From the Department of Chemistry, Brooklyn College
and the Graduate Center of the City University of New York, Brooklyn,
New York 11210-2889 and the § Department of Chemistry, New
York University, New York, New York 10003
Received for publication, August 13, 2002, and in revised form, December 26, 2002
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ABSTRACT |
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Mycobacterium tuberculosis
catalase-peroxidase (KatG) is a heme enzyme considered important for
virulence, which is also responsible for activation of the
anti-tuberculosis pro-drug isoniazid. Here, we present an
analysis of heterogeneity in KatG heme structure using optical,
resonance Raman, and EPR spectroscopy. Examination of ferric KatG under
a variety of conditions, including enzyme in the presence of fluoride,
chloride, or isoniazid, and at different stages during purification in
different buffers allowed for assignment of spectral features to both
five- and six-coordinate heme. Five-coordinate heme is suggested to be
representative of "native" enzyme, since this species was
predominant in the enzyme examined immediately after one
chromatographic protocol. Quantum mechanically mixed spin heme is the
most abundant form in such partially purified enzyme. Reduction and
reoxidation of six-coordinate KatG or the addition of glycerol or
isoniazid restored five-coordinate heme iron, consistent with
displacement of a weakly bound distal water molecule. The rate of
formation of KatG Compound I is not retarded by the presence of
six-coordinate heme either in wild-type KatG or in a mutant
(KatG[Y155S]) associated with isoniazid resistance, which contains
abundant six-coordinate heme. These results reveal a number of
similarities and differences between KatG and other Class I peroxidases.
Bacterial catalase-peroxidases
(KatG)1 are multimeric heme
enzymes with 80-81-kDa subunits having high sequence homology in their
N-terminal halves to cytochrome c peroxidase (CCP) and
ascorbate peroxidase, especially in the distal and proximal heme
regions (1). These enzymes fall into the category of Class I
peroxidases, according to the system used by Welinder based on
polypeptide structural parameters (2). In Mycobacterium
tuberculosis, KatG is important for virulence due to its role in
oxidative stress management (3, 4). KatG is also responsible for
activation of the pro-drug isoniazid (isonicotinic acid
hydrazide) (5-7), which has been in continual use since the early
1950s to treat tuberculosis infection (8). The mode of action of this
old antibiotic is of current interest especially in the context of INH
resistance mechanisms in clinically isolated strains, which appeared
not long after TB therapy with this drug began (9). Various mutations
in the gene (katG) that encodes this protein have more
recently been correlated with drug resistance (10, 11), although
identification of the structural or functional defects in the mutant
enzymes remains underexplored. The elucidation of altered drug binding,
altered heme binding, or other structural changes due to amino acid
substitutions is only in very early stages of detailed study, most of
which has been devoted to the commonly encountered mutant KatG[S315T]
(12-17). Altered drug binding may be an important factor in drug
resistance (18), rather than major changes in the enzyme's activity as
a catalase or peroxidase, although other mechanisms have been put
forth, including effects involving superoxide-initiated reactions
(19).
Although catalase-peroxidases from various microorganisms have been
purified and characterized, the relationship between the catalytic
function of wild type or mutant enzymes and the heme structural
features such as coordination number or spin state has not been
defined. Understanding such relationships is required if drug
resistance mechanisms in the growing number of mutant KatG enzymes are
to be characterized. Spectroscopic characterization of recombinant
M. tuberculosis KatG and mutant S315T reported elsewhere
showed that the purified enzyme contained a mixture of five-coordinate
(5-c) and six-coordinate (6-c) high spin (HS) heme iron as well as a
large component of low spin (LS) heme (12). Our earlier results (20)
suggested that the majority heme iron species in purified overexpressed
M. tuberculosis KatG was the 5-c HS form. A recent report on
overexpressed KatG from the cyanobacterium Synechocystis
(21) demonstrated the presence of 5-c HS and 6-c high and low spin heme
iron in this enzyme. In the present investigation, optical, resonance
Raman, and EPR spectroscopy were used to investigate the heterogeneity
of heme iron species in wild type M. tuberculosis KatG and
two mutants (S315T and Y155S), both identified as drug-resistant mutants (11, 22-24). Our results reveal that a 5-c heme form of KatG
may be isolated but that coordination and spin state changes are
unavoidable during handling of the enzyme under a variety of
conditions. Similar issues have appeared in the literature over the
years concerning yeast CCP (25-28).
The initiation of the peroxidase cycle, evaluated by determination of
the rate of formation of Compound I, is not altered greatly for
wild-type KatG samples varying in their content of 6-c heme iron. In
KatG[Y155S], formation of 6-c HS heme is facilitated and predominates
in the purified enzyme, although it is not inhibited compared with
wild-type KatG in its rate of Compound I formation either.
Interestingly, this mutation causes a significant change in heme
structure in the pure enzyme, whereas the common mutation S315T found
in INH-resistant bacteria does not.
Materials--
E. coli UM262 (pKAT II)
(overexpression system containing M. tuberculosis katG gene)
was a gift from Stewart Cole (Institut Pasteur, Paris). Mutagenesis was
performed as reported elsewhere (18). All reagents were from
Sigma-Aldrich. Commercial peroxyacetic acid contains hydrogen peroxide,
which was removed by incubation of PAA (10 mM) for 1 h
with bovine liver catalase (780 units/ml) in 20 mM
phosphate buffer, pH 7.2, followed by removal of the enzyme by ultrafiltration.
