-

[6]

CE

1625.

M.B. Arnao, M. Acosta, J.A. Del Rio, F. Garcia-Canovas, Inactivation of peroxidase by

AC

hydrogen peroxide and its protection by a reductant agent, Biochim. Biophys. Acta 1038 (1990) 85-89. [7]

M. Arnao, M. Acosta, J. Del Rio, R. Varon, F. Garcia-Canovas, A kinetic study on the suicide inactivation of peroxidase by hydrogen peroxide, Biochim. Biophys. Acta 1041 (1990) 43-47.

[8]

M. Paumann-Page, P.G. Furtmuller, S. Hofbauerb, L.N. Paton, C. Obinger, A.J. Kettle, Inactivation of human myeloperoxidase by hydrogen peroxide, Arch. Biochem. Biophys. 539 (2013) 51–62.

27

ACCEPTED MANUSCRIPT [9]

A. Hiner, J. Rodriguez-Lopez, M. Arnao, R.E. Lloyd, F. Garcia-Canovas, M. Acosta, Kinetic study of the inactivation of ascorbate peroxidase by hydrogen peroxide, Biochem. J. 348 (2000) 321-328. M. Huwiler, H. Jenzer, H. Kohler, The role of compound III in reversible and irreversible

T

[10]

[11]

RI P

inactivation of lactoperoxidase, Eur. J. Biochem. 158 (1986) 609-614.

G. Wu, C.E. Rogge, J.S. Wang, R.J. Kulmacz, G. Palmer, A.L. Tsai, Oxyferryl heme and

SC

not tyrosyl radical is the likely culprit in prostaglandin H synthase-1 peroxidase inactivation,

[12]

MA NU

Biochemistry 46 (2007) 534-542.

P. Rich, M. Iwaki, A comparison of catalytic site intermediates of cytochrome c oxidase and peroxidases, Biochemistry (Moscow) 72 (2007) 1047-1055.

[13]

H.B. Dunford, Mechanisms of horseradish peroxidase and alpha-chymotrypsin, Prog. React.

[14]

ED

Kinet. Mech. 38 (2013) 119-129.

D. Dolman, G.A. Newell, M.D. Thurlow, H.B. Dunford, Kinetic study of reaction of

K.J. Baynton, J.K. Bewtra, N. Biswas, K.E. Taylor, Inactivation of horseradish peroxidase

CE

[15]

PT

horseradish-peroxidase with hydrogen-peroxide, Can. J. Biochem. 53 (1975) 495-501.

by phenol and hydrogen peroxide: a kinetic investigation, Biochim. Biophys. Acta 1206

[16]

AC

(1994) 272-278.

C.J. Baker, K. Deahl, J. Domek, E.W. Orlandi, Scavenging of H2O2 and production of oxygen by horseradish peroxidase, Arch. Biochem. Biophy. 382 (2000) 232-237.

[17]

R. Nakajima, I. Yamazaki, The mechanism of oxyperoxidase formation from ferryl peroxidase and hydrogen peroxide, J. Biol. Chem. 262 (1987) 2576-2581.

[18]

A.N.P. Hiner, J. Hernandez-Ruiz, G.A. Williams, M.B. Arnao, F. Garcia-Canovas, M. Acosta, Catalase-like oxygen production by horseradish peroxidase must predominantly be an enzyme-catalyzed reaction, Arch. Biochem. Biophys. (2001) 295-302.

28

ACCEPTED MANUSCRIPT [19]

J. Hernandez-Ruiz, M. Arnao, A. Hiner, F. Garcia-Canovas, M. Acosta, Catalase-like activity of horseradish peroxidase: relationship to enzyme inactivation by H2O2, Biochem. J. 354 (2001) 107-114. D. Keilin, E.F. Hartree, Purification of horse-radish peroxidase and comparison of its

T

[20]

[21]

RI P

properties with those of catalase and methaemoglobin, Biochem. J. 49 (1951) 88-106. S. Bagger, R.J.P. Williams, Intermediates in the reaction between hydrogen peroxide and

J. Vlasits, C. Jakopitsch, M. Bernroitner, M. Zamocky, P.G. Furtmuller, C. Obinger,

MA NU

[22]

SC

horseradish peroxidase, Acta Chem. Scand. 25 (1971) 976-982.

Mechanisms of catalase activity of heme peroxidases, Arch. Biochem. Biophys. 500 (2010) 74-81. [23]

S.A. Adediran, Kinetics of the formation of p-670 and of the decay of compound III of

[24]

ED

horseradish peroxidase, Arch. Biochem. Biophys. 327 (1996) 279-284. B. Valderrama, M. Ayala, R. Vazquez-Duhalt, Suicide inactivation of peroxidases and the

U. Samuni, G. Czapski, S. Goldstein, Nitroxide radicals as research tools: Elucidating the

CE

[25]

PT

challenge of engineering more robust enzymes, Chem. Biol. 9 (2002) 555-565.

kinetics and mechanisms of catalase-like and "suicide inactivation" of metmyoglobin,

[26]

AC

Biochem. Biophys. Acta 1860 (2016) 1409-1416. A. Samuni, E. Maimon, S. Goldstein, Nitroxides catalytically inhibit nitrite oxidation and heme inactivation induced by H2O2, nitrite and metmyoglobin or methemoglobin, Free Rad. Biol. Med. 101 (2016) 491-499. [27]

K.G. Paul, T. Stigbrand, Four isoperoxidases from horse radish root, Acta Chem. Scand. 24 (1970) 3607-3617.

