From the
Resonance Raman spectroscopy has been used to investigate the
properties of cyanide-bound beef liver catalase (BLC) and
Aspergillus niger catalase (ANC) in the pH range
4.9-11.5. Evidence has been obtained for the binding of cyanide
to both BLC and ANC in two binding geometries. The first conformer,
exhibiting the
Catalase (EC 1.11.1.6; H
The catalytic cycle of
catalase involves the reaction with two molecules of hydrogen peroxide
to form water and oxygen. The first step is an oxidation process
involving the reaction with one molecule of hydrogen peroxide to form
an oxidized reactive intermediate, known as compound I, and water. The
second step is a reduction process which involves the reaction of
compound I with a second molecule of hydrogen peroxide to regenerate
the ferric catalase and molecular oxygen. Therefore, the mechanism of
catalase action can be written (Schonbaum and Chance, 1976) as
the following.
On-line formulae not verified for accuracy
Compound I of catalases, like that of HRP, is best described as
a ferryl
Resonance Raman (RR)
spectroscopy is a powerful technique for the investigation of
hemeprotein activesites (Spiro and Li, 1988) and axial-ligand
vibrations (Yu and Kerr, 1984; Yu et al., 1988). Previous RR
studies have concentrated on the investigation of the skeletal modes of
the native forms of various catalases, the enhancement of the axial
tyrosinate modes through excitation in the visible region (Chuang
et al., 1988; Sharma et al., 1989; Nagai et
al., 1989), and the detection of the reactive intermediates in the
catalytic cycle (Chuang et al., 1989; Chuang and Van Wart,
1992). The lack of RR studies on catalases ligated with various
exogenous ligands prompted us to carry out an investigation of the
ligated forms of catalases in order to probe the heme distal
environment and the effect of the endogenous tyrosinate ligation on the
vibrational modes of the axial ligand. Recently, this group reported
the results of an RR investigation of CO-ligated BLC (Hu and Kincaid,
1992). In other reports, we have demonstrated that RR investigation
(Al-Mustafa and Kincaid, 1993; Hu and Kincaid, 1992) of the cyanide
adducts of peroxidases can provide vital information about the
important structural elements of the active sites and reveal the role
of distal- and proximal-side heme environments on the catalytic cycle.
In the present work, we report RR studies of the cyanide adducts of two
catalases, a mammalian catalase (BLC) and a fungal catalase (ANC).
Aspergillus
niger catalase (ANC) suspension in 3.2 M ammonium sulfate
(pH 6) was obtained from Sigma. The ammonium sulfate was removed by
dialyzing twice against deionized water (6 h each) and then against 20
mM potassium phosphate at pH 7 (12 h). The ANC solution was
centrifuged and the deep green solution was chromatographed using the
same procedure described for BLC. The purified ANC solution (Rz
= 0.68) was used for the spectroscopic measurements. The buffer
exchange was achieved either by passing the catalase solution over a
Sephadex G-25 (Pharmacia) column or by ultrafiltration (Amicon). The
buffers used for the final samples were 250 mM sodium acetate
(pH 5), 250 mM potassium phosphate (pH 7), and 250 mM
sodium carbonate (pH 10.5 and 11.6).
