kinetic characterization of compound i formation in the ... · david g. kellner, shao-ching hung,...

Kinetic Characterization of Compound I Formation in the Thermostable

Cytochrome P450 CYP119

David G. Kellner, Shao-Ching Hung, Kara E. Weiss, and Stephen G. Sligar*

Departments of Biochemistry, Chemistry, and the College of Medicine

University of Illinois, Urbana, IL 61801, USA

*To whom correspondence should be addressed:Dr. Stephen G. SligarSchool of Molecular and Cellular Biology505 S. Goodwin AvenueUniversity of Illinois at Champaign-Urbana,Urbana, Illinois, 61801e-mail: [email protected]: (217) 244-7395Fax: (217) 265-4073

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

JBC Papers in Press. Published on January 17, 2002 as Manuscript C100745200 by guest on M

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Summary

The kinetics of formation and breakdown of the putative active oxygenating intermediate

in cytochrome P450, a ferryl-oxo-(π) porphyrin cation radical (Compound I), have been

analyzed in the reaction of a thermostable P450, CYP119, with meta-chloroperoxybenzoic acid

(m-CPBA). Upon rapid mixing of m-CPBA with the ferric form of CYP119, an intermediate

with spectral features characteristic of a ferryl-oxo-(π) porphyrin cation radical was clearly

observed and identified by the absorption maxima at 370, 610, and 690 nm. The rate constant

for the formation of Compound I was 3.20 (± 0.3) × 105 M-1 s-1 at pH 7.0, 4°C, and this rate

decreased with increasing pH. Compound I of CYP119 decomposed back to the ferric form with

a first order rate constant of 29.4 ± 3.4 s-1, which increased with increasing pH. These findings

form the first kinetic analysis of Compound I formation and decay in the reaction of m-CPBA

with ferric P450.

Keywords: Cytochrome P450; ferryl-oxo-( π) porphyrin cation radical; Compound I;

Compound I Formation in CYP119

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Introduction

P450 enzymes are ubiquitous in nature and carry out a wide range of important reactions

including the activation of carbon centers for catabolism, steroid metabolism, and detoxification

of xenobiotics (1). Due to the important physiological functions of P450 enzymes, the reaction

intermediates of P450 chemistry have been a subject of investigation for many years. Several

discreet steps occur in the hydroxylation chemistry of the cytochrome P450s including: substrate

binding, first electron transfer to the P450 from a physiological redox partner, oxygen binding (to

form oxy P450), a second electron transfer event (to form a reduced oxy or peroxo- state),

proton transfer to the distal oxygen (forming hydroperoxo), and an oxygen scission event

producing a putative high valent iron-oxo species that subsequently generates hydroxylated

product (2). The high valent iron-oxo has long been thought to be analogous to the activated

iron species characterized in other oxidative enzymes (horseradish peroxidase (HRP), catalase,

and chloroperoxidase (CPO)) and referred to as Compound I, a ferryl-oxo-(π) porphyrin cation

radical (3-5). The intermediates in the complex P450 reaction cycle have been gradually

divulged through the use of a wide range of spectroscopic techniques (6-8) and the use of

methods that allow access to the fast steps occurring after O2 binding (2,9). Recently the

peroxo- and hydroperoxo- intermediates have been observed through the use of cryo-

enzymology techniques combined with radiolysis, generating these intermediates through

gradual temperature annealing (2,10). Although there has been no identification of a Compound

I intermediate by these techniques, it has been inferred by indirect measurements (2,11,12). The

Compound I state as well as the peroxo- and hydroperoxo- states of the enzyme have been

proposed to be active in oxygenation events (13,14).


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In order to probe the nature of Compound I, meta-chloroperoxybenzoic acid (m-CPBA)

has been used as an oxidizing agent to produce this high valent iron-oxo through heterolytic

cleavage of the organic peroxide in CPO and HRP. The Compound I species of CPO and HRP

have sufficient half-lives for Resonance Raman and EPR studies and have been well

characterized (3,5,15-17). The same peroxy acid and its derivatives have been used with P450s

in attempts to generate intermediates. Evidence for the existence of Compound I in P450s was

demonstrated through substrate oxygenation or hydroxylation in the reaction of various organic

peroxides with either liver microsomal P450 or CYP101, respectively (18,19).

