effect of distal cavity mutations on the formation of compound i in

1

Effect of Distal Cavity Mutations on the Formation ofCompound I in Catalase-Peroxidases

Günther Regelsberger1, Christa Jakopitsch1, Florian Rüker2, Daniel Krois3,Günter A. Peschek4 and Christian Obinger1*

1 Institute of Chemistry, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna2 Institute of Applied Microbiology, University of Agricultural Sciences,Muthgasse 18, A-1190 Vienna3 Institute of Organic Chemistry, University of Vienna, Währingerstrasse 38, A-1090 Vienna4 Institute of Physical Chemistry, Molecular Bioenergetics, University ofVienna, Althanstrasse 14, A-1090 Vienna

* To whom correspondence should be addressed. Fax: +43-1-36006-6059;E-mail: [email protected]

Running title: Compound I Formation in Catalase-peroxidases

Abbreviations: PA, peroxoacetic acid; CPB, m-chloroperbenzoic acid

Keywords: Catalase-peroxidase, KatG, compound I, compound II, catalase activity,

peroxidase activity, Synechocystis PCC 6803

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 12, 2000 as Manuscript M002371200 by guest on February 12, 2018

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Abstract. Catalase-peroxidases have a predominant catalase activity but differ from

monofunctional catalases in exhibiting a substantial peroxidase activity and in having

different residues in the heme cavity. We present a kinetic study of the formation of the key

intermediate compound I by probing the role of the conserved distal amino acid triad Arg-

Trp-His of a recombinant catalase-peroxidase in its reaction with hydrogen peroxide,

peroxoacetic acid and m-chloroperbenzoic acid. Both the wild-type enzyme and six mutants

(R119A, R119N, W122F, W122A, H123Q, H123E) have been investigated by steady-state

and stopped-flow spectroscopy. The turnover number of catalase activity of R119A is 14.6%,

R119N 0.5%, H123E 0.03%, H123Q 0.02% of wild-type activity. Interestingly, W122F and

W122A completely lost their catalase activity but retained their peroxidase acitivity.

Bimolecular rate constants of compound I formation of the wild-type enzyme and the mutants

have been determined. The W122 mutants for the first time allowed to follow the transition

of the ferric enzyme to compound I by hydrogen peroxide spectroscopically underlining the

important role of W122 in catalase activity. The results demonstrate that the role of the distal

His-Arg pair in catalase-peroxidases is important in the heterolytic cleavage of hydrogen

peroxide (i.e. compound I formation) whereas the distal tryptophan is essential for compound

I reduction by hydrogen peroxide.

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Introduction

Catalase-peroxidases cover a growing group of enzyme. They are components of the

oxidative defense system of bacterial (1) and fungal (2, 3) cells and function primarily as

catalases to remove hydrogen peroxide before it can damage cellular components. They are

different from classical monofunctional catalases. On the basis of sequence similarities with

fungal cytochrome c peroxidase (CCP) and plant ascorbate peroxidases (APX), catalase-

peroxidases (KatG) have been shown to be members of class I of the superfamily of plant,

fungal, and bacterial peroxidases (4). Despite striking sequence homologies between class I

enzymes, there are dramatic differences in the catalytic activity and substrate specificity (5-7).

The most interesting feature of bifunctional catalase-peroxidases is the overwhelming catalase

activity with overall rate constants comparable with those of monofunctional catalases. In

both CCP and APX the catalase activity can be neglected. KatGs also function as broad

specificity peroxidases, oxidizing various electron donors, including NAD(P)H (8-10),

phenols and anilines (7), whereas typical substrates for APX and CCP (ascorbate and

cytochrome c, respectively) are extremely poor electron donors for KatGs (5-7, 11).

From both CCP and APX, the three-dimensional structures are known (12, 13) and exhibit

highly conserved amino acid residues at the active site. They indicate the presence of a

proximal histidine as well as the triad Arg-Trp-His at the distal side. Both physical

characterization as well as sequence analysis suggest the presence of these residues also in

catalase-peroxidases (6, 14). At the moment there is no structural basis to understand the

catalytic features of catalase-peroxidases. Thus - in developing ideas how protein structure

modifies heme reactivity - class I peroxidases are an extremely exciting field of research with

KatGs being the least understood type of peroxidase.

The initial step in the catalytic mechanism of a peroxidase or catalase is heterolysis of the

oxygen-oxygen bond of hydrogen peroxide. This reaction causes the release of one water

molecule (15) and coordination of the second oxygen atom to the iron center (16). Two


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electrons are transferred from the enzyme to the coordinated oxygen atom, one from the iron

and one from a second donor. In pea cytosolic APX the porphyrin serves as the second donor

(17) whereas in CCP the second donor is Trp191 (18-20). Recently, we have shown that the

spectrum of KatG compound I is reminiscent to APX compound I (7) with the important

difference (and this is caused by the overwhelming catalase activity) that compound I

formation of KatG could never be monitored with hydrogen peroxide. Instead of H2O2

peroxoacetic acid had to be used (7).

The triad Arg-Trp-His is located near the peroxide binding site. Histidine is suggested to

function as a general acid/base catalyst that assists in deprotonating the hydroperoxide and

protonating the departing water (18), whereas distal arginine is proposed to stabilize the

transition state for compound I formation by interacting with the developing negative charge

on the oxygen atom being reduced during the heterolytic cleavage of the peroxide bond and to

stabilize the resulting oxy-ferryl center of compound I (21). The role of distal tryptophan is

least clear. In CCP the mutant W51F has been shown to be hyperactive (22).

In the present work we undertook a detailed analysis of compound I formation of recombinant

catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803. Our goal was to

elucidate the role of the distal triad Arg-Trp-His in peroxide binding and cleavage of KatG

and to ascertain similarities or differences to both APX and CCP. The wild-type enzyme and

six mutants (R119A, R119N, W122F, W122A, H123Q, H123E) have been investigated by

both steady-state and presteady-state kinetic analysis. The most exciting finding of this work

is the role of tryptophan in catalase activity of KatG. The W122 mutants lost their catalase

activity completely but retained their peroxidase activity. The consequence was that, for the

first time, in these two proteins compound I formation could be followed spectroscopically

also with hydrogen peroxide. Mutation of distal arginine and histidine gave a 10-102 and 104-

fold decrease in catalase activity indicating a role in O-O heterolysis of hydrogen peroxide.