Purification of M. tuberculosis KatGs--
KatG was isolated and
purified from E. coli strain UM262 (katG minus)
expressing the M. tuberculosis katG gene or a mutated gene
for preparation of KatG[Y155S] and KatG[S315T]. The bacteria were
grown at 37 °C in LB medium containing ampicillin (100 µM) plus the heme biosynthetic precursor,
Solid sodium chloride, fluoride, or formate was added in large excess
(up to 1 M) to solutions of KatG for examination of spectroscopic changes. Ferric KatG-NO was prepared by exposing the
enzyme to NO gas under anaerobic conditions, monitoring the complete
formation of the complex according to optical features (Soret at 420 nm, Regeneration of 5-c KatG--
Reduction of KatG samples
containing a high proportion of 6-c heme, using a small excess of
sodium dithionite under anaerobic conditions, led to the formation of
ferrous KatG (confirmed according to its optical spectrum (29, 30)).
Exposure to air immediately converted ferrous KatG to the ferric
enzyme, based on optical spectra (29). Recovery of 5-c enzyme was also
attempted by adding glycerol (60%) to KatG in phosphate buffer (31,
32) or by titration with isoniazid.
Reconstitution of heme-deficient KatG yielded a mixture of specifically
and nonspecifically bound heme (both 6-c and 5-c), and this technique
was not pursued for evaluation of structural changes in the enzyme.
Electron Paramagnetic Resonance--
Low temperature (5-6 K)
EPR spectra were obtained at X-band using a Varian E-12 spectrometer
interfaced to a personal computer and equipped with a liquid helium
cryostat and Heli-Tran liquid helium transfer system (Advanced Research
Systems, Inc., Allentown, PA). Data acquisition made use of WinEPR
software (20). Assignment of g values was accomplished using difference
spectra and/or simulation for a large collection of spectra recorded
under varying conditions of pH, temperature, and/or microwave power. In
this way, the gy and gx partners could be
isolated when multiple signals were present. Quantitative EPR of low
spin heme (based on signal intensity of one rhombic low spin heme
species) was performed by double integration of signals recorded at 42 K, using the LS myoglobin-mercaptoethanol complex as a spin standard.
This sample was prepared from commercial ferric horse heart myoglobin
in potassium phosphate buffer, pH 7.0, by the addition of 1%
Resonance Raman Spectra of KatG--
Resonance Raman spectra
were obtained using a single spectrograph (TriAx 550; JY/Horiba) and a
liquid N2-cooled CCD detector (Spectrum One;
JY/Horiba) with a UV-enhanced 2048 × 512-pixel chip (EEV). The
Rayleigh scattering was removed using a 406.7-nm holographic notch
filter (Kaiser Optical). The samples were excited with the 406.7 nm
line from a Kr+ laser (Coherent, I-302). Samples were
placed in a spinning cell under an N2 atmosphere and kept
at 6 ± 2 °C during the experiments. Enzyme concentration was
40 µM, and the laser power incident on the sample was 10 mW. Background correction of spectra was performed by subtracting a
polynomial function from the data. The vibrational modes were labeled
according to Ref. 35, and the modes were assigned following published
analyses (36, 37). To determine relative peak intensities and
positions, a curve-fitting program was used to simulate experimental
spectra using Lorentzian line shapes. A bandwidth of 13.5 cm Kinetics of Compound I Formation--
The formation of KatG
Compound I was followed as previously described using a HiTech
Scientific model SF-61DX2 stopped-flow instrument equipped with a rapid
scanning diode array spectrophotometer and Kinet-Asyst software for
data acquisition and analysis (20). All reactions were thermostated at
25 °C. In a typical reaction, 10 µM KatG was mixed
with PAA (10 µM to 1 mM) in potassium
phosphate buffer, pH 7.2. Second order rate constants for Compound I
formation were determined based on absorbance changes in the Soret
region (20).
Optical Spectroscopy--
The absorption spectrum of M. tuberculosis KatG isolated and fully purified in
TEA-Cl2 for this work is
characterized by a Soret peak at 408 nm and a CT1 band at 629 nm (Table
I), as reported previously (20). Other
features of the spectrum, which are not discussed further, include a
shoulder near 545 nm and a maximum at 505 nm (the
The position of the Soret peak and the CT1 band (as well as absorbance
ratios at specific wavelengths defined below) were used to provide
clues about iron coordination number and possibly spin state, similar
to reports on CCP (27, 32). Whereas the purity of the enzyme varies
throughout purification, our approach, in which the enzyme was
monitored from the first through the last chromatographic procedure,
was considered viable, since KatG always represented the majority
protein (according to SDS-PAGE, not shown).
Table I summarizes the optical wavelength maxima for KatG recovered and
examined during and after purification. The spectrum of the enzyme
changes significantly during purification in TEA but much less so in
phosphate buffer.3 Catalytic
reactions stimulated in the presence of amine-containing buffer may be
partly responsible for these changes, as reported for CCP (39). Enzyme
having a Soret peak at or near 405 nm with a shoulder at 380 nm and a
CT1 band at or above 640 nm may be considered to represent mainly 5-c
HS heme, whereas peaks at 408 and 630 nm indicate abundant 6-c HS heme.