Contrary to the evidence for Compound I formation in cytochrome P450 using m-CPBA,

Blake and Coon reported that the reactions of several peroxy acids with CYP2B4 generated other

active species, which are neither Compound I nor its one electron reduced adduct (Compound II)

(20). However, in previous studies the generation of the Compound I intermediate in

cytochrome P450 (CYP101) required a large excess of peroxyacid, leading to the rapid

formation, then conversion of Compound I to other species. This inherent reactivity of CYP101

from Pseudomonas putida has made the identification of Compound I tenuous, and, despite

numerous efforts, kinetic analysis could not be completed in the 10 ms time regime of the

intermediate’s existence (21,22).

In order to provide a characterization of the spectral properties of Compound I in a

thiolate liganded heme protein, Harris et al. calculated the spectrum of methyl mercapto

oxyferryl protoporphyrin IX using density functional theory (DFT) (23). The calculated

spectrum had a split Soret blue shifted from the ferric Soret maximum by 60 nm with the Q

bands appearing at 690 nm. Recently, Mössbauer and EPR spectroscopic studies of freeze-

quenched samples from the reaction of m-CPBA with CYP101 demonstrated the existence of an


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organic radical and a ferryl species (24). The techniques employed were not fast enough to

capture the Compound I intermediate on the path to the observed ferryl-radical species. Again,

the high concentration of m-CPBA used in these studies leads to degradation of protein, which

further complicates data analysis.

CYP119 is a thermostable P450 isolated from Sulfolobus solfataricus with a melting

temperature of 91oC and remarkable stability to pressure denaturation (25,26). Thermostable

enzymes are thought to have more rigid active site structures at room temperature than their

mesophilic counterparts, which could lead to a slowing of individual steps in the catalytic cycle

(27). This alteration in relative reaction rates may enable the resolution of intermediates that

occur too rapidly to be characterized in their mesophilic counterparts.

CYP119 has proven to be an effective model system for the study of the intermediate

states of the reaction cycle of this P450. For instance, we recently employed cryoenzymology to

define the hydroperoxo- state resulting from the one electron reduction of the ferrous dioxygen

state (28). In this communication, we report the reaction kinetics of ferric CYP119 with m-

CPBA using stopped-flow spectroscopy, and document the formation of a spectral intermediate

with the characteristic spectrum of Compound I. The relevant time scales of formation and

breakdown of Compound I in this thermophilic P450 are such that a kinetic characterization of

these processes under various conditions can be obtained for the first time.

Experimental Procedures

Meta-chloroperoxybenzoic acid (m-CPBA) was purchased from Aldrich-Sigma

(Milwaukee, WI) and purified using the method described by Davies et al (29). Briefly, m-

CPBA was re-crystallized in a 1:3 ether/petroleum ether solution and characterized by NMR.


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Solutions of m-CPBA for kinetic studies were prepared by addition of an acetone stock. The

m-CPBA concentrations were determined by iodide oxidation to triiodide with the extinction

coefficient of 25.5 mM-1 cm-1 at 353 nm (30).

Cytochrome P450 CYP119 expression and purification from E. coli are as previously

published (26,31). Protein concentration was determined based on the Soret maximum at 415

nm (ε = 104 mM-1 cm-1). All steady-state UV-vis spectra were recorded on a Hitachi U3300

spectrophotometer. Single wavelength stopped-flow measurements were performed on a

KinTek (Austin, TX) model SF-2001 stopped-flow system at 4°C. For multiple-wavelength

absorption studies, a stopped-flow apparatus (model SX-18MV) and the associated computer

system from Applied Photophysics (UK) were used. Multiple-wavelength absorption studies

were carried out using a photodiode array detector and X-SCAN software (Applied

Photophysics, Ltd.). The dead-time of this instrument is about two milliseconds. In a typical

experiment one syringe contained CYP119 (6 µM heme) buffered in 100 mM phosphate (at

various pH), and the other contained various concentrations of m-CPBA. The reaction

temperature was controlled by a water bath. Spectral deconvolution and kinetic analysis were

performed by global analysis and numerical integration methods using PRO-K software

(Applied Photophysics, Ltd.).

Results

Reaction kinetics of m-CPBA with ferric CYP119 were first obtained by following the

decay of the ferric signal at 415 nm and a concurrent increase of absorption at 370 nm. Various

m-CPBA/CYP119 concentration ratios were investigated, as shown in Figure 1. At m-CPBA

concentrations less than half that of the ferric protein, the absorption kinetics clearly show the


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formation of an intermediate by a second order process. The spectral intermediate then decays in

a first order manner, with the spectrum returning to that of a ferric enzyme. At 21 µM CYP119

and an m-CPBA concentration of 1.25 µM, 2.5 µM, and 5 µM, the maximal observed

absorbance change corresponds to 1%, 2.1%, and 3.4% of the total enzyme concentration,

respectively. At higher m-CPBA concentrations, more complicated kinetic behavior was

observed, with a broadband decrease in absorbance indicating protein decomposition (see

discussion).