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On the contrary their role in compound I formation with peroxy acids was much less

pronounced.

Materials and Methods

Materials. Materials were from the following sources: PMSF, hemin, pepstatin A, leupeptin,

chloramphenicol, and ampicillin from Sigma; GFX PCR DNA and Gel Band Purification Kit,

Chelating Sepharose Fast Flow and HiPrep Sephacryl S-300 HR 16/60 gels from Amersham

Pharmacia Biotech; the Centriprep-30 concentrators from Amicon; KpnI from Stratagene,

BamHI and PstI restriction enzymes, T4 DNA ligase, and alkaline phosphatase from

Boehringer Mannheim, Pfu polymerases from Promega and Stratagene, respectively. All other

chemicals were of the highest purity grade available.

Methods. Mutagenesis. Oligonucleotide site-directed mutagenesis was performed using PCR-

mediated introduction of silent mutations as described (23). A pET-3a expression vector that

contained the cloned catalase-peroxidase gene from the cyanobacterium Synechocystis PCC

6803 (7, 14) was used as the template for PCR. At first unique restriction sites were selected

flanking the region to be mutated. The flanking primers were 5'-AAT GAT CAG GTA CCG

GCC AGT AAA TG-3' containing a KpnI restriction site and 5'-TGC ATA AAG GAT CCG

GGT GC-3' containing a BamHI restriction site. The internal 5'-primer was 5'-GCA CGC

TGC AGG CAC TTA TCG CAT TG-3' and possessed a PstI restriction site. The following

mutant primers with the desired mutation and a silent mutation introducing a restriction site

were constructed (point mutations italicized and restriction sites underlined): 5'-AGT GCC

TGC AGC GTG CCA GGC CAT AGC AAT CAT TAA TC-3' changed R119 to A, 5'-AGT

GCC TGC AGC GTG CCA GGC CAT GTT GAT CAG CCC ACC ATA ATG ACC-3'

changed R119 to N, 5'-AGT GCC TGC AGC GTG GAA GGC CAT AC-3' changed W122 to

F, 5'-AGT GCC TGC AGC GTG CGC GGC CAT AC-3' changed W122 to A, 5'-AGT GCC

TGC AGC CTC CCA GGC CAT AC-3' changed H123 to E, and 5'-AGT GCC TGC AGC


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CTG CCA GGC CAT AC-3' changed H123 to Q. The fragment defined by the KpnI and

BamHI restriction sites was replaced by the new construct containing the point mutation. All

constructs were sequenced to verify DNA changes using thermal cycle sequencing.

Expression and Purification. The mutant recombinant catalase-peroxidases were expressed in

E. coli BL21(DE3)pLysS and purified as previously described by Regelsberger et al. (7) and

Jakopitsch et al. (14) using the same conditions as for the wild-type enzyme.

Catalase Assay. Catalase activity was determined in 50 mM citrate/phosphate or phosphate

buffer adjusted to the corresponding pH in the range 3-9 both polarographically using a Clark-

type electrode (YSI 5331 Oxygen Probe) inserted into a stirred water bath (YSI 5301B) at

25°C and spectrophotometrically (24) using an extinction coefficient for hydrogen peroxide at

240 nm of 39.4 M-1 cm-1 (25). One unit of catalase is defined as the amount that decomposes 1

µmol of H2O2 per minute at pH 7 and 25°C.

Peroxidase Assay. Peroxidase activity was monitored spectrophotometrically using 1 mM

H2O2 and either 1mM o-dianisidine (ε460 = 11.3 mM-1 cm-1) or 20 mM pyrogallol (ε430 = 2.47

mM-1 cm-1) following the oxidation rate in 67 mM phosphate buffer, pH. 7.0. One unit of

peroxidase is defined as the amount that decomposes 1 µmol of electron donor per minute at

pH 7 and 25°C.

Circular Dichroism Measurements. CD spectra were recorded on a Jobin Yvon CD6

dichrograph equipped with a thermostated cell holder, and data were recorded on-line using a

personal computer. Spectra are averages of 4 accumulated scans with subtraction of the base

line. The quartz cuvette used had a path length of 1 mm. All samples were measured at 20°C

in 50 mM phosphate buffer, pH 7.0. Protein concentrations were 1 µM in all experiments.

Stopped-flow Measurements. Transient-state measurements were performed using an Applied

Photophysics instrument (Model SX-18MV) equipped with a 1-cm observation cell

thermostated at 15°C. Rate constants from experimental traces were calculated by the

SpectraKinetic Workstation v4.38 interfaced to the apparatus. Conventional stopped-flow


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analysis was used to investigate the oxidation of ferric enzyme by peroxides and formation of

compound I. At least three determinations of the pseudo-first-order rate constants, kobs, were

measured for each substrate concentration and the mean value was used to calculate the

second-order rate constants. To allow calculation of pseudo-first-order rates, the

concentrations of substrates were at least five times that of the enzyme. The slope of the linear

plot of the pseudo-first-order rate constant versus substrate concentration was used to obtain

the second-order rate constant for the reaction. All stopped-flow experiments were performed

in 50 mM phosphate buffer, pH 7.0, with the exception of pH dependence studies on reaction

rates for which the experimental traces were recorded in 50 mM citrate/phosphate buffers at

different pH values between 4.0 and 8.0. Reactions were also studied with a diode-array

detector (Model PD.1 from Applied Photophysics) as part of the stopped-flow machine as

well as in conventional steady-state spectroscopy using a Zeiss Specord S-10 diode-array

spectrophotometer.

Results

Recently, we have reported a high-level expression in Escherichia coli of a recombinant form

of a homodimeric catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803.

Both physical and preliminary kinetic characterization revealed its identity with the wild-type

protein (7, 14).

Spectral Properties. Figure 1 depicts CD spectra of ferric native recombinant KatG and of all

six mutants investigated in this study. The far-UV CD spectra give a measure of the protein

secondary structure. The signal observed for catalase-peroxidase was characteristic of a

protein composed primarily of α helices. Very little difference was observed between the CD

spectra of wild-type and the six mutant proteins indicating that there was no large scale

conformational change in the structure. If conformational changes did occur, they must be

very localized and minimal and thus went undetected by CD. Since the α-helical content


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seemed to be insensitive to the amino acid substitution at the distal Arg, Trp or His, we

assumed that the mutants were folded properly as is the wild-type enzyme.