The purity index
(ASoret/A280) for KatG
prepared in TEA-Cl was 0.65-0.7, whereas for KatG prepared in
potassium phosphate buffer, the ratio was consistently lower (0.47-0.5). This observation is not due to differences in purity but
demonstrates the greater abundance of 6-c heme when TEA buffer is used.
A greater Soret extinction coefficient occurs for 6-c HS heme, as
noted, for example, in CCP (32).
Also given (Table II) are absorbance
ratios in the Soret and CT1 band region that vary with the proportion
of 6-c, relative to 5-c heme. For these ratios, higher values
correspond to greater abundance of 6-c HS heme (27).
Close inspection of the CT1 band for the fresh enzyme after the first
chromatography protocol during numerous preparations provided evidence
that the enzyme even at this stage could be a mixture of at least two
components, since shoulders red- and blue-shifted from 640 nm could
occasionally be seen (not shown).
In order to evaluate the stability of 5-c heme in KatG, the enzyme
recovered after partial purification (step 1) in potassium phosphate
buffer was stored for 3 weeks at 4 °C. During this time, the
proportion of 6-c HS enzyme increased (Fig.
1 and Table II), and higher
A404/A380 and
A614/A645 ratios were
found.
We wanted to ensure that the length of the period for bacterial
overexpression of KatG had not allowed changes in structure before
isolation of the enzyme. To address this issue, we compared partially
purified (step 1) KatG isolated from cells grown for 6 or 20 h
after induction of enzyme overexpression. No differences in optical
spectra were found following elution from the first chromatographic
column (not shown). This result demonstrates that if any structural
changes occur during overexpression, they do not continue during the
extended period from 6 to 20 h. For this reason, we suggest that
the characteristics of the fresh, partially purified enzyme represent
or closely resemble a "native" structure.
In order to determine whether 6-c heme could be converted back to a 5-c
form, we evaluated the effects of reducing and reoxidizing KatG, the
effect of glycerol, and the effect of organic ligand binding. The first
approach was based on the behavior of certain reversibly formed 6-c LS
hemichromes identified in hemoglobin that can be converted back to high
spin methemoglobin by reduction/reoxidation (40). Glycerol has been
shown to modify the coordination state of heme iron in CCP (31, 32).
For KatG, these two methods produced changes in optical spectra
indicating conversion toward 5-c enzyme (Fig. 1, Table II). The
reoxidation results also demonstrate the instability of KatG Compound
III (oxyferrous KatG) as reported previously based on optical
stopped-flow kinetic measurements (20), in which it had been generated
from the ferric enzyme in the presence of excess hydrogen peroxide
(31).
The addition of INH and analogous ligands to 6-c KatG regenerated 5-c
enzyme (not shown), confirming earlier reports (15, 18). Overnight
dialysis to remove INH restored the spectrum of the starting, 6-c
enzyme. No optical changes were detected when INH was added to the
fresh, partially purified enzyme.
Whereas formation of 6-c heme due to water coordination under different
conditions was considered the most likely origin of the heterogeneity
in KatG heme structure, the potential formation of a 6-c KatG-chloride
complex during purification was also investigated. Chloride binding to
heme iron was considered possible because it is used in a relatively
high concentration (0.3 M) to elute the enzyme from ion
exchange media, and chloride binding to heme iron in CCP has been
reported (26). However, no significant differences were found in the
optical spectrum of KatG when the first chromatography procedure was
performed using a potassium phosphate concentration gradient (at pH
6.5) in place of the usual sodium chloride gradient for protein elution
(not shown). This demonstrates that exposure of the enzyme to 0.3 M sodium chloride for periods of 20-24 h does not produce
heme structural changes. This also suggests that the enzyme eluted from
the first column can be assigned to a "native" form with more
confidence. We then deliberately attempted to produce a chloride
complex in fresh partially purified KatG using higher concentrations of
sodium chloride (1 M).4 Here, a
Soret peak at 408 nm without the shoulder at 380 nm, a new shoulder
around 358 nm, and a CT1 band at 640 nm (Fig.
2) were found. Whereas the changes in
optical features suggest formation of 6-c iron, the shift in the CT1
band is a small fraction of that seen for 6-c HS KatG anion complexes
(KatG-fluoride or KatG-formate (Table III
and Fig. 2), and only a small shift in the Soret band occurred. For
comparison, in CCP plus chloride, the Soret peak occurs at 413 nm, and
the CT1 band occurs at 640 nm (26).
Fluoride binding to fresh, partially purified enzyme was also examined.
Interestingly, two CT1 bands are found in the optical spectrum, one
around 615 nm (typical of 6-c HS anion complexes) and another close to
650 nm (Fig. 2, inset). In contrast to this, the
KatG-fluoride complex prepared under the same conditions, using KatG
that contained abundant 6-c heme, exhibited a single CT1 band at 616 nm
(not shown). These results are consistent with a low affinity for
fluoride in enzyme that lacks a specific water molecule in the distal
pocket required for stabilization of the ligand (41).
In order to evaluate the amount of LS iron in KatG, we used optical
difference spectroscopy (and EPR presented below) based on the report
of an endogenous LS KatG form exhibiting peaks at 540 and 570 nm (12).