[28]

D.P. Nelson, L.A. Kiesow, Enthalpy of decomposition of hydrogen peroxide by catalase at 25 oC (with molar extinction coefficients of H2O2 solutions in the UV). Anal. Biochem. 49 (1972) 474-478.

29

ACCEPTED MANUSCRIPT [29]

N.V. Klassen, D. Marchington, H.C.E. McGowan, H2O2 Determination by the I3- method and by KMnO4 titration, Anal. Chem. 66 (1994) 2921-2925.

[30]

S.A. Adediran, A.M. Lambeir, Kinetics of the reaction of compound II of horseradish

T

peroxidase with hydrogen peroxide to form compound III, Eur. J. Biochem. 186 (1989) 571-

[31]

RI P

576.

J.B. Wittenberg, R.W. Noble, B.A. Wittenberg, E. Antonini, M. Brunori, J. Wyman, Studies

SC

on the equilibria and kinetics of the reactions of peroxidase with ligands, J.Biol. Chem. 242

[32]

MA NU

(1967) 626-634.

A.N. Hiner, J. Hernandez-Ruiz, M.B. Arnao, F. Garcia-Canovas, M. Acosta, A comparative study of the purity, enzyme activity, and inactivation by hydrogen peroxide of commercially available horseradish peroxidase isoenzymes A and C, Biotechnol. Bioengineer. 50 (1996)

[33]

ED

655-662.

I. Weinryb, The behavior of horseradish peroxidase at high hydrogen peroxide

A.J. Kettle, C.C. Winterbourn, A kinetic analysis of the catalase activity of

CE

[34]

PT

concentrations, Biochemistry 5 (1966) 2003-2008.

myeloperoxidase, Biochemistry 40 (2001) 10204-10212. M.I. Gonzalez-Sannchez, F. Garcia-Carmona, H. Macia , E. Valero, Catalase-like activity of

AC

[35]

human methemoglobin: a kinetic and mechanistic study, Arch. Biochem. Biophys. 516 (2011) 10-20. [36]

A.N. Hiner, J. Hernandez-Ruiz, F. Garcia-Canovas, A.T. Smith, M.B. Arnao, M. Acosta, A Comparative study of the inactivation of wild-type, recombinant and two mutant horseradish peroxidase isoenzymes C by hydrogen peroxide and m-chloroperoxybenzoic acid, Eur. J. Biochem. 234 (1995) 506-512.

[37]

S. Goldstein, A. Samuni, A. Russo, Reaction of cyclic nitroxides with nitrogen dioxide: The intermediacy of the oxoammonium cations, J. Am. Chem. Soc. 125 (2003) 8364-8370.

30

ACCEPTED MANUSCRIPT [38]

S. Goldstein, G. Merenyi, A. Russo, A. Samuni, The role of oxoammonium cation in the SOD-Mimic activity of cyclic nitroxides, J. Am. Chem. Soc. 125 (2003) 789-795.

[39]

J.S. Stillman, M.J. Stillman, H.B. Dunford, Photochemical reactions of horseradish

T

peroxidase compounds I and II at room Temperature and 10oK, Biochem. 14 (1975) 3183-

[40]

RI P

3188.

H.B. Dunford, J.S. Stillman, On the function and mechanism of action of peroxidases,

M.C. Krishna, A. Samuni, J. Taira, S. Goldstein, J.B. Mitchell, A. Russo, Stimulation by

MA NU

[41]

SC

Coord. Chem. Rev. 19 (1976) 187-251.

nitroxides of catalase-like activity of hemeproteins - Kinetics and mechanism, J. Biol. Chem. 271 (1996) 26018-26025. [42]

B.J. Reeder, F. Cutruzzola, M.G. Bigotti, R.C. Hider, M.T. Wilson, Tyrosine as a redox-

ED

active center in electron transfer to ferryl heme in globins, Free Radic. Biol. Med. 44 (2008) 274-283.

S.M. Vaz, O. Augusto, Inhibition of myeloperoxidase-mediated protein nitration by tempol:

PT

[43]

[44]

CE

Kinetics, mechanism, and implications, Proc. Natl. Acad. Sci. USA 105 (2008) 8191-8196. A. Israeli, M. Patt, M. Oron, A. Samuni, R. Kohen, S. Goldstein, Kinetics and mechanism of

AC

the comproportionation reaction between oxoammonium cation and hydroxylamine derived from cyclic nitroxides, Free Radic. Biol. Med. 38 (2005) 317-324. [45]

R. Nakajima, I. Yamazaki, The conversion of horseradish peroxidase C to a verdohemoprotein by a hydrogen peroxide derived enzymatically from indole-3-acetic acid and by m-nitroperoxybenzoic acid, J. Biol. Chem 255 (1980).

[46]

F.R. Queiroz, S.M. Vaz, O. Augusto, Inhibition of the chlorinating activity of myeloperoxidase by tempol: revisiting the kinetics and mechanisms, Biochem. J. 439 (2011) 423-431.

[47]

B.H.J. Bielski, D.E. Cabelli, R.L. Arudi, A.B. Ross, Reactivity of superoxide radicals in aqueous solution, J. Phys. Chem. Ref. Data 14 (1985) 1041-1100. 31

AC

CE

PT

ED

MA NU

SC

RI P

T

ACCEPTED MANUSCRIPT

33

ACCEPTED MANUSCRIPT Highlights HRP catalyzes H2O2 dismutation while undergoing heme inactivation



Nitroxides catalytically protect HRP against suicide inactivation



Nitroxides reduce compound I, II and oxidize compound III



Nitroxide reduce P940, which is the precursor of the inactivated form P670



Protection by nitroxides against heme inactivation appears to be general

AC

CE

PT

ED

MA NU

SC

RI P

T



34