Potassium cyanide was from
Aldrich. The K
On-line formulae not verified for accuracy
The results corresponding to the best fit for ANC-CN
at pH = 4.9 and BLC-CN at pH 7.0, obtained by the computer
program described above, are summarized in . In the process
of simulation it has been assumed that different isotopic substitutions
in the CN fragment influence the absolute frequencies of the peaks only
and that the band widths and intensities do not change upon these
substitutions. Various initial estimates and perturbation steps have
been used in the process of fitting. In all cases the frequencies
obtained were within experimental error ± 2 cm
Identification of the Fe-C-N Axial Modes
The presence of a fourth isotope-sensitive mode
( i.e. a second bending mode) is less apparent in both the
absolute and the difference spectra shown in Figs. 1 and 2,
respectively. However, careful examination of the difference spectra in
Fig. 2
, especially traces a-b, a-d, and c-d,
which are expected to show all of the stretching and bending modes,
reveals an asymmetrical difference feature near 430
cm
Studies
carried out on the cyanide adducts of the one electron-reduced sulfite
reductase (Han et al., 1989), ferric HRP (Al-Mustafa and
Kincaid, 1993), ferric myeloperoxidase (Lopez-Garriga et al.,
1990), and ferric lactoperoxidase (Hu et al., 1993) revealed
that the bending of the Fe-C-N linkage causes a reversal of the
ordering of the
Inspection of Figs. 1
and 2 reveals the presence of several other isotope-sensitive features
which are not readily discernable in Fig. 1owing to their small
shift and their overlap with isotope-insensitive heme deformation
modes. The region near 380 cm
Fig. 7
shows the low frequency RR
of the isotopomeric CN adducts of BLC at pH 11.5. The most noticeable
changes in the frequencies of the axial modes is in the
In
addition to the distal histidine (His-74), the crystal structure of BLC
reveals the presence of a distal asparagine residue (Asn-147), an amino
acid known to be capable of acting as a hydrogen bond donor to the
bound cyanide (Murthy et al., 1981; Fita and Rossman, 1985a).
Therefore, it is reasonable to suggest that a hydrogen bonding
interaction of the bound cyanide with the distal asparagine (known to
be displaced further from the heme normal than the histidine) produces
the bent Fe-C-N linkage which exhibits its characteristic
The presence of hydrogen bonding between these
amino acid residues and the bound cyanide may impart sensitivity of the
Fe-C-N vibrations to changes in pH, since raising the pH above the
p K
RR investigation of the
ANC-CN in the pH range 4.9-11.5 did not reveal any significant
shift in the axial mode frequencies of either the linear or the bent
conformers (Figs. 5 and 9). The lack of a pH-dependent shift in ANC-CN
presumably reflects a greater stability of the hydrogen bond between
the distal histidine and the bound cyanide, although no structural
argument for this increased stability can be made in the absence of
definitive active site structural data.
These differences in behavior between
catalase and HRP can be reasonably explained in terms of the variation
in active site environments between catalase and HRP. Many factors
might contribute to this unusually low
The difference in
response to pH changes between catalase and HRP is also consistent with
active site structural variations between the two proteins. It is
generally agreed that deprotonation of the distal histidine in HRP
produces a corresponding alteration of the trans
Fe-N
The following
abbreviations are used: BLC, bovine liver catalase; ANC,
Aspergillus niger catalase; HRP, horseradish peroxidase;
SiR-HP
[Fe-CN] stretching mode at a higher
frequency than the
[Fe-C-N] bending mode, exists as an
essentially linear Fe-C-N linkage. For both BLC-CN and ANC-CN, the
[Fe-CN] and
[Fe-C-N] frequencies of this
conformer were practically identical and observed at
434 and
413 cm
, respectively. The second conformer
exhibits a
[Fe-CN] mode at lower frequency than the
[Fe-C-N] mode, and is thus characteristic of a bent
Fe-C-N linkage. The
[Fe-CN] and
[Fe-C-N]
modes were identified at 349 and 445 cm
,
respectively, for BLC-CN, and at 350 and 456 cm
,
respectively, for ANC-CN. The two conformers persist in the pH range
4.9-11.5. Furthermore, upon raising the pH to 11.5, the
[Fe-CN] mode of the linear conformer of BLC-CN
downshifts to 429 cm
while that of the bent
conformer remains unchanged. The observed pH-dependent shift is
attributed to the deprotonation of a distal-side amino acid residue,
probably a distal histidine. The Fe-C-N axial vibrations of the two
conformers identified for ANC-CN did not show any significant
pH-dependent shifts, indicating a more stable hydrogen bonding
interaction relative to BLC-CN.
