heme oxygenase paper

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Formation and Decay of Hydroperoxo-Ferric Heme Complex in Horseradish Peroxidase Studied by Cryoradiolysis.

Ilia G. Denisov#,*,1, Thomas M. Makris+, Stephen G. Sligar#,+,*

Department of Biochemistry#, Center for Biophysics and Computational Biology+, Beckman

Institute*, University of Illinois, Urbana-Champaign, IL, 61801.

Keywords: Horseradish peroxidase; hydroperoxo-ferric intermediate; peroxo-ferric intermediate;

radiolytic reduction; Compound I; EPR spectra; UV/Vis spectra

Running Title: Hydroperoxo-ferric HRP

Correspondence should be addressed to:

Dr. Stephen G. Sligar

University of Illinois

Department of Biochemistry

505 S. Goodwin Avenue

Urbana, IL, 61801

E-mail: [email protected]

Phone: 217-244-9872

Fax: 217-265-4073

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 4, 2002 as Manuscript M207949200 by guest on A

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SUMMARY

Using radiolytic reduction of the oxy-ferrous horseradish peroxidase (HRP) at 77 K, we observed

the formation and decay of the putative intermediate, the hydroperoxo-ferric heme complex, often

called “Compound 0”. This intermediate is common for several different enzyme systems as the

precursor of the “Compound I” (ferryl-oxo π-cation radical) intermediate. EPR and UV/Vis

absorption spectra show that protonation of the primary intermediate of radiolytic reduction, the

peroxo-ferric complex, to form the hydroperoxo-ferric complex is completed only after annealing

at temperatures 150 – 180 K. After further annealing at 195 - 205 K, this complex directly

transforms to ferric HRP, without any observable intervening species. The lack of Compound I

formation is explained by inability of the enzyme to deliver the second proton to the distal oxygen

atom of hydroperoxide ligand, shown to be necessary for dioxygen bond heterolysis on the

“oxidase pathway”, which is non-physiological for HRP. Alternatively, the physiological substrate

H2O2 brings both protons to the active site of HRP, and Compound I is subsequently formed via

rearrangement of the proton from the proximal to the distal oxygen atom of the bound peroxide.

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INTRODUCTION

Horseradish Peroxidase (HRP)2 is a highly characterized heme enzyme, which has historically

served as a paradigm for evaluating the rules of formation of redox active heme-oxygen

intermediates, including the ferryl-oxo heme complexes commonly known as “Compound I” and

“Compound II.” A detailed mechanism for Compound I formation in the reaction of H2O2 with

peroxidases was formulated by Poulos and Kraut on the basis of structural analysis of cytochrome c

peroxidase active center (1), and has been successfully applied to other enzymes (2-5):

The essential features of this mechanism also provide a framework for the analysis of new systems,

which involve formation of redox-active heme-oxygen intermediates (6-8). A crucial point in the

formation of the ferryl-oxo species is the second protonation of the distal oxygen atom of the bound

hydroperoxo species, followed by the heterolytic scission of O-O bond. The importance of these

elementary steps has been confirmed by quantum chemical methods (9-11). A vast amount of

kinetic data obtained in mutagenesis studies of the distal pocket (5,12-15), provide an additional

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support for the evaluation of the second protonation of the distal oxygen as the most important, and

in some cases, the rate-limiting step in catalysis.

Despite many efforts, experimental characterization of this step, as well as the detailed properties of

the hydroperoxo-ferric heme (Fe3+P-OOH-) complex in peroxidases, is still far from complete. One

reason is due to the low stability of such complexes (16), as their transient character leads to a lack

of accumulation during the reaction course. Several studies of these intermediates in wild-type and

mutant HRP (17-19) and microperoxidase (20-22) by means of stopped-flow methods have

produced absorption spectra that could be tentatively assigned to Fe3+P-OOH- (hydroperoxo- anion)

or Fe3+P- H2O2 (neutral hydrogen peroxide) complex via comparison with theoretical estimates

(23). Even so, we are aware of yet only one successful isolation of an (Fe3+P-OOH-) intermediate in

an H2O2 driven heme protein system, which was obtained using a distal pocket mutant of Mb (24).