The absorption spectrum of the recombinant enzyme in the resting state exhibited the typical

bands of a heme b-containing ferric peroxidase in the visible and near ultraviolet region.

Figure 2(A) shows the Soret peak to be at 406 nm and the two charge-transfer bands at 500

(CT2) and 631 nm (CT1). Both the Soret and the CT1 band suggested the presence of a five-

coordinate high-spin heme coexisting with a six-coordinate high-spin heme. The A406/A280

ratio (i.e. Reinheitszahl) varied between 0.63 and 0.65 and was similar to that of wild-type

KatG (6). The spectral parameters of the mutants are summarized in Table 1. Mutations of

Arg, Trp and His caused shifts of 1-4 nm at the Soret region and more pronounced shifts at

both CT1 and CT2. Mutation of distal histidine led to a marked red-shift observed for the CT1

band which (together with an increase of the shoulder at 380 nm) indicated an increase of the

five-coordinate high-spin heme at the expense of six-coordinate high-spin heme. A similar but

less pronounced transition was observed for R119A and R119N.

With the exception of His123 mutants both the protein yield (40-60 mg recombinant KatG

from 1 L of E. coli culture) and the Reinheitszahl of the mutants were comparable with those

of the wild-type enzyme. The lower heme content of H123Q and H123E could reflect that

heme binding in these variants was disrupted.

Catalase and Peroxidase Activity. Recombinant KatG exhibited an overwhelming catalase

activity. The polarographically measured specific catalase activity in the presence of 1 mM

hydrogen peroxide was 553-624 units per mg of protein. The presence of classical peroxidase

substrates influenced the oxygen release from hydrogen peroxide only to a small extent. With

1 mM H2O2 and either 5 mM o-dianisidine or 20 mM pyrogallol the peroxidase activity was

determined to be 2.0-2.8 units/mg and 5.4-6.1 units/mg, respectively. Although it is incorrect

to treat catalase kinetics in a Michaelis-Menten way (and therefore the definition of Km and

kcat should be avoided) we have calculated apparent Km and kcat values as has been done in the


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literature in recent years (5-7, 11, 14). With wild-type KatG (1 nM heme) there was a linear

correlation in a Lineweaver-Burk plot between 50 µM and 10 mM hydrogen peroxide

allowing determination of both apparent Km and vmax. At H2O2 concentrations higher than 10

mM, a progressive inhibition of enzyme activity was seen. For the wild-type protein the

corresponding apparent kcat value with H2O2 as the sole substrate was determined to be 3500

s-1 and the apparent Km was 4.9 mM. The kcat/Km ratio was 7.1 × 105 M-1 s-1.

Table 1 contains some of the kinetic constants for KatG and the investigated mutants. It

shows how the mutations decrease the affinity for hydrogen peroxide as well as the turnover

rates and thus confirms that the changed residues are involved in catalase activity of KatG.

Interestingly, both W122 mutants lost their catalase activity completely. Mutation of R119

and H123 gave partially active forms. Mutation of histidine decreased the kcat value by a

factor of 3000-4000, whereas the kcat value of catalase activity was 14.6% for R119A and

0.5% for R119N compared with the wild-type protein. The catalase activity of the variants

was measured polarographically and was proportional to hydrogen peroxide concentrations in

the range 2-20 mM. Corresponding to the effect of mutation on catalytic activity the

concentration of the mutants in the assays had to be 30-300 nM (per heme). At higher

peroxide levels (> 20 mM) the reaction rate again lost proportionality to H2O2 concentration.

Generally, inactivation of mutants at high H2O2 concentration was more dramatic than

inactivation of the wild-type protein.

Both the wild-type protein and the R119 and H123 variants exhibited only a small decrease in

absorbance (hypochromicity) of ∼0-2% at the Soret region. Assuming that compound I would

give a Soret absorbance ∼60% that of the ferric enzyme (7) these hypochromicities fitted a

steady-state compound I concentration between 0-4%. This calculation was based on a

hypothetical catalase reaction mechanism involving Reactions 1a,1b and 2a,2b in Figure 6.

During catalase activity the ratio of free enzyme to compound I is a constant determined by

the rate constants of compound I formation, k1(app), and compound I reduction, k2(app). From


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[KatG]/[compound I] = k2(app)/ k1(app) follows that during catalase activity k2(app) >> k1(app). Our

experiments demonstrated that mutation of distal Arg and His did not alter this ratio. On the

contrary mutation of distal Trp dramatically shifted this ratio. Addition of hydrogen peroxide

to both catalatically inactive mutants W122F and W122A led to compound I accumulation.

Apparently, in the W122 variants Reaction 2a,2b was blocked.

The peroxidatic activity of the R119 and H123 mutants was reduced proportionally to the

catalase activity. On the contrary, in both W122 variants the peroxidatic to catalatic ratio

dramatically increased. Though having lost the catalase activity, the specific peroxidase

activity of W122F was still 0.5 units/mg (1 mM H2O2, 5 mM o-dianisidine) and 0.83 units/mg

(1 mM H2O2, 20 mM pyrogallol).

Compound I Formation. The initial event in the catalytic mechanism of a peroxidase or

catalase is a two-electron oxidation of the enzyme by hydrogen peroxide to an intermediate

called compound I (Reactions 1a and 1b). Compound I of peroxidases normally exhibits the

same Soret band maximum as the corresponding native peroxidase but a hypochromicity of

about 40-50%. We have recently demonstrated that bifunctional catalase-peroxidases exhibit

similar spectral changes upon addition of peroxides (7). In contrast to its homologous

members of class I peroxidases, APX and CCP, monitoring of compound I in KatGs is

impossible because of its high catalase activity (k2(app) >> k1(app)). Consequently, peroxy acids

had to be used to form a stable KatG compound I.

From the single-mixing stopped-flow experiments on rates of compound I formation using

excess peroxy acids the following results were obtained. Reaction of wild-type KatG with

both peroxoacetic acid (PA) and m-chloroperbenzoic acid (CPB) exhibited single exponential

curves, indicating pseudo-first-order kinetics (inset of Figure 2(B)). Plots of the first-order

rate constants, kobs, versus peroxide concentration were linear with very small intercepts (<

0.5 s-1) (Figure 2(B)). These small intercepts fitted well with the observation that wild-type

compound I was stable for more than 30 seconds even in the presence of excess PA. With PA


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and CPB the rate constants for compound I formation, k1(app), were (3.9 ± 0.4) × 104 M-1 s-1

and (5.3 ± 0.2) × 104 M-1 s-1, respectively (pH 7.0, 15°C). Figure 2(A) shows the spectrum of

KatG compound I produced by mixing 20 µM recombinant enzyme with 200 µM

peroxoacetic acid. Its spectrum was distinguished from the resting state by a 40%

hypochromicity at 406 nm and two distinct peaks at 604 and 643 nm. Isosbestic points

between compound I and the resting enzyme were determined to be at 357, 430, and 516 nm.