Figure 2 shows intermediate formation by the increase of absorption at 370 nm in the

reaction of ferric CYP119 with a sub-stoichiometric concentration of m-CPBA. The data (o)

are easily fit by a mechanism involving second order formation and first order decay (solid line,

Equation 1). Moreover, to assess the order of reactions (inset), the initial velocity of the

intermediate formation was plotted against m-CPBA concentration, clearly showing the linear

increase in velocity with increasing substrate concentration, indicative of a second order reaction.

On the other hand, the decay rate constants obtained by the mechanism described above are

within the range of 27-33 s-1 and independent of m-CPBA concentration for the reactions of 21

µM ferric CYP119 with 1.25, 2.5, and 5 µM of m-CPBA. It is evident that the decomposition

of this intermediate goes through a first order process under sub-stoichiometric amount of m-

CPBA. Note that at high m-CPBA concentrations there appears to be a departure from first

order decomposition process, indicating a combination of protein destruction and side oxidation

processes. In order to better characterize the first intermediate formed in this reaction, multi-

wavelength kinetic measurements were carried out at low m-CPBA concentrations.

Rapid-scan stopped-flow spectrophotometry, together with singular value


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decomposition (SVD), was used to provide spectral identification of the observed intermediate.

In this case, full spectra as a function of time were analyzed using the following model:

(1)

The spectra of the ferric protein and Intermediate I as well as the kinetic trace of each

species involved were resolved (Figure 3). As indicated, rapid mixing of m-CPBA with

CYP119 produced a spectral intermediate (solid line, Figure 3(a)) with absorbance maxima at

370, 610 and 690 nm. This spectrum is indicative of a Compound I state of the enzyme, as

shown by the DFT calculations from Harris et al (23).

The formation of Compound I at a ratio of m-CPBA to protein of 1:2.5 (pH 7.0) had a

second order rate (k1) of 3.20 (± 0.3) × 105 M-1 s-1, and a first order decomposition rate (k2)

of 29.4 ± 3.4 s-1. When m-CPBA concentrations were higher than the concentration of

CYP119, protein decay occurred in multiple complicated kinetic processes.

In order to probe the role of proton concentration on Compound I formation, the reaction

between CYP119 and m-CPBA was studied as a function of pH by means of multi-wavelength

measurements. The rates of formation and decay for compound I in CYP119 at different pH are

shown in Table I. Additionally, the rates of formation and decay for Compound I were increased

with an increase in reaction temperature. The Arrhenius plot (data not shown) for the Compound

I formation reaction (k1) equates to an Ea = 14.1 kcal/mole and the decay rate (k2) Ea =18.1


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kcal/mole.

Discussion

The thermostable P450 CYP119 has proven to be an excellent system for investigating

the nature of intermediates with higher oxidation states of this important class of heme proteins.

Previous attempts to form and isolate a Compound I intermediate in CYP101 and other P450s

were hampered by the use of a large excess of peroxyacids, leading to rapid progression from

Compound I to other intermediates and concomitant protein destruction (21,24). The formation

of Compound I in CYP119 was slowed both by the use of lower concentrations of m-CPBA, and

by the temperature at which the experiments were carried out (4oC), which was well below the

physiological temperature for this thermophilic enzyme (80oC). This allowed the detection of an

intermediate clearly defined as Compound I well within the time scale accessible by stopped-

flow spectroscopy. Protein degradation, which can often complicate kinetic analysis, was largely

avoided. These properties of Compound I formation in CYP119 have made the spectral and

kinetic characterization of this previously elusive chemical species in P450s possible.

CYP119 is capable of forming an intermediate with the spectral characteristics of a

Compound I, ferryl-oxo-(π) porphyrin cation radical when rapidly mixed with the peroxy acid

m-CPBA. The spectral similarities of the CYP119 Compound I to the optical spectra of

Compound I in other heme proteins are summarized in Table II. The Soret band of this

Compound I was asymmetric and single-peaked at 370 nm (Figure 3a), while asymmetric Soret

bands of both CYP101 and CPO are at 367 nm (21,32). This asymmetric feature has been

deconvoluted using Gaussian-type transition bands and assigned as the splitting Soret band of

the d-type hyper-porphyrin in the recent spectroscopic studies of CPO Compound I (16).