The difficulties in the kinetic resolution of the (Fe3+P-OOH-) intermediate on this pathway may be

caused by the fact that both protons necessary for the Compound I formation are brought into the

active site of the enzyme with the H2O2 molecule itself. Apparently, the subsequent 1-2 proton shift

is fast and not rate limiting under most conditions (5,12-15,19).

An alternative way of generating (Fe3+P-OOH-) intermediates with high yield is the cryoradiolytic

reduction of the corresponding oxy-ferrous complexes using ionizing radiation (25,26). This

method mimics the oxidase/oxygenase pathway of oxygen activation, which utilizes molecular

oxygen instead of H2O2, and includes the reduction of the bound oxygen and delivery of two

external protons to form Compound I (6,27). As a result of the one-electron reduction of oxy-

ferrous precursors at 77 K, peroxo/hydroperoxo- ferric heme enzyme complexes can be stabilized

at low temperatures (26,28-30). The (Fe3+P-OOH-) intermediates of cytochromes P450 (31-34),


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heme oxygenase (35), Mb (26,29,30), and Hb (25,28) have been isolated and characterized using

EPR and optical absorption spectroscopy. Several X-ray structures of intermediates produced by

radiolytic reduction of the crystals of corresponding oxy-ferrous precursors, including Compound

III in HRP (36), are also available (36-38), although none of these studies was able to detect the

(Fe3+P-OOH-) complex. Earlier investigations using cryoradiolysis (30), or pulse radiolysis at

ambient conditions (39) for reduction of oxy-ferrous HRP, have not provided unambiguous

assignment of observed intermediate states. Detailed studies of the reductive pathway of oxygen

activation in HRP, where the associated Compound I state is relatively stable, could provide critical

input into the mechanism for oxygen-oxygen bond scission and the requirements for specific distal

pocket residues in generating higher valent metal – oxo complexes thought to be operating in the

heme-thiolate oxygenases such as cytochrome P450. To investigate this path, we used the

cryogenic radiolytic reduction of oxy-ferrous HRP to prepare the peroxo/hydroperoxo ferric

intermediate and monitor its protonation and possible Compound I formation, using EPR and

optical absorption spectroscopy. As a reference point, we also document the cryoradiolytic

reduction of Compound I, prepared using the classic reaction of the ferric enzyme with hydrogen

peroxide.

METHODS

Ferrous HRP was prepared by anaerobic reduction of the ferric HRP (Type XII, Sigma) with

dithionite and subsequent anaerobic chromatography on a Sephadex G25 column (Pharmacia) to

remove the excess dithionite and decomposition products. The enzyme solution was mixed with

buffered deoxygenated glycerol (GC grade, Sigma) to give a final concentration of 25 - 50 µM of

HRP in 70% (v/v) glycerol and 0.1 M phosphate-citrate buffer, pH 6.2, oxygenated at 270 K by


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bubbling air and stirring for 30 s, and cooled to 77 K, as described (33,34). All samples for optical

spectroscopy were prepared in UV-enhanced methacrylate semimicro spectroscopic cells (Fisher

Scientific, Pittsburgh, PA) and used with 4.3 mm path. The ferric HRP samples used for the control

were prepared in the same fashion, cooled to 77 K and irradiated together with other samples.

Compound I and Compound II were prepared according to published procedures (3, and references

therein). Compound I was obtained by manual mixing 70% glycerol/buffer solutions of the ferric

HRP and 1.2 molar excess of H2O2 at low temperature (260 - 270 K). After mixing for 20 - 30 sec,

the sample was placed into a thermostat and quickly cooled to 200 K. The sample was then slowly

cooled to 77K, utilizing UV/Vis spectroscopy to monitor the purity of the preparation. Compound

II was prepared by mixing of 75% glycerol/buffer solutions of the ferric HRP, containing fourfold

molar excess of the substrate, 3,5-dimethylphenol, with a fourfold molar excess of H2O2 in the

similar conditions. The spectra of Compound I and Compound II showed weak temperature

dependence and were similar to those reported in the literature (40,41).