When PA or CPB was added to R119A and R119N a similar exponential decrease of

absorbance at the Soret maximum was observed. However, the exponential phase

(representing ∼90% of total hypochromicity) was followed by a relatively slow decrease in

absorbance of this mutant, and this decrease could be attributed to a decay of compound I.

The pseudo-first-order rate constants, kobs, were calculated from the first exponential phase.

The k1(app) values determined from plots of these first-order rate constants, kobs, versus

peroxide concentration are summarized in Table 2. There was a distinct influence of

mutations of R119 on the reaction with PA whereas the reactivity towards CPB seemed to be

unaffected. With PA the intercepts were relatively small (0.2 s-1 with R119A and 1.1 s-1 with

R119N) whereas with CPB the corresponding values were 3.6 s-1 and 4.5 s-1, respectively.

This fitted well with the observation that CPB always caused a faster decrease in absorbance

during the second (linear) phase than PA.

Mutation of H123 and W122 resulted also in a biphasic reactivity towards both PA and CPB

and calculation of k1(app) was performed by fitting the first exponential phase of reaction.

Replacement of the distal His with Gln led to a complete depression of reaction with PA,

whereas reaction with CPB gave a k1(app) comparable with the wild-type protein. Replacement

of H123 with Glu gave a substantial effect on compound I formation with CPB. Compared

with the wild-type protein, the k1(app) value increased by a factor of 140 whereas the reactivity

towards PA was unaffected. Table 2 depicts the calculated apparent bimolecular rate

constants.


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The calculated rate constant of the reaction between W122F or W122A and peroxoacetic acid

showed a ∼5-fold increase compared with wild-type KatG. On the contrary, the stability of

compound I of these variants decreased which is underlined by increased intercepts of the

plots kobs versus peroxide concentration (12.2 s-1 with W122F and 14.6 s-1 with W122A).

A relatively small increase in reactivity was measured upon reaction of W122F with CPB,

whereas compound I formation of W122A with CPB was shown to be 170 times faster that of

the wild-type enzyme. This increase in reactivity towards CPB could result from the increased

accessibility of the bulky peroxide to the heme in the W122A mutant. Again the biphasic

behaviour of compound I formation of the W122 mutants was more pronounced with CPB

underlined by an increase of the intercepts of the plots of kobs versus CPB concentration (67

s-1 and 176 s-1).

The most interesting observation of mutational analysis was the fact that both W122 mutants

lost their catalase activity completely. According to the suggested reaction mechanism based

on Reactions 1 and 2 this behaviour could be caused by inhibition of either Reaction 1 or 2.

Our experiments demonstrated unequivocally that Reaction 2 was suppressed when W122

was exchanged with either Phe or Ala, because, firstly, the mutants retained their peroxidase

activity and, secondly, we were able to monitor compound I formation by addition of

hydrogen peroxide. We obtained single-exponential decays at 409 nm and finally linear plots

of the first-order rate constants, kobs, versus hydrogen peroxide concentration (Figure 3(C)).

The calculated rate constants were (8.2 ± 0.5) × 104 M-1 s-1 (W122F) and (8.8 ± 0.4) × 104 M-1

s-1 (W122A) and the intercepts 29 s-1 and 34 s-1 (pH 7.0, 15°C). Using a diode-array detector,

the stopped-flow experiments revealed a compound I spectrum (Figure 3(A)) with absorbance

bands at 409 nm (25-30% hypochromicity), 537 nm and 650 nm. The isosbestic points

between compound I and the ferric protein were determined to be at 355 nm, 426 nm, 476 nm

and 538 nm. Reaction of W122F with PA resulted in similar spectral changes. The difference

spectra (compound I minus ferric KatG) were independent of the type of peroxide used in


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these transient-state experiments (Figure 3(B)). Similar spectral changes were obtained when

W122A reacted with H2O2 or PA (not shown).

pH-Dependence of Compound I Formation. The overall catalase activity of recombinant wild-

type KatG showed a significant pH dependence, with a maximum activity at pH 6.5 (Figure

4(A)). The apparent bimolecular rate constant for the reaction between wild-type KatG and

PA as well as between the W122F mutant and hydrogen peroxide depended on pH and

exhibited a similar bell-shaped pattern with a maximum at pH 6 (Figure 4(B) and 4(C)). Since

both peroxides caused a similar dependence on pH, it is reasonable to assume that two

enzyme ionizations influence the formation of compound I.

Decay of Compound I. Inactivation of mutants at high hydrogen peroxide concentrations was

more pronounced than inactivation of the wild-type protein. This correlated with compound I

stability. Wild-type compound I was stable for more than 30 s whereas in the mutants

formation of compound I was followed by a slow phase of spectral transition. At low

hydrogen peroxide concentrations (< 100 µM) a very slow decay of compound I back to the

ferric enzyme was observed (not shown) whereas at higher peroxide concentrations a red-shift

of the spectrum occurred. The spectrum shown in Figure 5(A) was recorded 4 s after W122F

was mixed with 50 mM H2O2. The absorbance maxima were at 417 nm, 544 nm and 578 nm,

respectively. Longer incubation times resulted in a continuous decrease of absorbance

indicating heme destruction (Reaction 5 in the scheme of Figure 6). With PA and above all

with CPB these spectral transitions were faster.

Compound I suffered a completely different fate when a one-electron donor was offered.

Addition of ascorbate to wild-type compound I resulted in the formation of an intermediate

with a Soret maximum at 407 nm and a distinct peak at 626 nm (Figure 5(B), spectrum 3).