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Moreover, the visible spectrum of CYP119 Compound I has almost identical absorption intensity

at 610 and 690 nm, whereas the intensity at 610 nm of CPO Compound I is lower than its

absorption at 688 nm. Overall, these findings indicate the similarity between the Compound I

entity of these two thiolate-ligated heme proteins and the minor differences in the absorption

spectra can be attributed to their electronic structure differences. Similar spectral characteristics

of P450 Compound I have also been generated from DFT calculations by Harris et al (23). The

calculated spectra of the methyl mercapto oxy-ferryl protoporphyrin IX model structure are blue

shifted by 50-60 nm in both the ferric resting state and Compound I, however, the other features

and the peak shapes are quite similar to the results presented here. The major peak in the split

Soret band of the calculated Compound I spectrum is blue shifted from the ferric Soret by 60 nm,

similar to the 45 nm shift in the CYP119 Compound I. Additionally, the Q-bands of the

calculated Compound I are shifted to 690 nm, with the equivalent bands in the same location for

CYP119 Compound I.

The reaction of CYP101 with m-CPBA was shown by Egawa et al. to form an

intermediate with spectral characteristics similar to those of CYP119 Compound I (21).

However, this intermediate was formed in the dead-time of the stopped-flow instrument used

and decomposed rapidly into a series of unidentified intermediates. The high concentration of

peracid used also caused breakdown of the protein. The experimental conditions necessary to

generate the putative Compound I in CYP101 included the use of a 30-fold excess of m-CPBA,

increasing the formation rate to the point that the intermediate was fleetingly formed and rapidly

decayed. The Compound I, formed under the conditions presented here, in CYP119 has a half-

life more than an order of magnitude longer than the apparent CYP101 half-life with high

concentrations of m-CPBA. This longer half-life has allowed the first kinetic characterization


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of the formation and decomposition processes for Compound I in a P450. To compare the

reaction kinetics of CYP119 Compound I with other heme proteins, rate constants for Compound

I formation and decay in various systems are summarized in Table III. While the Compound I

states of the other three proteins are stable enough to detect at room temperature, characterization

of a P450 Compound I species necessitated the utilization of a thermophilic protein and low

temperature (4°C).

The kinetic competence in the conversion of the observed intermediate to an oxygenated

product would be a valuable piece of information. Unfortunately, the physiological substrate for

this enzyme is not known. However, the addition of laurate to the reaction mixture in a double

mixing configuration after the addition of m-CPBA leads to complete quenching of the observed

spectral intermediate within the dead-time of the instrument. The reaction of a Compound I

intermediate with juxtaposed substrate has been suggested to be very fast, and hence we do not

expect to directly observe the kinetic conversion to product. Analysis of the reaction mixture

following addition of laurate in this double-mix experiment demonstrated the presence of

hydroxylaurate as a product (data not shown), suggesting the coupling of intermediate decay to

substrate oxygenation.

The pH dependence for Compound I formation reflects the pK of m-CPBA of 7.4.

Previous data have shown that the protonated form of m-CPBA is the active species for

Compound I formation, leading to increased rates of Compound I formation at lower pH (20).

The pH-dependence of the m-CPBA reaction with CYP119 is similar to the behavior of catalase

reacting with peroxyacetic acid (33). In both cases the results are most simply interpreted as a

reaction between the enzyme and non-ionized peroxy-acid molecules. This is substantiated by

similar pH dependence for the reaction between peroxybenzoic acid and horseradish peroxidase


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(29). Additionally, the Ea for Compound I formation (14.1 kcal/mol) in CYP119 is relatively

high compared to the reaction of hydrogen peroxide with peroxidases (2.5-5 kcal/mol) that

utilize hydrogen peroxide as a substrate (34). This is in agreement with the recent findings for

the reaction of prostaglandin endoperoxide synthase with hydrogen peroxide, which also has an

elevated Ea (~ 24 kcal/mol) of its Compound I formation and does not use hydrogen peroxide as

a native substrate (35).

The disappearance of CYP119 Compound I is a first order process. This makes it

unlikely that the degradation of Compound I involves O2 formation by nucleophilic attack of a

second m-CPBA molecule as observed in chloroperoxidase (32). Similarly, the possibility of

the formation of Compound II from the reaction of Compound I with a second peroxy acid

molecule as in HRP can be ruled out. One possibility is that the oxene oxygen is transferred to

the protein. Alternatively, it has been suggested that the slow regeneration of the ferric state in

catalase, from Compound I, is carried out by a reducing equivalent originating from the protein

matrix (33,36). The CYP119 Compound I decays to the low-spin ferric form at a rate about 105

times faster than that of catalase Compound I. These active species can be reduced by any

adventitious reducing equivalent from the protein matrix (4). The pH dependence of the

Compound I decomposition rate is similar to the decay of ferryl intermediate in cytochrome c

oxidase, which was explained by an auto-reduction pathway (37). Taken together, the

mechanism of degradation of CYP119 Compound I, and the regeneration of the ferric enzyme

can only be determined with more experimental analysis.