No new maxima could be detected between 530 and 580 nm in difference
spectra recorded for enzyme containing abundant 5-c versus
6-c heme (data not shown). We also examined the first derivative of the
optical spectrum of pure KatG and did not identify features indicating
the presence of bands due to LS iron. Furthermore, the well formed CT1
band and the ratio of absorbances at 570 nm compared with 640 nm in the
optical spectrum of KatG argue against the presence of LS heme in the
pure enzyme. This ratio correlates well with the relative abundance of
6-c LS heme according to inspection of the data for
Synechocystis KatG, CCP, and ascorbate peroxidase presented
in Ref. 21. Also, when LS heme is abundant enough to contribute a
feature near 570 nm in spectra of these Class I peroxidases, the Soret
peak is usually found at or above 410 nm (12, 21), in contrast to the
significantly lower Soret wavelengths for partially purified and pure
KatG presented above. No 6-c LS heme was found in the optical spectra
of KatG at pH 10.
The optical spectrum of purified KatG[S315T] was characterized by
features close to those of the wild type enzyme lacking 6-c heme (Soret
peak at 403 nm and a CT1 band at 642 nm), whereas for KatG[Y155S] the
spectrum resembled that of 6-c wild type enzyme (not shown).
EPR Spectra--
EPR spectroscopy was applied to confirm and
extend the results described above wherever possible. The partially
purified enzyme (from potassium phosphate buffer) had an EPR spectrum
dominated by a rhombic species (signal r1) with other
features at g = 6.0 and ~5.6 assigned to signal
r2 (Fig.
3; g-values of signals summarized in
Table IV). An additional rhombic species
is assigned to signal r3. The broadest rhombic signal
(r3) is assigned to 5-c HS heme, whereas signals
r1 and r2 are assigned to other 5- and 6-c
species, respectively. The predominating rhombic signal r1
exhibited by the partially purified enzyme confirms the idea that the
"native" structure of KatG may be exclusively 5-c. The EPR spectrum
of the enzyme after complete purification exhibits a small increase in
the intensity of signal r2. Samples of pure KatG exhibit
some weak intensity near g = 2 that may be due to LS heme (Fig. 3, inset).
The EPR spectrum of the purified enzyme was recorded after storage of a
sample (in potassium phosphate buffer) for 3 weeks at 4 °C (Fig. 3).
Here, the changes indicate increased abundance of 6-c heme and loss of
5-c heme, consistent with the optical and Raman results (see below).
Quantification of low spin heme based on the intensity of a feature at
g = 2.2, considered to represent the gmid for one or more low spin heme species, provided estimates that ~10% of the total heme could be present as LS heme in pure KatG examined weeks after purification. Barely detectable amounts of LS heme signal are
present in the fresh (partially purified) enzyme.
The effect of chloride or fluoride addition to predominantly 5-c KatG
was also monitored. KatG in the presence of excess fluoride exhibits a
nearly axial signal (g
The g values for signals r1 and r2 fit the
"rule of thumb" definition for the presence of intermediate spin
iron used by many authors in analyses of rhombic EPR signals in other
heme proteins and enzymes (42-45) in that the gav
((g1 + g2)/2) values fall between 4 and 6. The high g1 value for signal r1,
however, is just beyond the range usually associated with rhombic EPR
signals thought to represent quantum mechanically mixed spin (QS) heme
and may be best assigned to 5-c HS heme. The gav value, as
well as the g1 and g2 values for signal
r2, fall within the range of those assigned to 6-c QS heme
species in Class III peroxidases (45, 46). Thus, some agreement is
found between our results for KatG and observations in Class III
peroxidases containing 5- and 6-c QS heme (see "Discussion").
The EPR spectrum of the purified mutant KatG[Y155S] is also shown
(Fig. 5). Here, the predominant signal
represents a 6-c HS species assumed to have water as the sixth ligand
by analogy to similar EPR signals reported for lignin peroxidase and
other heme proteins (47, 48). This mutant enzyme was considered useful
to examine Compound I formation starting from an endogenous 6-c resting
form of the enzyme (see below). EPR spectra of the purified mutant
KatG[S315T] (not shown) were nearly identical to the pure wild-type
enzyme and were not analyzed further.
Resonance Raman Spectra of KatG--
We turned to Raman
spectroscopy to help define the heterogeneity observed in the optical
and EPR spectra of KatG described above. Here, we will be focusing on
the
The high frequency region of resonance Raman spectra of pure KatG in
Hepes buffer (not shown) was indistinguishable from that in potassium
phosphate. This suggests again that formation of the 6-c species
involves a molecule of water rather than coordination of a buffer component.
A
The lowest frequency
In order to obtain a reference marker for 6-c HS KatG, the Raman
spectrum of KatG-fluoride was examined. Here, a band typical for a 6-c
HS peroxidase anion complex was found (1482 cm
The resonance Raman spectrum of partially purified KatG was also
examined in the presence of a large excess of NaCl. Here, no increase
in intensity was found at a frequency indicating formation of a typical
6-c HS anion complex. Instead, a broad Rate of KatG Compound I Formation--
In earlier work on purified
M. tuberculosis KatG (20), the rate of Compound I formation
from the resting enzyme and peroxides was found to be significantly
slower than that for horseradish peroxidase, and we considered that a
reason for this could be the presence of 6-c heme iron in KatG. We
therefore measured the rate constants for Compound I formation from
fresh KatG (partially purified) and the same enzyme after storage for 4 weeks. The values for KatG examined weeks after purification
were somewhat greater than that for the fresh enzyme (6.5 ×103 M
We also monitored the effects of peroxide (PAA and CPBA) on the
heterogeneity of KatG starting from partially purified enzyme and
enzyme that had been stored for a few weeks. The optical spectra of the
starting forms were recovered in both cases after the spontaneous return to the ferric state that occurs when excess peroxide is consumed, with no observable changes due to heme degradation. In the
case of the fresh enzyme, resonance Raman results demonstrated that the
6-c form is found immediately after cycling of the enzyme with
peroxide, whereas the features assigned to 5-c KatG, including the QS
heme, slowly return after a longer time period has elapsed (not shown).