O
:
H
O
-oxidoreductase) is a heme protein found in
nearly all aerobically respiring organisms. Although catalases can
catalyze the peroxidative oxidation of a wide range of organic and
inorganic substrates, its primary function is believed to be to protect
the cell components from the hazardous effects of hydrogen peroxide by
catalyzing its dismutation according to the reaction: 2
H
O
2 H
O +
O
. Catalases have been isolated from a wide range of
organisms, including mammals (Sumner and Dounce, 1937), plants
(Galston, 1957), fungi (Scott and Hammer, 1960), and bacteria (Herbert,
1948). Most isolated catalases are tetramers of noninteracting subunits
with one heme group per subunit. The crystal structure has been solved
and refined for three catalases: bovine liver catalase
(BLC)
(
)
(Murthy et al., 1981),
Penicillium vitale catalase (PVC) (Vainshtein et al.,
1986), and Micrococcus lysodeikticus catalase (MLC) (Murshudov
et al., 1992) at 2.5-, 2.0-, and 1.5-Å resolution,
respectively. The crystal structure of the three catalases are very
similar, the heme groups of each subunit being deeply buried in a
nonpolar cavity formed by single polypeptide chains. The most
distinctive feature of the catalase structure is the coordination of a
phenolate group of the proximal tyrosine residue to the heme iron as a
fifth ligand. Interestingly, attempts to model catalase activity with
simple iron porphyrin compounds indicate that axial ligands with an
oxygen donor ( e.g. phenolate, tyrosinate, alcoholate ligands)
are less efficient in catalyzing the dismutation of hydrogen peroxide
than ligands with nitrogen donors ( e.g. imidazole) (Belal
et al., 1989; Robert et al., 1991). However, the
coordination of these nitrogen donor ligands to the heme enhances not
only the catalase activity but also the oxygenase activity, which is
destructive for the distal amino acid residues of the protein (Belal
et al., 1989; Robert et al., 1991). This
oxygenase activity is quite small for oxygen donors, but they still
maintain reasonable catalase activity.
-cation radical, which is produced by the heterolytic
cleavage of the O-O bond of hydrogen peroxide and it is well
established that the active sites of peroxidases and catalases contain
structural elements which facilitate the transition state charge
separation and promote the heterolytic O-O bond cleavage (Fita and
Rossman, 1985a; Finzel et al., 1984; Poulos, 1987). Recently,
it has been reported that BLC binds four molecules of NADPH very
tightly (Kirkman and Gaitani, 1984; Fita and Rossman, 1985b). Although
the removal of the bound NADPH molecules does not eliminate the
catalase activity, a significant loss of activity was observed upon
lengthy exposure to H
O
. It has been proposed
that binding of NADPH circumvents the inactivation of catalase by its
natural substrate, hydrogen peroxide, by preventing the accumulation of
compound II (Kirkman et al., 1987).
Materials and Methods
BLC powder was purchased
from Sigma and purified by standard methods (Browett and Stillman,
1979; Eglinton et al., 1983; Kikuchi-Torii et al.,
1982). BLC was dissolved in deionized water and centrifuged. The deep
green solution was dialyzed for 6 h against deionized water and for 12
h against 20 mM potassium phosphate buffer (pH 7). The BLC
solution was then loaded onto a DE-52 (Whatman) column (2.5 25
cm) equilibrated with 20 mM potassium phosphate buffer (pH 7).
The column was washed with 100-200 ml of the same buffer and
eluted with a linear gradient consisting of 300 ml of 20 mM
and 300 ml of 300 mM potassium phosphate buffer (pH 7). The
BLC fractions having an Rz
( A
/ A
) value greater than
0.9 were pooled and used for spectral measurements.
C
N,
K
C
N, and K
C
N at 99%
enrichment were obtained from Isotech Inc. (Miamisburg, OH). The
various isotopomeric forms of cyanoferric ANC and BLC were prepared by
mixing 0.15 ml of catalase solution (approximately 0.1 mM in
heme) with 0.05 ml of the isotopomeric cyanide solution (160
mM).