The isosbestic points between this intermediate and the ferric enzyme were at 345 nm, 426

nm, 489 nm and 531 nm. Recently, we have proposed that this intermediate is KatG

compound II which is still one oxidizing equivalent above that of the native enzyme


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(containing an oxidized amino acid residue which is not electronically coupled to the heme)

(7). Upon addition of ascorbate to W122F compound I an intermediate with a Soret

absorbance between that of compound I and the ferric protein accumulated (Figure 5(C),

spectrum 3). Recently, we have shown that ascorbate is a very poor substrate for both wild-

type and recombinant KatG from Synechocystis PCC 6803 (8). On the contrary, preliminary

(unpublished) studies about the peroxidase activity have shown that the W122F mutant

exhibited an enhanced peroxidase activity with ascorbate compared to the wild-type protein.

Discussion

The availability of significant amounts of fully intact recombinant catalase-peroxidase and

properly folded mutants for the first time permitted a comprehensive study of the role of

active site residues in compound I formation of a catalase-peroxidase. The amino acid triad

Arg-Trp-His is found at the distal heme site of all members of class I peroxidases (CCP, APX

and KatG), however, these three peroxidases exhibit dramatic differences in enzyme

reactivities. The key intermediate in the peroxidase cycle is compound I and its kinetic and

spectral investigation is a prerequisite for understanding the bifunctional behaviour of

catalase-peroxidases.

With CCP and APX, the reaction between the resting enzyme and hydrogen peroxide could be

followed by conventional stopped-flow spectroscopy. For the transient-state kinetics of

compound I formation of pea cytosolic APX two second-order rate constants were published,

namely 8.3 × 107 M-1 s-1 (26) and 4.0 × 107 M-1 s-1 (27). The rate for compound I formation of

yeast CCP with hydrogen was shown to be in the range of 107-108 M-1 s-1 (28-30). On the

contrary, we failed in assessing the formation of KatG compound I by adding hydrogen

peroxide even by rapid-scanning stopped-flow spectroscopy. The characteristic spectral

transitions which occur when APX (24) and CCP (15) are mixed with H2O2 were not

observed. This could be caused by one of the following two reasons: (i) compound I is not the


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intermediate in catalase reaction or (ii) the catalase reaction cycle of a KatG is similar to

monofunctional catalases thus involving Reactions 1a,1b and 2a,2b with k2(app) >> k1(app)

(Figure 6). The results presented in this paper give substantial evidence that the catalase

reaction of KatG involves compound I. Firstly, the spectrum of the intermediate formed upon

addition of peroxoacetic acid to the wild-type protein is reminiscent of what occurs with

other peroxidases when PA is added (Figure 2(A)). Secondly, the mutants W122F and

W122A for the first time allowed to monitor the direct reaction of a catalase-peroxidase with

hydrogen peroxide resulting in an enzyme intermediate with spectral features also similar to

the classical plant peroxidase compound I (including APX) (Figure 3(A)) which has been

shown to contain two oxidizing equivalents (13) and forms a porphyrin π-cation radical in

combination with an iron(IV) center (17, 27). Since the Trp mutants completely lost their

catalase activity but retained their peroxidase activity it is reasonable to assume that Trp122 is

the crucial residue in compound I reduction by H2O2 (Reaction 2a,2b in Figure 6). Thirdly,

under steady-state turnover conditions the ferric KatG has been shown to dominate which fits

well with k2(app) >> k1(app) and finally, we have been able to show that reduction of this

intermediate by monosubstituted anilines and phenols resulted in an intermediate which was

not a stable end product but was converted to ferric KatG in a following reaction (thus

demonstrating that KatG compound I could be reduced to ferric enzyme by two one-electron

reductions) (7).

The reaction rate between W122F or W122A and hydrogen peroxide was determined to be

about 2 orders of magnitude smaller relative to the value of k1(app) of a typical peroxidase (26-

30). Since W122A and W122F exhibited a similar reactivity towards H2O2, but extend

significantly different side chain volumes into the cavity one could speculate that the missing

indole-ferryl interaction is likely to be responsible for the reduced reactivity of both W122

mutants towards H2O2 and not changes in the distal cavity volume. From mutational analysis

of CCP reactivity it is known that mutations at distal Trp (W51, CCP numbering)


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significantly affected the coordination and functional properties of the enzyme (31-34). W51

in CCP forms part of the distal active-site cavity near the open coordination position of the

heme iron. In CCP the tryptophan side chain is in van der Waals contact with the heme and

forms a hydrogen bond with one of the water molecules which is displaced on binding H2O2

(12). Other peroxidases such as lignin peroxidase (35) and horseradish peroxidase (36) have a

Phe in this position. Studies on the CCP variants W51F and W51A have shown that the

bimolecular rate constants of compound I formation were relatively unaffected by these

mutations. This seems to be a further discrepancy between CCP and catalase-peroxidases.

The distal histidine is highly conserved in peroxidases and has been considered to play a

major role as a general acid-base catalyst for the peroxidase reaction cycle (12, 17, 18, 37).

Poulos and Kraut (1980) proposed that the distal His assists in the formation of the initial Fe-

OOH complex by deprotonating an approaching H2O2 as a base and the following heterolytic

cleavage of the O-O bond by protonating the departing distal oxygen as an acid (37). Recent

studies with mutant peroxidases have been conducted to investigate the role of the distal His.

The replacement of His52 by Leu in CCP has profound effects on the rate of compound I

formation, the apparent bimolecular rate constant being decreased by 5 orders of magnitude

relative to the value for native enzyme (38). In HRP, the mutants in which the distal His was

replaced by Ala, Val, and Leu also exhibited extremely low reactivity to H2O2 (39, 40).

Similar to the wild-type KatG, a direct monitoring of compound I formation with hydrogen

peroxide was impossible in the R119 and His123 mutants because reduction of compound I

apparently seemed to be much faster than its production (k2(app) >> k1(app)). Thus, the

significantly reduced overall catalase activities of these variants unequivocally demonstrate

that in catalase-peroxidases the distal His and Arg exhibit a similar role which can be

described by the Poulos-Kraut mechanism (37). Going from H2O2 to PA and CPB the role of

distal His and Arg became less pronounced in compound I formation.


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Whether Trp plays a role in compound I formation is not clear. It is known from studies with

horseradish peroxidase that small structural perturbations around the distal His can seriously

decrease the peroxidase activity (41). One can speculate that the exchange of distal Trp

caused similar structural perturbations of distal His and finally a decreased reactivity towards

hydrogen peroxide. It is much easier to relate an important role in the two-electron reduction

of compound I by hydrogen peroxide (Reaction 2) to distal Trp. The present work

demonstrates that the indole ring of distal Trp is essential for the catalase activity of these

bifunctional enzymes. Interestingly in both CCP and APX this indole ring is also present but,

nevertheless, these homologous enzymes exhibit no catalase activities. The actual structural

differences between CCP and APX with catalase-peroxidase are unknown because at the

moment the crystal structure of a KatG protein is not available.