UV/Vis spectra were measured on a Cary 3 spectrophotometer (Varian, Australia) in an unsilvered

dewar (33) using liquid nitrogen as a cooling agent. ESR spectra were obtained at the University of

Illinois EPR Resource Center on a Varian E-122 X-Band (9.08 GHz) spectrometer with a protein

concentration of 0.4 mM. Typical conditions include a microwave power of 0.5mW with

modulation amplitude of 12.5 Gauss at 100 kHz. An Air Products (Allentown, PA) liquid helium

flow system was used for measurements at 4 to 20K. Samples for EPR were prepared in a similar

manner as those for UV/Vis experiments, with a final glycerol concentration of 20% (v/v).


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The radiolytic reduction of the enzyme and reference samples was achieved by exposing to gamma-

radiation from a 60Co source (dose rate 11 kGy/hour, for 4 or 8 hours) (Notre Dame Radiation Lab,

Notre Dame University, South Bend, IN). The samples were fully immersed in liquid nitrogen

during irradiation. Background absorption due to the radiolysis products was subtracted as

described (33) using irradiated buffer/glycerol reference samples. These samples were irradiated at

77 K together with the protein samples, and 40 absorption spectra were taken at different

temperatures from 80 K – 240 K. The background spectra for any arbitrary temperature in this

range were then calculated using linear interpolation.

RESULTS AND DISCUSSION

The spectra of oxy-HRP before and after irradiation at 77 K are shown in Figure 1. The radiolytic

reduction of the oxy-ferrous heme complex in HRP results in formation of a primary intermediate,

peroxo/hydroperoxo - ferric intermediate, as has been observed with other heme proteins (26,29-

35,42,43). The yield of this intermediate (35% - 50%) was estimated from the dose dependent

absorption spectra, using the spectra of oxy-HRP as the reference. The spectrum of the pure (Fe3+P-

OOH-) HRP complex, calculated from multiple experiments with different yields, is also shown in

Figure 1. The absorption spectrum of this intermediate has a red-shifted Soret band (maximum at

419 nm, compared to 416 nm for oxy-ferrous HRP) and blue-shifted Q-bands (556 and 526 nm).

This spectrum is in a very good agreement with the spectrum of the (Fe3+P-OOH-) complex of

heme oxygenase3 , and with the kinetically resolved spectrum of the same intermediate obtained in

stopped-flow studies of the reaction of Mb mutant H64Q with H2O2 (24). This intermediate of the

reaction of H2O2 with heme enzymes is very unstable at ambient conditions, and previously was


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characterized only via decomposition of transient absorption spectra in stopped-flow studies or via

freeze-quench techniques (12 – 21, 24). The same (Fe3+P-OOH-) complex is firmly identified as an

intermediate of the reductive activation of the bound dioxygen in heme proteins, as demonstrated

by cryoradiolytic reduction studies (26,29,30,32-35) and quantum chemical calculations (10,11,23).

Unfortunately, it was impossible to resolve the spectra of (Fe3+P-OOH-) complex at wavelengths

shorter than 360 nm due to the high absorption of the products of radiolysis of glycerol and the

protein matrix. Thus, we cannot confirm the true hyperporphyrin shape of the Soret band of

(Fe3+P-OOH-) complex, as calculated by Harris and Loew (23) and often used as a criterion to

distinguish between the neutral peroxide – ferric (Fe3+P-HOOH) and hydroperoxo-anion – ferric

(Fe3+P-OOH-) complexes based on their absorption spectra in the Soret region. (19,44).

Figure 2 shows the annealing of the (Fe3+P-OOH-) complex monitored by EPR and UV/Vis spectra.

In the EPR spectra, the presence of two species in the primary intermediate is clearly resolved.

Based on the analogous studies of other heme proteins (25,29,42,43), these are assigned as the

unprotonated peroxo-ferric heme complex (Fe3+P-OO2-, g3=2.27), and the protonated, hydroperoxo-

ferric heme complex (Fe3+P-OOH-, g3=2.32). Gradual annealing at 140 – 160 K results in

disappearance of the feature at g3=2.27 and increase at g3=2.32, as the first protonation of peroxo-

ferric complex and formation of hydroperoxo- intermediate is completed. These results are in

agreement with EPR spectra obtained using cryoradiolytic reduction studies of oxy-HRP in 50%

ethylene glycol (30).