The KatG mutant enzyme, KatG[Y155S], which is nearly completely 6-c
HS in its resting state according to optical and EPR spectra, did not
show a decreased rate of Compound I formation compared with the
wild-type enzyme. In contrast to these results, ferric KatG-NO was
strongly inhibited in Compound I formation under similar conditions.
For example, kobs for Compound I formation,
using 10-fold excess PAA, was 1.23 s The aims of this study include characterization of heme structural
heterogeneity in M. tuberculosis KatG and possibly
definition of a "native" form of this enzyme isolated from
overexpression in E. coli. Our approach has relied, in part,
on examination of KatG after only partial purification and within
36 h of isolation from the E. coli overexpression
culture system. We reported earlier (20) that pure KatG seemed to be
stable after purification, when, instead, the use of TEA-Cl buffer for
its purification at that time had accelerated the loss of 5-c, or what
we now consider "native" enzyme. Summarized in Fig.
7 are the different forms of KatG and the
processes and factors that allow for interchange between them. The
apparent "evolution" of 6-c species from the 5-c form is due to
coordination of a molecule of water to iron and is apparently
unavoidable. The water ligand is weakly bound and dissociates in
ferrous KatG and under other conditions (e.g. by the
addition of glycerol or organic ligands like INH). One interesting
hypothesis concerning the accumulation of the specifically bound water
molecule in 6-c heme may be from basal level catalytic turnover of the
enzyme, which produces 2 mol of water in the active site from the decay
of Compounds I and II.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminolevulinic acid (300 µM), which eliminated the
large proportion of heme-deficient enzyme isolated in its absence.
Cells were cultured for various time periods (routinely 6-8 h and up
to 20 h, as indicated). The enzyme was purified by fast protein
liquid chromatography according to a published procedure (29), using
either 20 mM potassium phosphate buffer (pH 7.2) or 20 mM triethanolamine-HCl (pH 7.8) (TEA-Cl). Sodium chloride
gradients were generally used for enzyme elution from ion exchange
media (Amersham Biosciences Q-Sepharose FastFlow, MonoQ), whereas
ammonium sulfate from 1 M down to 0 M was used in the gradient for elution of KatG from phenyl-Sepharose media (Amersham Biosciences phenyl-Sepharose). Optical spectroscopy was
performed using a model NT14 UV-visible spectrophotometer (Aviv
Associates, Lakewood, NJ) interfaced to a personal computer running
14DS software. All spectra were recorded for enzyme in potassium
phosphate buffer, pH 7.2, at 25 °C, except where noted otherwise.
and
bands at 570 and 536 nm). No evidence for reduction to
the ferrous enzyme was found under these conditions.
-mercaptoethanol (33). Final heme concentration in the standard was
based on the Soret absorbance of the starting aquometmyoglobin (34),
assuming complete conversion to the LS form.
1 was used for the simulation of the
3
region bands.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and CT2 bands,
respectively). The wavelength maxima for the Soret and CT1 bands are
very close to those reported elsewhere for overexpressed M. tuberculosis KatG prepared in other buffers, either at pH 7.0 or
8.0 (15, 30, 38). These maxima, which are most often associated with
6-c heme in peroxidases, however, were found to be notably different
from those seen early in the KatG purification protocol, an observation
suggesting that published spectra did not represent "native" KatG
and that a more detailed investigation was warranted.
Absorption maxima in the optical spectrum of M. tuberculosis KatG after
each step in the purification protocol
Absorption maxima and optical ratios sensitive to coordination state in
various forms of M. tuberculosis KatG
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Fig. 1.
Electronic absorption spectra of fresh
partially purified KatG, KatG examined after 3 weeks' storage, and
KatG after reduction/reoxidation. Inset, expanded
spectra showing CT1 bands. The spectra are offset for presentation
purposes.
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Fig. 2.
Electronic absorption spectra of fresh
partially purified KatG and this enzyme in the presence of 1 M NaCl or NaF. Inset, expanded spectra
showing CT1 bands. The spectra are offset for presentation
purposes.
Absorption maxima of fresh partially purified KatG in the presence of
weak field ligands
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Fig. 3.
Low temperature EPR spectra of fresh
partially purified KatG, pure KatG, and KatG after 3 weeks'
storage. Experimental conditions were as follows: temperature, 5 K; microwave power, 5 mW; frequency 9.2446 GHz. Inset, EPR
spectrum of pure KatG recorded at 16 K and 5-mW microwave power.