Spectroscopic Measurements
The RR spectra were
acquired with a Spex 1269 spectrometer (mechanical slit width of 75
µm) equipped with a Princeton Instruments ICCD-576 uv-enhanced
detector and a 413.1-nm notch filter (Kaiser, Ann Arbor, MI). The
413.1-nm laser line was from a Coherent Innova model 100-K3 Krypton ion
laser. The spectra were collected with the back scattering geometry
from 5-mm NMR tubes. The NMR tube containing the sample was spun during
illumination to avoid local heating. In all cases a gradually
increasing background was observed which became more serious at high pH
values, especially for BLC. The frequencies of the severely overlapping
isotope-sensitive features were estimated using the comparison of the
difference spectra obtained by subtraction of absolute experimental
spectra for different isotopes with computer-simulated difference
spectra (see below).
Computer Simulations
To obtain a more reliable
estimate of the actual vibrational parameters of individual modes from
the difference spectra, the following approach has been used.
Initially, the idealized (built of ideal, 50/50, Gauss-Lorentzian
peaks) absolute spectra are simulated by computer. These simulated
spectra are then subtracted and the difference spectra obtained in this
way are compared with their experimental analogues. In the next step
the idealized absolute spectra are slightly perturbed (by allowing
frequency, band width, and intensity of peaks to vary) and the new set
of simulated difference spectra is compared with the set of
experimental difference spectra. The perturbation process is repeated
iteratively until the discrepancy between the simulated and
experimental difference spectra is minimized. The computer program
based on this strategy has been locally generated.(
)
In this program the quantitative measure of the difference
between the simulated and the experimental data ( D) is defined
by the following equation.
of those summarized in .
BLC-CN
Fig. 1
shows the RR spectra of the various
isotopomeric forms of BLC-CN. To help identify the Fe-C-N modes, we
digitally generated difference spectra (Fig. 2) by the
subtraction of the various pairs of spectra shown in Fig. 1.
Inasmuch as the Fe-C-N axial modes overlap, and the difference features
in Fig. 2therefore do not necessarily correspond to the absolute
frequencies, we have developed a simulation procedure described above
which allows us to estimate the absolute frequencies of the peaks
contributing to the experimental difference spectra. Fig. 3shows
the results of computer simulation of experimental difference spectra.
The peak positions estimated from the fitting of the difference spectra
between 340 and 460 cm for the CN
adducts of BLC are summarized in .
(
)
An easily identified isotope-sensitive mode in the spectrum
of natural abundance BLC-CN can be seen at 433 cm
in
Fig. 1
, a mode that is absent in the RR spectra of the native
BLC. This mode exhibits a monotonic isotope shift pattern (Yu et
al., 1984) as the total mass of the cyanide isotopomer is
increased, shifting to 431 cm
(
C
N
), 430
cm
(
C
N
),
and 429 cm
(
C
N
). A feature that
corresponds to this mode can be seen in Fig. 2. On the basis of
this isotope shift pattern we assign this mode to the
[Fe-CN] stretching vibration in BLC-CN (Yu et
al., 1984). In Fig. 1, there is an apparent
isotope-sensitive intensity fluctuation observed in the region between
400 and 416 cm
. Upon labeling the carbon atom of the
bound cyanide, the mode at 416 cm
decreases in
intensity and shifts 4-5 cm
to higher
frequency. Simultaneously, a shoulder at 404 cm
appears only when the carbon atom of the bound cyanide is
labeled. The digitally subtracted difference spectra, given in
Fig. 2
, clearly document the presence of this isotope-sensitive
mode. Traces `` c-b,'' ``a-b,'' ``a-d,'' and `` c-d'' of Fig. 2,
which are obtained by the subtraction of spectra of BLC-CN isotopomers
in which the carbon atom of the ligated cyanide is labeled from spectra
of cyanide adducts in which the carbon atom is not labeled, show
difference features at 412 and 402 cm
. This mode
cancels out completely in traces ``a-c'' and
``b-d,'' which are obtained by the subtraction of a
pair of spectra corresponding to the isotopomers in which the carbon
atoms have the equal masses. As is clear from inspection of Figs. 2 and
3 this behavior results from the presence of a band which exhibits a
zigzag isotopic shift pattern, shifting (based on the simulation) from
413 cm
(
C
N
), to 402
cm
(
C
N
),
412 cm
(
C
N
), and 401
cm
(
C
N
).
Accordingly, we assign this mode to a
[Fe-C-N] bending
vibration (Yu et al., 1984).