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The analysis of absorption spectra of the similar samples measured at 90K – 160 K does not reveal

any significant changes that can be assigned to the protonation of peroxo-ferric intermediate. Thus,

at least under the conditions of our experiments, the optical spectra in this region are not sensitive

enough to distinguish clearly between the unprotonated and protonated peroxo/hydroperoxo ferric

heme complex of HRP. Recently we obtained EPR and UV/Vis spectra of peroxo/hydroperoxo

intermediates of the distal mutants of cytochrome P450 CYP101, in which the first protonation of a

peroxo-ferric heme complex could be detected in optical absorption spectra as a three nanometer

blue shift of Soret, and a similar blue shift of the Qα band from 561 nm to 558 nm4. However, the

relative spectral differences between oxy-ferrous and peroxo/hydroperoxo- ferric heme complexes

in the Soret region are weaker in HRP and in heme oxygenase than in P450 enzymes (33,34). This

difference, which is caused by the different proximal ligands in these enzymes,

imidazole/imidazolate in HRP and heme oxygenase versus thiolate in P450, also results in different

sensitivity of the optical absorption spectra to the protonation of the distal peroxo- ligand.

Further annealing by increasing the sample temperature from 180K to 210K shows the decay of

(Fe3+P-OOH-) complex with no evidence for the formation of a Compound I intermediate. EPR

spectra of the samples annealed at 195K, 200K, and 210K, show gradual decrease of the signal

from (Fe3+P-OOH-) complex and formation of the ferric HRP with no other observable

intermediates. This result is consistent with two scenarios. One would involve the transient

formation of Compound I, which is quickly reduced to generate the EPR-silent Compound II, and

after the second reduction returns to the ferric enzyme. Another possibility is the direct decay of

(Fe3+P-OOH-) intermediate through dissociation of the ligand and return to the ferric resting state of

HRP. Because EPR-silent species may be involved in the evolution of this system, EPR


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spectroscopy does not provide complete characterization of the (Fe3+P-OOH-) fate in HRP. Thus,

the optical absorption spectra of the similar samples were studied, and the reference experiments

using cryoradiolytic reduction of Compound I were performed to assign the annealing pathway of

(Fe3+P-OOH-) complex.

Figure 2B shows the absorption spectra of the radiolytically reduced oxy-ferrous HRP measured at

190 – 210 K. At temperatures above the glass transition of the solvent matrix, the hydroperoxo-

ferric complex gradually disappears, as clearly seen by simultaneous decrease of the sharp

maximum at 557 nm and of the Soret band at 420 nm. No increase of absorption at 650 nm

characteristic for Compound I porphyrin π-cation radical was detected in annealing experiments.

On the contrary, the bands at 407 nm and 635 nm are observed, corresponding to the spectrum of

the resting form of HRP, possibly perturbed by the presence of the dissociated hydroperoxo-anion/

hydrogen peroxide at the distal pocket of the enzyme. At higher temperatures this spectrum, as well

as the spectrum of the remaining oxy-HRP disappears, and the characteristic spectrum of the

carbonmonoxy-ferrous complex of HRP emerges with Soret band at 421 nm and Q bands at 540

nm and 572 nm (not shown). The CO adduct is the result of the nonspecific reduction of the heme

enzyme by the products of radiolysis, and binding of CO, which is one of the products of glycerol

radiolysis (45), and is always observed after thawing of irradiated frozen solutions of the heme

enzymes (33,34,46-48). In a control experiment, the dissolution of nonirradiated metMb into a

glycerol/buffer reference sample, which was irradiated at 77 K and thawed, resulted in formation of

fully reduced Mb-CO.