EPR and resonance Raman (RR) features in various forms of KatG
= 5.8), with rhombic component r1 still present (Fig. 4). In
contrast to this, the spectrum in high sodium chloride contains a well
resolved r2 signal and a lower intensity of signal
r1 compared with the same enzyme without sodium chloride,
consistent with some conversion of 5-c heme into 6-c heme. When excess
NaCl was added to a sample of KatG that had been stored for 3 weeks, a
nearly axial signal (g
~ 5.8) typical of a 6-c HS
complex was found (Fig. 4, inset). The results from optical
and resonance Raman spectroscopy (see below) do not suggest chloride
coordination under these conditions and are more consistent with water
coordination being facilitated in the presence of 1 M
sodium chloride.
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Fig. 4.
Low temperature EPR spectra of fresh
partially purified KatG alone and in the presence of 1 M
NaCl or NaF. Inset, EPR spectrum of KatG (after 3 weeks' storage) plus excess NaCl. Experimental conditions were the
same as in Fig. 3 at 5 K.
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Fig. 5.
Low temperature EPR spectrum of
purified mutant KatG[Y155S]. Experimental conditions were the
same as in Fig. 3 at 5 K.
3 bands (spin and coordination state markers) in the
high frequency region for evidence in support of coordination number
and spin state assignments (36, 37). The
3 frequencies
for the different forms of KatG are summarized in Table IV. The
resonance Raman spectrum of M. tuberculosis KatG purified in
TEA-Cl buffer was previously reported to contain a
3
band centered at 1490 cm
1 (20) assigned to 5-c HS heme,
whereas a mixture of high and low spin forms was described
in another report for KatG purified and examined in Tris-Cl buffer
(12). Here, the fresh partially purified enzyme (from phosphate buffer)
exhibited two bands of nearly equal intensity; the 1495 cm
1 band corresponds to a 5-c HS species, whereas the
1503 cm
1 band can be assigned either to a 6-c LS or a 5-c
QS species (Fig. 6). A band near 1487 cm
1 also contributes to the breadth of the
3 region, indicating the presence of a low abundance
component due to 6-c heme (but not low spin heme). The relative
abundance of these species based on deconvolution of the bands is 8%
6-c HS (or QS) form (1487 cm
1), 38% 5-c HS form (1495 cm
1), and 54% 5-c QS form (1503 cm
1). The
Raman spectrum of pure KatG contains higher intensity near 1487 cm
1 associated with 6-c heme (Fig. 6) and a decrease to
30% in the proportion of the band at 1503 cm
1. This is
consistent with optical and EPR spectra, which also revealed a greater
abundance of 6-c heme in the pure enzyme. Enzyme examined after storage
showed further loss of 5-c QS heme and small increases in 6-c heme (but
not LS heme), whereas the central band remained relatively constant in
intensity (not shown). No increased intensity due to 6-c LS species was
detected in the high frequency region of spectra for KatG examined at
pH 10.
View larger version (24K):
[in a new window]
Fig. 6.
Resonance Raman spectra of fresh partially
purified KatG, pure KatG, and the partially purified enzyme in the
presence of 1 M NaCl or NaF. See "Experimental
Procedures" for details.
3 band near 1503 cm
1 has been assigned
elsewhere to the unusual QS state heme in Class III peroxidases (44,
46, 49, 50). However, a similar
3 band was assigned to
6-c LS heme in KatG containing LS heme signatures in EPR and/or optical
spectra (12), whereas a nearby band at 1505 cm
1 was
assigned to 6-c LS heme in Synechocystis KatG (21). Here, we
prefer to assign the 1503 cm
1 band to 5-c QS heme based
on the lack of LS species in the optical spectrum and the near absence
of LS heme in the EPR, contrasted by the high proportion of this
feature in the Raman spectrum. Furthermore, and most importantly, the
relative intensity at 1503 cm
1 decreases on going from
the partially purified to the pure enzyme and finally to enzyme
examined after storage for a few weeks, whereas the EPR data indicate
some increase in LS components, the other form that may give rise to
the 1503 cm
1 band in such samples.
3 band (1487 cm
1) is
assigned to 6-c heme, and its high frequency suggests QS rather than HS
heme (44, 46, 49, 50). Formation of 6-c QS heme from 5-c QS heme is reasonable, since the heme distortions thought to be required for
stabilization of the mixed spin state are not removed upon conversion
to a 6-c form, at least in Class III peroxidases (45).
1),
although residual
3 bands due to the starting enzyme
were evident. Incomplete conversion of the fresh enzyme to the 6-c fluoride complex was also found in EPR and optical spectra described above.
3 feature centered at 1495 cm
1 with no resolved feature at 1503 cm
1 was seen, along with an increase in intensity around
1487 cm
1 (according to deconvolution estimates). These
observations demonstrate that high concentrations of sodium chloride
reduce the abundance of the 5-c QS species and may increase the
abundance of 6-c QS heme, the latter based on similarities to 6-c QS
peroxidase-benzhydroxamic acid complexes (41, 45). These results are
also in agreement with the EPR data in that an increase in coordination
number was suggested, although an axial signal characteristic of 6-c HS
anion complexes was not found for similar samples. Removal of excess NaCl by extensive dialysis restored the Raman spectrum (and the optical
spectrum) of the "native" enzyme (not shown).
1 s
1
versus 5.6 × 103
M
1 s
1). Whereas these rates are
lower than those reported previously for the purified enzyme, the
results indicate that the presence of 6-c heme iron species is not
inhibitory in the reactions leading to Compound I formation in
KatG.
1 for pure KatG and
0.08 s
1 for KatG-NO.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
[in a new window]
Fig. 7.