Figure 1:
Low frequency RR spectra of the
different isotopomeric forms of the cyanide adducts of bovine liver
catalase at pH 7.
Figure 2:
Difference spectra generated by the
digital subtraction of the various spectra shown in Fig.
1.
Figure 3:
Difference
spectra of the BLC-CN at pH 7 generated by the simulation
procedure.
Recently, we reported that
cyanide binds to ferric HRP in two geometries (Al-Mustafa and Kincaid,
1993). Therefore, we examined the spectra for evidence of heterogeneity
in binding of cyanide to ferric BLC. Fig. 2reveals the presence
of an isotope-sensitive mode hidden by the strong porphyrin mode
() at 349 cm
. This mode can be seen
in all traces of Fig. 2with the exception of trace c-b.
These difference spectra were generated by the subtraction of spectra
of isotopomeric forms which differ in the total mass of the ligated
cyanide, while it cancels out completely in trace c-b, which
was obtained by the subtraction of the spectrum of the
C
N
adduct from that of the
C
N
adduct ( i.e. those which have equal total masses). These data thus document the
presence of a mode which shifts monotonically with increase of the
total mass of the cyanide ligand, regardless of the position of
labeling (Yu et al., 1984). The overlap of this band with an
intense porphyrin mode (
) hinders the determination of
the exact frequency of this mode. However, with the help of the
simulation procedure and using the difference spectra shown in
Fig. 2
, we estimate that this mode occurs at 349 cm
for the
C
N
adduct and
shifts to 345 cm
for the
C
N
adduct. Accordingly, we
assign this mode to the
[Fe-CN] stretching mode of a
second conformer of BLC-CN (Han et al., 1989; Lopez-Garriga
et al., 1990; Hu et al., 1993; Al-Mustafa and
Kincaid, 1993).
. On the other hand, traces a-c and
b-d of Fig. 2, which should show only the stretching
modes (the bending modes should cancel out) yield a symmetrical
difference feature in the same region. Therefore, we conclude that the
asymmetry of the difference features in traces a-b, a-d, and
c-d of Fig. 2, in the region around 430
cm
, is due to the combination of two overlapping
difference features; a feature that is ascribable to the monotonically
shifting mode at 433 cm
mentioned earlier and
assigned to a stretching of
[Fe-CN], and a second mode
of slightly higher frequency that exhibits a zigzag isotopic shift
pattern. In trace c-b of Fig. 2, which is obtained by
the subtraction of isotopomeric forms that have equal total masses but
differ in the mass of the carbon atoms, should reveal only the bending
vibration ( i.e. the stretching vibrations should cancel out).
This trace clearly reveals the presence of two bending vibrations; the
feature near 410 cm
mentioned earlier, and another
bending mode near 440 cm
. The numerical fitting
yielded two simulated bands, having a zigzag pattern, in these regions.
One mode at 413 cm
(
C
N
) shifting to 401
cm
(
C
N
)
and a second mode at 445 cm
(
C
N
) that shifts to
435 cm
(
C
N
).
[Fe-CN] and
[Fe-C-N]
vibrations and drives them apart relative to the linear form.
Therefore, we assign the stretching mode at 434 cm
and the bending mode at 413 cm
to a conformer
of ferric BLC-CN which possesses an essentially linear Fe-C-N linkage
and the stretching and the bending modes at 349 cm
and 445 cm
, respectively, to a second
conformer having a more bent Fe-C-N linkage.
in
Fig. 1
appears to be composed of two modes; one of which is a mode
at 380 cm
which exhibits a small (2
cm
) but definite downshift. The observation of sharp
difference features near 380 cm
in the difference
spectra in Fig. 2confirms the isotope sensitivity of this mode.
The behavior of this component upon isotopic substitution is similar to
a mode observed at 378 cm
in the RR spectra of
cyanide adducts of HRP (Al-Mustafa and Kincaid, 1993). We
assign this vibration, as it was assigned in the case of HRP-CN, to an
out-of-plane heme core mode or possibly a peripheral substituent mode
and we attribute the observed shifts to the coupling of this mode to
the Fe-C-N vibrations, which are also out-of-plane in nature. The
second component can be seen as a shoulder at 385 cm
which does not show any isotope dependence.