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In order to probe the reactivity of Compound I under radiolytic conditions we separately generated

this intermediate using H2O2, as described in Methods. Figure 3 shows the spectra of Compound I

before and after radiolytic reduction. The spectrum of Compound I obtained at low temperature is

in a good agreement with earlier reports (40,41). Radiolytic reduction of Compound I at 77 K

results in formation of a new species with the absorption spectra similar to those reported for

Compound II. Direct formation of Compound II confirms the chemical competence of

cryoradiolysis on HRP intermediates, and the true one-electron reduction as a result of cryogenic

radiolytic treatment of heme enzyme intermediates. At temperatures below 190 K, the shape of the

spectrum of Compound II does not change significantly, which implies that it is relatively stable

and does not undergo further reduction by products of radiolysis until annealed above the glass

transition temperature. At 198 K, this intermediate begins to disappear gradually, with concomitant

formation of ferric HRP (Fig. 3, spectra at 198 K – 205 K). The latter process is consistent with

one-electron reduction of Compound II, as occurs in the peroxidase enzymatic cycle under ambient

conditions.

Results shown in Fig. 3 suggest that the annealing of (Fe3+P-OOH-) complex in HRP (Fig. 2B) to

form ferric HRP does not follow a path through Compound I and Compound II. Comparison of Fig.

2B and Fig. 3 reveals substantial differences in observed spectra and annealing behavior of reduced

oxy-HRP and Compound II. Although the spectrum of Compound II has almost the same peak

positions (418 nm, 526 nm and 558 nm, Fig. 3) as the (Fe3+P-OOH-) intermediate (419 nm, 526 nm

and 556 nm), the spectra of these two species are not identical. The Qα band at 556 nm has

substantially higher amplitude than the Qβ band at 526 nm in the spectrum of (Fe3+P-OOH-)

complex, while in the spectrum of Compound II these two bands are almost of the same intensity.


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Thus, the appearance of Compound II spectra in the optical annealing studies of (Fe3+P-OOH-)

species would have been detected, if the pathway through Compound I and Compound II was the

operative one. Instead, in all such experiments, the spectrum of ferric HRP emerged at the expense

of (Fe3+P-OOH-) spectrum, and the direct decay of hydroperoxo-ferric complex, or the so-called

peroxide shunt, was observed.

The lack of Compound I formation from its immediate precursor, (Fe3+P-OOH-) complex, may be

considered as an unexpected result, because HRP is known for its facile formation of the stable

Compound I species. However, there are important mechanistic differences between the enzymatic

cycle of this enzyme, which uses H2O2 as the oxygen donor, and the oxidase/oxygenase path

explored in the current studies. The (Fe3+P-OOH-) complex is formed as the precursor of

Compound I in both systems, and the second protonation of the β oxygen atom in this complex is

the decisive step to form Compound I. The specific delivery of this second proton is crucial for

Compound I formation from (Fe3+P-OOH-) complex (10,11,23,52), since the latter is very unstable

and quickly disappears even at low temperatures (16-18). The perturbation of this proton delivery

system caused by mutations at the active site was shown to result in nonproductive reduction of

oxygen and formation of hydrogen peroxide, or “uncoupling” in the cytochrome P450 catalytic

cycle (49,50). In HRP, the (Fe3+P-OOH-) complex is formed from H2O2 via deprotonation of the

neutral hydrogen peroxide at the distal pocket of the enzyme, and the same proton can be used for

the second protonation of the β oxygen via the rearrangement catalyzed by the distal His42 and

Arg38 side chains (1,51,52). If dioxygen is used instead of H2O2, both protonation steps have to be

provided by the enzyme, as occurs in the P450 monooxygenases (32,53,54). Since this path is non-


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physiological for HRP, the enzyme fails to deliver the second proton required for Compound I

formation, and an uncoupling is observed.

In conclusion, we prepared the main redox intermediates of the HRP enzymatic cycle and

performed the radiolytic reduction of these species. The (Fe3+P-OOH-) intermediate, the precursor

of Compound I formation, was isolated via radiolytic reduction of oxy-HRP. Absorption and EPR

spectra of this intermediate are reported with much higher resolution and precision than in previous

studies. The first protonation of the peroxo-ferric heme complex, the primary product of the

reduction of oxy-ferrous HRP, is completed only at the temperatures higher than 150 K, as was

observed in D251N mutant of P450 CYP101 (32,43), where the proton delivery mechanism is

impaired (50,53,55). In wild-type P450 CYP101, the primary intermediate of radiolytic reduction

of oxy-ferrous enzyme at 77 K is the fully protonated hydroperoxo-ferric complex (32,43).