The kinetic characterization of Compound I formation in CYP119 has allowed for the

optimization of conditions for the maximal production of this putative reaction intermediate in


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time regimes accessible to other spectroscopic methods. The information thus obtained will aid

in the further determination of the nature of Compound I in P450 reactions.


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Acknowledgements

The authors wish to thank the National Institutes of Health for research funding (GM 31756 and

GM 33775) as well as Prof. Robert Gennis and Dr. Joel Morgan for the use of their Applied

Photophysics rapid scanning stopped-flow spectrophotometer.

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Figure Legends

Figure 1. Absorbance at 370 nm (A) and 415 nm (B) upon mixing ferric CYP119 with various

m-CPBA concentrations, pH 7.0, and 4oC. The final concentration of CYP119 after mixing was

21 µM, and the m-CPBA concentrations are: (n) 20 µM, (o) 10 µM, (l) 5 µM, (•) 2.5 µM, and (p)

1.25 µM.

Figure 2. Time dependent formation of the spectral intermediate upon rapid mixing of m-CPBA

with CYP119. Stopped-flow data is shown (o), along with curve fit of second order formation,

first order decay mechanism (solid line); [CYP119] = 21 µM, [m-CPBA] = 5 µM. Initial

velocity of Compound I formation (n) as a function of m-CPBA concentration is shown in the

inset.

Figure 3. (a) Calculated spectra of ferric CYP119 (dashed line) and Intermediate I (solid line)

using the Pro-K fitting program (Methods). Shown is the average of 24 individually calculated

spectra. Experimental conditions: [CYP119] = 16.25 µM, [m-CPBA] = 7 µM, pH 7, 4°C. (b)

The corresponding kinetic traces of ferric CYP119, m-CPBA, and Compound I obtained from

global fitting are shown in dashed line, dotted line, and solid line, respectively.


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TablesTable I. The observed second order formation (k1) and first order decomposition (k2) rate

constants of CYP119 Compound I at various pH values. pH k1 (M-1 s-1) k2 (s-1)6.24 4.28 (± 1.5) × 105 22.0 ± 3.1

6.64 3.69 (± 0.4) × 105 27.5 ± 5.2

7.02 3.20 (± 0.3) × 105 29.4 ± 3.4

7.43 2.49 (± 0.3) × 105 32.1 ± 3.6

7.8 1.86 (± 0.3) × 105 37.6 ± 3.1

Table II. Comparison of the absorption maxima (in nm) of Compounds I and II for various heme proteins.

Compound I Compound II Reference

CYP119 370, 610, 690 This work

CYP101367, 694

(21)

Chloroperoxidase 367, 688 438, 542, 571 (32)

Catalase 405, 660 429, 536, 568 (4)Horseradish Peroxidase

400, 577, 622, 651420, 527, 554 (29)

Table III. The formation (k1) and decomposition (k2) rate constants of Compounds I of several

heme proteins. k1 k2CYP119 a 3.20 × 105 M-1 s-1 29.4 s-1

CPO b 3.8 × 106 M-1 s-1 0.5 s-1 e

Catalase c 1.44 × 104 M-1 s-1 1.6 × 10-4 s-1

HRP d 3.5 × 107 M-1 s-1 1.1 × 106 M-1 s-1 f

a. pH 7.0, 4°C, m-chloroperoxybenzoic acid, this work.b. pH 4.7, 25°C, peroxyacetic acid (38).c. pH 7, 25°C, peroxyacetic acid, ox liver catalase (33).d. pH 7, 25°C, m-chloroperoxybenzoic acid (39).e. Obtained by first-order approximation, the decay of CPO Compound I goes to either the ferric form or CPO Compound II. f. The HRP Compound I reacts with a second m-CPBA molecule so that the decay is a second-order reaction.


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Figures and Tables

Figure 1 should be inserted after “Results” paragraph 1

Figure 2 after “Results” paragraph 2

Figure 3 after “Results” paragraph 4

Table I should be inserted following the final paragraph of the “Results” section

Table II after paragraph 2 of the “Discussion”

Table III after the third “Discussion” paragraph


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David G. Kellner, Shao-Ching Hung, Kara E. Weiss and Stephen G. SligarP450 CYP119

Kinetic characterization of compound I formation in the thermostable cytochrome

published online January 17, 2002J. Biol. Chem.

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