Our experiments have also demonstrated that the mechanism of H2O2 degradation in

bifunctional catalase-peroxidases and monofunctional catalases is different. Figure 6 shows a

schematic summary of our findings. Reaction of ferric KatG with hydrogen peroxide leads

two a two-electron oxidation of the ferric enzyme to compound I (Reactions 1a and 1b).

Distal His and Arg seem to play a similar role as in other peroxidases. In monofunctional

catalases the equivalent function of Arg is taken over by Asn (42). Compound I is involved in

both the catalase cycle and the peroxidase cycle. In the catalase cycle a second H2O2 molecule

is used as a reducing agent of compound I (Reactions 2a and 2b in Figure 6). In this two-

electron reduction step of the enzyme the indole ring of Trp is essential. Its actual role is not

clear at the moment but one could speculate that the indole nitrogen is involved in both the

binding and finally oxidation of the second H2O2 molecule. In monofunctional catalases again

His and Asn are necessary to catalyze this step (42).

Based on our experiments with the Trp mutants in the absence of one-electron donors we have

included a third pathway for KatG compound I in the scheme of Figure 6 (Reaction 5) which


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shows that compound I can also decay to an intermediate (designated X in the scheme). This

intermediate was formed by long-time exposure of the Trp mutants with hydrogen peroxide or

by addition of high concentrations of H2O2. It seems to be not a stable end product but leads

to irreversible enzyme inactivation and finally heme destruction.

In the peroxidatic cycle compound I is reduced in two one-electron steps via compound II

back to the ferric enzyme (Reactions 3 and 4). Recently, based on its spectral features we

have proposed that the single oxidizing equivalent in KatG compound II is contained on an

amino acid which is not electronically coupled to the heme (7). We have also shown that

ascorbate is a very poor substrate for cyanobacterial catalase-peroxidases (5-7, 11). In the

system KatG/PA/ascorbate compound II accumulated (11). Interestingly, for the W122

mutants ascorbate seemed to be a better substrate underlining preliminary experiments which

indicated that the distal Trp is not involved in the one-electron reduction of compound I (i.e.

in the peroxidase activity). This fitted well with our observation that the spectrum of the

accumulating intermediate formed upon addition of ascorbate to W122F compound I was a

mixture of compound I and a compound II (assuming similar spectral features of W122

compound II and wild-type compound II) (Figure 5(C), spectrum 3) . The actual ratio of

[compound II]/[compound I] in W122 during ascorbate oxidation suggested that k3,app ≈ 2 ×

k4,app, whereas in the wild-type KatG k3,app >> k4,app.

Summing up, nature has evolved two heme enzymes that function primarily in hydrogen

peroxide degradation: monofunctional catalases and catalase-peroxidases. Both types of

enzyme use hydrogen peroxide as oxidant and reductant thereby producing water and

molecular oxygen. Our work has shown that in the two classes different active-site residues

are involved: catalase-peroxidases use the distal His-Arg pair in the oxidation stage and Trp in

the reduction stage, whereas monofunctional catalases utilize the distal His-Asn pair for both

stages (42).


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In further experiments we plan to perform a comprehensive EPR and Resonance Raman study

of the mutants as well as of the postulated KatG intermediates. And we shall look at the effect

of the distal-site mutations on the kinetics of both compound I and compound II reduction by

typical one-electron donors.

Acknowledgments. This work was supported by the Austrian Science Fund FWF (grant

numbers P12374-MOB and P8208) and the Jubiläumsfond of the Österreichische

Nationalbank (Project 7554).

References

(1) Loewen, P. C. (1997) in Oxidative Stress and the Molecular Biology of Antioxidant

Defenses (Scandalios, J. G., ed.) pp 273-308, Cold Spring Harbour Press. New York.

(2) Fraaije, M. W., Roubroeks, H. P., Hagen, W. R., and Van Berkel, W. J. H. (1996) Eur.

J. Biochem. 235, 192-198.

(3) Levy, E., Eyal, Z., and Hochman, A. (1992) Arch. Biochem. Biophys. 296, 321-327.

(4) Welinder, K. G. (1992) Curr. Opin. Struct. Biol. 2, 388-393.

(5) Obinger, C., Regelsberger, G., Strasser, G., Burner, U., and Peschek, G. A. (1997)

Biochem. Biophys. Res. Commun. 235, 545-552.

(6) Regelsberger, G., Obinger, C., Zoder, R., Altmann, F., and Peschek, G. A. (1999) FEMS

Microbiol. Lett. 170, 1-12.

(7) Regelsberger, G., Jakopitsch, C., Engleder, M., Rüker, F., Peschek, G. A., and Obinger,

C. (1999) Biochemistry 38, 10480-10488.

(8) Marcinkeviciene, J. A., Magliozzo, R. S., and Blanchard, J. S. (1995) J. Biol. Chem.

270, 22290-22295.

(9) Nagy, J. M., Cass, A. E. G., and Brown, K. A. (1997) J. Biol. Chem. 272, 31265-31271.

(10) Johnsson, K., Froland, W. A., and Schultz, P. G. (1997) J. Biol. Chem. 272, 2834-2840.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

20

(11) Obinger, C., Regelsberger, G., Furtmüller, P. G., Jakopitsch, C., Rüker, F., Pircher, A.,

and Peschek, G. A. (1999) Free Rad. Res. 31, S243-249.

(12) Finzel, B. C., Poulos, T. L., and Kraut, J. (1984) J. Biol. Chem. 259, 13027-13036.

(13) Patterson, W. R., and Poulos, T. L. (1995) Biochemistry 34, 4331-4341.

(14) Jakopitsch, C., Rüker, F., Regelsberger, G., Dockal, M., Peschek, G. A., and Obinger,

C. (1999) Biol. Chem. 380, 1987-1096.

(15) Schonbaum, G. R., and Lo, S. (1972) J. Biol. Chem. 247, 3353-3360.

(16) Hager, L. P., Doubek, D. L., Silverstein, R. M., Harges, J. H., and Martin, J. C. (1972)

J. Am. Chem. Soc. 94, 4364-4366.

(17) Patterson, W. R., Poulos, T. L., and Goodin, D. B. (1995) Biochemistry 34, 4342-4345.