ANC-CN
The RR spectra of ferric ANC-CN obtained at
pH 4.9 and their difference spectra are shown in Figs. 4 and 5,
respectively. Fig. 4shows an isotope-sensitive mode at 433
cm (
C
N
),
432 cm
(
C
N
), 431
cm
(
C
N
),
and 429 cm
(
C
N
). This band
corresponds to the 434 cm
mode observed for BLC-CN
and accordingly is assigned also to the
[ Fe-CN] mode of
a linear conformer of ANC-CN. A second isotope-sensitive mode that
exhibits a zigzag shift pattern can be seen near 410 cm
in both Figs. 4 and 5. Similarly to BLC-CN, simulations suggest
that this mode occurs at 412 cm
(
C
N
) and at 402
cm
(
C
N
)
and is assigned to the
[Fe-C-N] bending vibration of the
linear conformer of ANC-CN.
Figure 4:
Low frequency RR spectra of the different
isotopomeric forms of the cyanide adducts of A. niger catalase
at pH 4.9.
As in the case of BLC-CN, the spectra of
ANC-CN reveal the presence a second conformer with a reversed ordering
of the stretching and bending frequencies. The difference spectra in
Fig. 5
reveal the presence of a monotonically shifting mode near
350 cm (
C
N
), shifting to 347
cm
(
C
N
)
and it is assigned to the
[Fe-CN] stretching vibration
of a second, bent, conformer of ANC-CN. The major difference between
the spectra of BLC-CN and ANC-CN is the frequency of the bending mode
identified at 445 cm
in BLC-CN. This mode appears to
shift to a slightly higher frequency in ANC-CN as is indicated by the
appearance of a new positive component at about 456 cm
in traces a-b, a-d, and c-d of
Fig. 5
. Also trace c-b of Fig. 5, which is
expected to exhibit only the difference features associated with the
bending vibrations of ferric ANC-CN, shows a difference feature at 455
and 443 cm
slightly higher than BLC-CN (444 and 436
cm
). The absolute spectra of ANC-CN in
Fig. 4
show a weak feature at 456 cm
(
C
N
,
C
N
) that apparently
disappears, most likely shifting underneath the strong band at
431-429
cm
(
C
N
,
C
N
). Accordingly, it is
assigned to the
[Fe-C-N] bending vibration of the second
form of ferric ANC-CN. We assign the features at 350 and 456
cm
to the stretching and bending vibrations of a
second conformer of ANC-CN which exists in a bent Fe-C-N geometry. The
results of simulation (Fig. 6) of the experimental difference
spectra suggest another significant difference in the spectral behavior
of BLC-CN and ANC-CN besides the upshifting of the bending mode from
445 cm
to 456 cm
; i.e. the isotope shift upon
C substitution for the
[Fe-C-N] of the bent conformer has been found to be
10 cm
for BLC-CN and
15 cm
in the case of ANC-CN. The
10 cm
isotopomeric shift found in the case of BLC-CN is in reasonable
agreement with the experimental data reported for related systems,
where the shift varied between 5 and 8 cm
(Lopez-Garriga et al., 1990; Han et al., 1989;
Hu et al., 1993). pH Effect
Figure 5:
Difference spectra generated by digital
subtraction of the various spectra in Fig.
4.
Figure 6:
Difference spectra of ANC-CN at pH 4.9
generated by the simulation procedure.
In order to investigate the hydrogen bonding interactions of the
bound cyanide with the active site distal environment, we have studied
the RR spectra of BLC and ANC in the pH range 4.9-11.5. Analysis
of the high and low frequency RR spectra of the native form (spectra
not shown) and the cyanide-ligated forms indicates no significant
denaturation for either protein. However, at pH values slightly below
5, BLC starts to precipitate while ANC remains soluble (possibly
because ANC is a glycoprotein containing about 9% carbohydrate while
BLC does not contain any carbohydrates) (Kikuchi-Torii et al.,
1982). The fluorescence from the catalase samples increases
significantly as the pH of the medium is raised above 10, especially
for BLC. Nevertheless RR of adequate quality spectra can be obtained at
pH values as high as 11.5.