Diagram representing the changes in
coordination and spin state of KatG under different conditions.
The history of sample handling may in some undetermined manner be a
factor in the nature of the active site heterogeneity in all
circumstances. *, Storage is used to indicate the time
period during which unidentified slow processes generate changes in
heme structure.
The 6-c form is converted back to 5-c enzyme upon binding of INH, although this is nearly fully reversed during overnight dialysis to remove the drug. This reversal is considered significant, because it may be evidence for permanent modification of the enzyme that prevents reestablishing the heme pocket structure found in the fresh partially purified enzyme. Relevant to this point is the finding that pure KatG isolated in TEA-Cl buffer, having the optical spectrum characteristic of abundant 6-c heme, exhibited a molecular weight (using matrix-assisted laser desorpton ionization mass spectrometry) within 0.04% of that calculated for the KatG dimer (based on translation of the M. tuberculosis katG gene (51); two runs, average mass 162,540 ± 167 kDa; calculated mass 162,470 kDa). This may be taken to indicate that if permanent modification of KatG occurred and was responsible for maintaining 6-c heme, the modification is not large enough to yield a detectable change in the overall mass of the enzyme.
In the absence of coordination to iron of distal residues, the occupancy and architecture of water in the distal pocket of KatG is considered an important feature producing heterogeneity in KatG and other peroxidases. This water may or may not be directly coordinated to heme iron and may participate in hydrogen bonding with the side chains of distal residues including the conserved Trp, Arg, and His found in KatG and other Class I peroxidases. The distal residues are important for establishing catalytic behavior (as has been shown for KatG (52, 53)) and, along with water molecules, for governing ligand binding to heme iron (21, 54). The hydrogen bonding interactions between these residues (water and bound fluoride, for example) influence fluoride affinity and the wavelength of the CT1 bands in the respective 6-c HS fluoride complexes. Since KatG-F and CCP-F have very similar optical spectra, including nearly identical wavelengths for their CT1 bands (616 and 617 nm, respectively), similar hydrogen bonding environments for the sixth ligands in these two enzymes may be reasonably assumed. Based on these arguments, the fractional binding of fluoride to KatG that had been minimally treated after its isolation from cells and contained a majority of 5-c heme, suggests the absence of a specific water molecule in the distal pocket being responsible for the low affinity form. Once water has accumulated in the pocket, the affinity for fluoride is enhanced, because all of the binding requirements for this ligand can then be satisfied.
The behavior of KatG in the presence of very high concentrations of sodium chloride or in the presence of INH also indicates changes in the occupancy and/or orientation of distal pocket water. Can we draw some analogy between INH binding to KatG and binding of organic donor ligands to Class III peroxidases? The binding of benzhydroxamic acid to horseradish peroxidase and ascorbate peroxidase is known to involve hydrogen bonding interactions between the exocyclic substituent of the ligand and the side chains of distal Arg and His residues as well as stabilization of water coordination to iron (55, 56). Similar interactions are expected for binding of hydrazides such as INH (55). Nevertheless, in the case of INH binding to KatG, water coordination in the 5-c fresh enzyme is not induced, and dissociation of water from iron in the 6-c enzyme occurs. The origin of this opposite effect is not understood at this time, and little is known about the details of INH binding to KatG other than the proximity between heme iron and nitrogen sites of the ligand in the complex (15, 57). The effects of high sodium chloride also remain poorly understood at this time.
Other results presented here support the finding of the QS heme in
M. tuberculosis KatG. The structural origin of this spin state in Class III peroxidases, which arises when intermediate spin
iron (S = 3/2) is present along with high spin iron, may reside in
deformations of the heme macrocycle, with saddling considered important
but not a sole requirement for the QS state (44). Thus, there is no
a priori reason for the QS spin state not being accessible
in a Class I peroxidase; nor can we state that KatG possesses a known
feature that would predict its presence. Although the Raman spectrum of
fresh KatG exhibits a 3 band analogous to that found in
Class III peroxidases containing QS heme (44-46), we have not found an
accompanying EPR signal that closely resembles the axial one assigned
to 5-c QS heme (in barley peroxidase) (44). Rhombic EPR signals with
gav values below 6 have often been assigned to QS heme (42,
43), but the r1 signal of KatG representing the predominant
species in the fresh enzyme has a high g1 value more
consistent with 5-c HS heme. The correlation between gav values and spin state may even be of questionable reliability, since,
for example, magnetic susceptibility measurements reported in a recent
investigation of R. capsulatus cytochrome c' were most consistent with high spin iron (59) despite the rhombic EPR
spectrum meeting the accepted criteria for QS heme in this protein.
Therefore, the most cautious interpretation of our results that takes
into account the Raman and EPR observations on KatG is that close
structural analogs, both of which are 5-c, represent QS heme at room
temperature and HS heme in frozen solution at 5 K. Little additional
insight into the assignment of spin states in KatG is found by
examination of optical spectra, since CT1 bands may overlap for the
various forms in question, and KatG under the conditions we have
employed here is apparently always a mixture of species. Ongoing
studies of KatG, including low temperature resonance Raman
spectroscopy, are expected to shed more light on this matter. We also
note that temperature-dependent structural changes that
alter spin state and coordination number have been documented for
peroxidases (60-62), warning against making strict correlations
between the present Raman and EPR results for KatG. Nevertheless, we
have found general agreement between room temperature and frozen
solution results when following changes in coordination number observed
in KatG. The assignment of the r1 signal as the predominant
species in partially purified KatG to a 5-c HS species, while more
satisfying in terms of the nature of the EPR spectrum, would mean that
temperature- or freezing-induced structural changes favored a shift in
the spin state distribution from 3/2 toward 5/2.