(18) Erman, J. E., Vitello, L. B., Mauro, J. M., and Kraut, J. (1989) Biochemistry 28, 7992-

7995.

(19) Scholes, C.P., Liu, Y., Fishel, L. A., Farnum, M. A., Mauro, J. M., and Kraut, J. (1989)

Isr. J. Chem. 29, 85-92.

(20) Sivaraja, M., Goodin, D. B., Mauk, A. G., Smith, M., and Hoffman, B. A. (1989)

Science 245, 738-740.

(21) Vitello, L. B., Erman, J. E., Miller, M. A., Wang, J., and Kraut, J. (1993) Biochemistry

32, 9807-9818.

(22) Roe, J. A., and Goodin, D. B. (1993) J. Biol. Chem. 268, 20037-20045.

(23) Kohli, R. M. (1998) BioTechniques 25, 184-188.

(24) Beers, R. F., and Sizer, I. W. (1952) J. Biol. Chem. 195, 133-140.

(25) Nelson, D. P., and Kiesow, L. A. (1972) Anal. Biochem. 49, 474-478.

(26) Mandelman, D., Jamal, J., and Poulos, T. L. (1998) Biochemistry 37, 17610-17617.

(27) Marquez, L. A., Quitoriano, M., Zilinskas, B. A., and Dunford, H. B. (1996) FEBS Lett.

389, 153-156.

(28) Balny, C., Anni, H., and Yonetani, T. (1987) FEBS Lett. 221, 349-354.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

21

(29) Loo, S., and Erman, J. E. (1975) Biochemistry 14, 3467-3470.

(30) Ohlsson, P. I., Yonetani, T., and Wold, S. (1986) Biochim. Biophys. Acta 874, 160-166.

(31) Goodin, D. B., Mauk, A. G., and Smith, M. (1987) J. Biol. Chem. 262, 7719-7724.

(32) Wang, J. M., Mauro, J. M., Edwards, S. L., Oatley, S., Fishel, L. A., Ashford, V. A.,

Xuong, N. H., and Kraut, J. (1990) Biochemistry 29, 7160-7173.

(33) Smulevich, G., Wang, Y., Mauro, J. M., Wang, J., Fishel, L. A., Kraut, J., and Spiro, T.

G. (1990) Biochemistry 29, 7174-7180.

(34) Goodin, D. B., Davidson, M. G., Roe, J. A., Mauk, A. G., and Smith, M. (1991)

Biochemistry 30, 4953-4962.

(35) Edwards, S. L., Raag, R., Wariishi, H., Gold, M. H., and Poulos, T. L. (1993) Proc.

Natl. Acad. Sci. U. S. A. 90, 750-754.

(36) Henriksen, A., Welinder, K. G., Gajhede, M. (1998) J. Biol. Chem. 273, 2241-2248.

(37) Poulos, T. L., and Kraut, J. (1980) J. Biol. Chem. 255, 8199-8205.

(38) Erman, J. E., Vitello, L. B., Miller, M. M., Shaw, A., Brown, K. A., and Kraut, J. (1993)


(39) Newmyer, S. L., and Ortiz de Montellano, P. R. (1995) J. Biol. Chem. 270, 19430-

19438.

(40) Rodriguez-Lopez, J. N., Smith, A. T., and Thorneley, R. N. F. (1996) J. Bioinorg.

Chem. 1, 136-142.

(41) Tanaka, M., Ishimori, K., Mukai, M., Kitagawa, T., and Morishima, I. (1997)


(42) Fita , I., and Rossmann, M. G. (1985) J. Mol. Biol. 185, 21-37.


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Legends to figures.

FIG. 1. Circular dicroism spectra of wild-type (thick line) and 6 mutants at the distal heme

region (thin lines) of Synechocystis PCC 6803 catalase-peroxidase. Molar ellipticities [θ] in

the far-UV region are plotted versus wavelength. Samples were prepared in 50 mM phosphate

buffer, pH 7.0, at a protein concentration of 1 µM.

FIG. 2. Absorption spectra and stopped-flow measurements of wild-type catalase-peroxidase

from Synechocystis PCC 6803. (A) Spectra of 20 µM ferric enzyme (1) and compound I (2)

formed by mixing 20 µM enzyme with 200 µM peroxoacetic acid and waiting for 20 s. (B)

Plot of pseudo-first-order rate constants between wild-type enzyme and peroxoacetic acid.

The inset shows a typical stopped-flow time trace of 1 µM enzyme with 100 µM peroxoacetic

acid monitored at 406 nm and 15°C in 50 mM phosphate buffer, pH 7.0.

FIG. 3. Absorption spectra and stopped-flow measurements of the mutant W122F of

Synechocystis PCC 6803 catalase-peroxidase. (A) Spectra of 12 µM ferric enzyme (1) and

compound I (2) formed by mixing 12 µM enzyme with 100 µM hydrogen peroxide and

waiting for 4 s. (B) Difference spectra of 12 µM compound I minus ferric enzyme; left,

compound I was formed with 100 µM hydrogen peroxide; right, compound I was formed with

100 µM peroxoacetic acid. (C) Plot of pseudo-first-order rate constants between the mutant

W122F enzyme and hydrogen peroxide. The inset shows a typical stopped-flow time trace of

1 µM enzyme with 100 µM hydrogen peroxide recorded at 409 nm and 15°C in 50 mM

phosphate buffer, pH 7.0.

FIG. 4. pH dependence of catalase activity and second-order rate constants, kapp, of catalase-

peroxidase determined in 50 mM citrate/phosphate or phosphate buffer. (A) pH profile for the

catalase activity with hydrogen peroxide determined photometrically at 240 nm and 22°C. (B)


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Effect of pH on the rate constants of the reaction of wild-type enzyme and peroxoacetic acid

recorded at 406 nm and 15°C. (C) Effect of pH on the rate constants of the reaction of mutant

W122F and hydrogen peroxide recorded at 409 nm and 15°C.