[Fe-CN] stretching frequency of the linear conformer.
The feature which occurs at 433 cm
at pH 7.0
(Fig. 1) shifts to 429 cm
at pH 11.5.
Therefore, we assign the feature at 429 cm
to the
[Fe-CN] mode of the alkaline form of the linear
conformer. It is clear that changing the pH in the range 4.9-11.5
does not eliminate the heterogeneity as indicated by the observation of
two difference features near 430 and 350 cm
in the
difference traces shown in Fig. 8. Two additional minor
downshifts can be noticed comparing the difference traces at pH 11.5
(Fig. 8) to the traces at pH 7.0 (Fig. 2). One is the small
2-4 cm
downshift of the positive feature at
about 440 cm
and the second is the
4
cm
downshift of the negative feature at about 400
cm
.
Figure 7:
Low
frequency RR spectra of the ANC-CN adducts at pH 11.5 in 250 mM carbonate buffer.
Figure 8:
Difference spectra generated by the
digital subtraction of the various spectra shown in Fig.
7.
RR investigation of the cyanide adducts of
ferric ANC at pH 11.5 (not shown) yield spectra practically identical
to those obtained at pH 4.9. Also the difference spectra (Fig. 9)
show no observable differences compared to traces obtained at pH 4.9
(Fig. 5). Thus, we detect no significant shifts in axial
vibrational modes of ANC-CN in the pH range 4.9-11.5.
Figure 9:
Difference
spectra generated by the digital subtraction of the low frequency RR
spectra (not shown) of the different isotopomeric forms of ANC-CN
adducts at pH 11.5.
The
absolute frequencies for the CN adducts of both BLC-CN and ANC-CN, at
both high and low pH, are summarized in . The values of
frequencies, band widths, and relative intensities at low pH for both
adducts have been obtained from the computer simulation of the
experimental difference spectra. The frequencies shown for both BLC-CN
and ANC-CN at pH 11.5 have been estimated by comparing their difference
traces with corresponding low pH analogue and using the information
from the low pH simulation data.
Spectral Behavior
The RR data presented for
BLC-CN and ANC-CN unambiguously demonstrate the presence two conformers
characterized by the reversal of the ordering of their
[Fe-CN] stretching and
[Fe-C-N] bending
frequencies. In the first conformer, the observed frequency of
[Fe-CN] (434 cm
) is higher than the
[Fe-C-N] (413 cm
) frequency. This
behavior has been observed previously for the cyanide adducts of
several heme proteins and model compounds (Yu et al., 1984; Yu
and Kerr, 1988; Han et al., 1989; Lopez-Garriga et
al., 1990; Tanaka et al., 1987; Uno et al.,
1988; Al-Mustafa and Kincaid, 1993) and is considered to be indicative
of an essentially linear Fe-C-N linkage. In the second conformer the
[Fe-CN] mode (349 cm
) occurs at a
lower frequency than the
[Fe-C-N] mode (445
cm
for BLC-CN and 456 cm
for
ANC-CN), behavior which also has been observed in the cyanide adducts
of several heme proteins ( e.g. myeloperoxidase (Lopez-Garriga
et al., 1990), lactoperoxidase (Hu et al., 1993),
sulfite reductase (Han et al., 1989), and HRP (Al-Mustafa and
Kincaid, 1993)) and is characteristic of a nonlinear Fe-C-N linkage.
The essential from all of these previous studies are summarized in
.