Comparison with similar studies on other heme proteins (25,26,28-30,35,42) shows that the ability

to deliver a proton to the peroxo-ferric heme complex correlates with the function of the protein, as

this protonation is most effective in P450s and least effective in Mb, which does not carry out

dioxygen bond scission during its normal function.

Annealing of the reduced oxy-ferrous complex of HRP results in completion of the first protonation

of the peroxide ligand and formation of the ferric hydroperoxo intermediate. Formally, this

intermediate is the same as the one formed in the reaction of H2O2 with heme proteins, which is a

precursor for the formation of Compound I in HRP and other peroxidases. However, when obtained

by reduction of the oxy-ferrous heme complex, the peroxo-ferric intermediate via this path still

needs two protons provided by the enzyme to affect O-O bond scission. This difference between


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peroxidase (using H2O2) and oxygenase/oxidase (using O2) pathways is essential in the subsequent

evolution of this intermediate. Annealing at higher temperatures of (Fe3+P-OOH-) intermediate in

HRP did not result in Compound I formation. We conclude, that Compound I is not formed on this

pathway, because the delivery of the second proton to the distal (β) oxygen atom of the

hydroperoxo-anion ligand is not optimized in HRP. These results confirm the importance of

protonation mechanisms in heme enzymes, which use dioxygen for the formation of the redox

active heme oxygen intermediates.

ACKNOWLEDGMENTS

We gratefully appreciate the help provided by Dr. John Bentley while using the 60Co source in the

Notre Dame Radiation Laboratory (Notre Dame University, IN). Irradiations were conducted at the

Notre Dame Radiation Laboratory, which is a facility of the U.S. Department of Energy, Office of

Basic Energy Sciences. Useful discussions with Dr. S.-C. Hung are gratefully acknowledged. We

thank Ms. Aretta Weber for the expert editorial assistance. NIH Research Resources Grant P-41-

RR01811 is acknowledged for the use of IERC resources. The work was supported by a merit

award from the National Institutes of Health R37GM31756.


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FOOTNOTES

1 On leave from the Institute of Macromolecular Compounds, Russian Academy of Sciences,

St.Petersburg, Russia.

2 The abbreviations used are: HRP, horseradish peroxidase; (Fe3+P-OOH-), ferric

protoporphyrin IX, complex with hydroperoxo-anion; Mb, myoglobin; Hb, hemoglobin;

3 Denisov, I.G., Ikeda-Saito, M., Yoshida, T., and Sligar, S.G., submitted

4 Makris, T.M., et.al., in preparation


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LEGENDS

Figure 1 Absorption spectra of oxy-HRP before irradiation (1), and after irradiation at 77 K,

with doses 44 kGy (2) and 88 kGy (3). Spectrum of pure hydroperoxo-ferric

intermediate, calculated and averaged from multiple experiments with yields 35% -

50% (4). Spectra were measured at 80 K – 140 K.

Figure 2. (A) EPR spectra of oxy-HRP irradiated at 77 K and annealed at the indicated

temperatures. All spectra were measured at 77 K. (B) Absorption spectra of oxy-

HRP irradiated at 77 K and annealed at the same temperatures as the EPR samples.

The arrow shows the direction of spectral changes with increasing temperatures.

Spectra are separated for clarity.

Figure 3. Absorption spectra of Compound II, formed by γ-irradiation of Compound I at 77 K,

and measured at the indicated temperatures (see text). Arrows show the directions

of spectral changes with increasing temperatures. Full lines, spectra of radiolytically

reduced at 77 K Compound I, measured below 195 K. Dashed lines, spectra of the

same samples, measured at 198 K – 205 K indicating gradual transition to ferric

HRP. (– . – .) spectrum of Compount I before irradiation, measured at 100 K.


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Ilia G. Denisov, Thomas M. Makris and Stephen G. Sligarstudied by cryoradiolysis

Formation and decay of hydroperoxo-ferric heme complex in horseradish peroxidase

published online September 4, 2002J. Biol. Chem.

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