Another aim here was to attempt to correlate heme structural heterogeneity with functional or mechanistic changes in the peroxidase cycle of KatG. This was approached through the measurement of the rate of Compound I formation for KatG samples containing 5-c or 6-c enzyme as a majority species and a mutant enzyme, in which 6-c heme was present in high abundance. If we assume that the similarity between KatG and CCP fluoride complexes extends to the structures in the 6-c species having water bound and that the presence of this water is responsible for the slow formation of Compound I in "aged" CCP as reported previously (39), similar behavior for aged KatG would be predicted. Instead, no inhibition of Compound I formation was found for 6-c KatG or in a purified mutant enzyme containing mainly 6-c heme. The finding that ferric KatG-NO is strongly inhibited in its rate of Compound I formation indicates that a feature such as a slow sixth ligand off-rate interfering with coordination of peroxide could contribute to inhibition. These results then suggest that the water molecule occupying the sixth ligand position in 6-c KatG may be readily exchangeable with peroxide.
A remaining interesting question is the origin of heterogeneity in spin states of even 5-c KatG. If we consider that the partially purified enzyme represents a "native" conformation of the KatG dimer, the spin state heterogeneity may reflect different heme pocket structures in each monomer. Consistent with this idea is the reported cooperative nature of INH binding to KatG (15, 18), which also implicates subunit heterogeneity. In contrast to this is the heterogeneity of spin state and also coordination number in purified monomeric Class I and Class III peroxidases (26, 44, 46, 49, 50, 63). Interestingly, horseradish peroxidase even exhibits heterogeneity when examined by EPR spectroscopy in situ (64). In addressing this issue, it was also noted that the simple act of repipetting purified horseradish peroxidase from a test tube into an EPR tube could result in alterations in the observed EPR signals (64).
Here, the evolution of 5-c to 6-c KatG has been observed through its
straightforward isolation and purification. At this point, it is not
clear how generally true the phenomenon might be in which heterogeneity
evolves in a peroxidase as a function of time due to unaccounted for
processes, as originally suggested for CCP (26). (Note that aging
phenomena do not occur in crystals of CCP stored over 1 year at
40 °C (27).) One idea that has not received attention is that
accumulated irreversible changes in the polypeptide may be responsible
for heme structural changes in KatG and for the heterogeneity of
structure in other peroxidases as well. Such permanent changes might
result from amino acid side chain oxidation due to reactions catalyzed
by Compound I formed during turnover of the enzymes with endogenous
peroxide. Whereas the binding of INH to KatG can displace the sixth
ligand water molecule and regenerate 5-c enzyme, water rapidly
recoordinates when the drug is removed. This behavior would be likely
under circumstances in which the properties of the distal pocket in the
"native" enzyme are not reestablished simply by the reversible binding of an organic ligand. Ongoing studies are expected to shed some
light on these speculations.
The crystal structure of a catalase-peroxidase from Haloarcula
marismortui, reported during review of this manuscript (58), shows
covalent linkages between the 3'- and 5'-phenol ring positions of a
conserved distal tyrosine residue (Tyr208) and nearby
conserved tryptophan and methionine side chains. The high homology
between this enzyme and M. tuberculosis KatG raises the interesting possibility that tyrosine modification is a
factor contributing to structural modification surrounding the heme in
KatG, an issue we are actively addressing.
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ACKNOWLEDGEMENTS |
---|
We thank Irina Kovatch and Nicolas Carrasco for assistance in data collection.
![]() |
FOOTNOTES |
---|
* This work was supported by NIAID, National Institutes of Health, Grant AI-43582 (to R. S. M.) and start-up funds from New York University (to J. P. M. S. and S. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Chemistry, Brooklyn College and the Graduate Center of the City University of New York, 2900 Bedford Ave., Brooklyn, NY 11210-2889. E-mail: rmaglioz@brooklyn.cuny.edu.
Published, JBC Papers in Press, December 28, 2002, DOI 10.1074/jbc.M208256200
2 TEA and other cationic amine buffers are recommended for use with strong anion exchangers such as FastFlow Q-Sepharose and MonoQ (Amersham Biosciences).
3 The major change in optical features occurred as a result of ammonium sulfate precipitation in this buffer but not in potassium phosphate buffer. Direct titration of the enzyme in potassium phosphate buffer with ammonium sulfate to 1 M (23%) did not cause any change in the optical spectrum.
4 Optical changes with chloride, fluoride, and formate addition to KatG were very slow. A very high concentration of these ligands was used to effect optical changes in a reasonable time frame and to achieve maximal conversion of the enzyme into a new form.
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ABBREVIATIONS |
---|
The abbreviations used are: KatG, catalase-peroxidase; CCP, cytochrome c peroxidase; CPBA, 3-chloroperoxybenzoic acid; HS, high spin; INH, isonicotinic acid hydrazide; LS, low spin; PAA, peroxyacetic acid; QS, quantum spin; TEA, triethanolamine; 5-c, five-coordinate; 6-c, six-coordinate.
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