FIG. 5. Absorption spectra of wild-type and mutant W122F enzyme of Synechocystis

PCC6803 catalase-peroxidase at their ferric and intermediate states in 50 mM phosphate

buffer, pH 7.0. (A) Spectra of 18 µM ferric mutant W122F (1) and an intermediate (2) formed

by mixing 18 µM enzyme with 50 mM hydrogen peroxide and waiting for 4 s. (B) Spectra of

16 µM ferric wild-type enzyme (1), compound I (2) formed by mixing 16 µM enzyme with

200 µM peroxoacetic acid and waiting for 20 s, and compound II (3) produced by mixing of

16 µM enzyme with 200 µM peroxoacetic acid, after 20 seconds adding of 200 µM ascorbate

and waiting for 5 seconds. (C) Spectra of 12 µM ferric mutant W122F (1), compound I (2)

formed by mixing 12 µM enzyme with 100 µM hydrogen peroxide and waiting for 4 s, and an

intermediate (3) produced by mixing of 12 µM enzyme with 100 µM hydrogen peroxide, after

25 seconds adding of 2 mM ascorbate and waiting for 10 seconds.

FIG. 6. Putative reaction scheme. In the first step H2O2 is used for compound I formation

(1a,1b). Compound I is two oxidizing equivalents above that of the native enzyme with a

porphyrin π− cation radical in combination with an iron(IV)-center. Compound I (Cpd I) can

react with a second H2O2 reducing the enzyme back to the ferric state (catalase reaction;

2a,2b). In the peroxidase reaction Cpd I is transformed in a first one-electron reduction to

compound II (Cpd II) containing an amino acid radical (R.) and Fe(III) (3). At last Cpd II is

reduced back to the ferric catalase-peroxidase in a second one-electron reduction (4). Enzyme

inactivation occurs at millimolar concentrations of H2O2 (5).


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TABLE 1. Absorption maxima (ASoret, Aββ, Aαα), Reinheitszahl (ASoret/A280nm), apparent Km, kcat

and kcat/Km values (with H2O2) for wild-type (WT) and mutants (R119A, R119N, W122F,W122A, H123Q, H123E) of recombinant catalase-peroxidase from Synechocystis PCC 6803.

WT R119A R119N W122F W122A H123Q H123E

ASoret (nm) 406 410 408 409 409 404 409

Aβ (nm) 500 527 505 511 521 508 515

Aα (nm) 631 639 637 634 628 645 640

RZ (ASoret/A280nm) 0.64 0.68 0.63 0.60 0.64 0.46 0.48

Km (mM) 4.9 7.1 60 n.d. n.d. 15.5 6.2

kcat (s-1) 3500 513 17.3 n.d. n.d. 0.83 1.17

kcat/Km (s-1M-1) 7.1 × 105 7.2 × 104 2.9 × 102 n.d. n.d. 5.4 × 101 1.9 × 102

n.d., not detectable


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TABLE 2. Apparent bimolecular rate constants, kapp, of the reaction of ferric catalase-peroxidase with various hydroperoxides. Wild-type (WT) and six mutants (R119A, R119N,W122F, W122A, H123Q, H123E) of recombinant enzyme from Synechocystis PCC 6803 weremeasured at 15°C in 50 mM phosphate buffer, pH 7.0.

kapp (M-1 s-1) WT R119A R119N W122F W122A H123Q H123E

Hydrogen peroxide n.d. n.d. n.d. 8.2 × 104 8.8 × 104 n.d. n.d.

Peroxoacetic acid 3.9 × 104 1.9 × 103 4.5 × 102 1.8 × 105 2.1 × 105 n.d. 1.1 × 105

m-Chloroperbenzoicacid

5.3 × 104 5.0 × 104 1.4 × 104 7.3 × 104 9.0 × 106 7.3 × 104 7.5 × 106

n.d., not detectable


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Figure 1

-100

-50

0

50

100

150

195 215 235 255

Wavelength (nm)

[ θ] (

103 d

egre

es c

m2 d

mol

-1)


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Figure 2

0

0.5

1

1.5

2

2.5

350 400 450

Abs

orba

nce

(A)

Wavelength (nm)

1

2

450 550 650 7500

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

1

2

(B)

0123456789

10

0 100 200 300 400

Peroxoacetic acid (µM)

kob

s (s

-1)

-0.05

-0.04

-0.03

-0.02

-0.01

0.0 0.5 1.0 1.5 2.0Time (s)

Rel

.A

bsor

banc

e

(B)


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Figure 3

0

20

40

60

80

0 200 400 600 800 1000Hydrogen peroxide (µM)

kob

s (s

-1)

0

0.5

1

350 400 450

(A)A

bsor

banc

e1

2

2

1

Wavelength (nm)

0.0350.04

0.0450.05

0.0 0.1 0.2 0.3 0.4 0.5Time (s)

Rel

.A

bsor

banc

e

450 550 650 7500

0.05

0.1

0.15

0.2

0.25

-0.30

-0.20

-0.10

0.00

0.10

300 500 700 300 500 700

Wavelength (nm)

Abs

orba

nce

(B)

(C)


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Figure 4

0200400600800

100012001400

2 3 4 5 6 7 8 9 10pH

Act

ivity

(µ

mol

/[min

.mg]

)

0

25000

50000

75000

100000

125000

150000

3 4 5 6 7 8 9

pH

kap

p (M

-1 s-1

)

(C)

(A)

0

10000

20000

30000

40000

50000

60000

3 4 5 6 7 8 9

pH

kap

p (M

-1 s-1

)

(B)


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Figure 5

0

0.5

1

350 400 450

0

0.5

1

1.5

350 400 450

Abs

orba

nce

0

0.5

1

1.5

2

350 400 450

Abs

orba

nce

450 550 650 7500

0.1

0.2

0.3

0.4

Wavelength (nm)

Wavelength (nm)450 550 650 750

0

0.1

0.2

0.3

0.4

450 550 650 7500

0.1

0.2

Wavelength (nm)

Abs

orba

nce

(A)

(B)

(C)

1

1

1

2

2

2

3

1

23

1

2

3

1 2

3


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FeIII FeIII

FeIV

FeIV

O

O +

1a

2a

1bFerri

Cpd I

FeIIIR

Cpd II

2b

[ ]X

AH+ H

2+

H O2 2

H O2

H O2 2

H O2 2

AH + H O2AH2

AH+ H+

4

3

5

H O2 2

H O+ O

22

Figure 6

+


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Peschek and Christian ObingerGünther Regelsberger, Christa Jakopitsch, Florian Rüker, Daniel Krois, Günter A

peroxidaseEffect of distal cavity mutations on the formation of compound I in catalase

published online May 12, 2000J. Biol. Chem.

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effect of distal cavity mutations on the formation of compound i in

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