Structural Interpretation
The crystal structures
of BLC, PVC, and MLC (Murthy et al., 1981; Vainshtein et
al., 1986; Murshudov et al., 1992) reveal the presence of
a conserved essential histidine residue on the distal-side of the heme
active sites. Furthermore, kinetic studies of cyanide binding (Chance,
1952; Schonbaum and Chance, 1976) indicate that catalases bind HCN
rather than CN with the proton presumably protonating
the distal histidine, as in the case of HRP and cytochrome
c-peroxidase (Thanabal et al., 1988; Banci et
al., 1991). The observed heterogeneity can reasonably be
attributed to distinct hydrogen bonding interactions between the Fe-C-N
linkage and two different distal-side amino acid residues. Thus, the
bound cyanide can participate in a hydrogen bonding interaction with
the imidazolium group of the distal histidine, giving rise to a linear
conformer, which exhibits the characteristic stretching and bending
vibrations (434 and 413 cm
, respectively). The bent
conformer presumably arises from an off-axis hydrogen bonding
interaction with another residue. Apparently because of slight
differences in the distal-side environment, the hydrogen bonding
interactions which give rise to the bent conformers of BLC-CN and
ANC-CN appear to be somewhat different as indicated by the variation in
the
[Fe-C-N] frequency of the bent conformer (observed
at 445 and 456 cm
for BLC-CN and ANC-CN,
respectively) and the increased isotopic shift for ANC relative to BLC.
The
10 cm
upshift in the bending frequency of
the bent conformer of ANC-CN relative to that of BLC-CN can be
attributed to variation in binding geometry of the cyanide or in the
strength of the hydrogen bonding interaction with the heme distal
environment. This difference in behavior between the cyanide adducts of
ANC and BLC is consistent with the known structural differences in heme
distal between mammalian and fungal catalases (Murthy et al.,
1981; Vainshtein et al., 1986) as is discussed below.
[Fe-CN] and
[Fe-C-N] frequencies (350 and
445 cm
, respectively). Comparison of the available
crystal structures of the various catalases reveals a high degree of
similarity in their heme distal environments (Murthy et al.,
1981; Vainshtein et al., 1986). The asparagine residue
(Asn-147) found in BLC (Murthy, 1981) is conserved in both PVC
(Asn-132) (Vainshtein et al., 1986) and MLC (Asn-129)
(Murshudove et al., 1992).
pH Effects
It is generally agreed that the
distal-side of the heme active sites of catalases and peroxidases
contain two or more amino acids capable of participating in hydrogen
bonding interactions with the natural substrate, hydrogen peroxide
(Finzel et al., 1984; Fita and Rossman, 1985a; Sakurada et
al., 1986; Thanabal et al., 1988). Furthermore, it has
been suggested that these residues promote the heterolysis of the O-O
bond of HO
and the formation of the reactive
intermediate by stabilizing the developing charge separation and
facilitating the proton transfer to the terminal oxygen of
H
O
(Finzel et al., 1984; Fita and
Rossman, 1985a).
of the amino acid residue would
eliminate the hydrogen bond and consequently decrease the axial mode
frequencies (Lopez-Garriga et al., 1990; Hu et al.,
1993; Al-Mustafa and Kincaid, 1993). Comparison of Figs. 1
( trace a) and 7 shows that raising the pH from 7 to 11.5
causes a 4 cm
downshift in the
[Fe-CN] stretching frequency of the linear conformer of
BLC-CN. This response to pH changes is analogous to the downshift
observed for the linear conformer of cyanoferric HRP, and can
reasonably be ascribed to the deprotonation of the imidazolium group of
the distal histidine (Al-Mustafa and Kincaid, 1993). In contrast to the
behavior observed for cyanoferric HRP, the
[Fe-CN]
vibration of the bent conformer does not show any significant
pH-dependent shift in this pH range. This behavior is not only
consistent with hydrogen bonding interaction with the distal asparagine
(which is not deprotonated at this pH), but also indicates that the
distal- and the proximal-sides of the heme active site in catalases
function independently. This behavior is different from that observed
in HRP and other peroxidases wherein deprotonation of the distal
histidine causes a corresponding change in the trans-axial bond
strength (Teroaka and Kitagawa, 1981).
Comparison with Cyanide Adducts of HRP and Other Heme
Proteins
Recently we reported the results of a RR investigation
of the cyanide adducts of HRP (Al-Mustafa and Kincaid, 1993). Although
HRP and the two catalases studied in this report exhibit evidence for
the existence of two conformers (linear and bent), several significant
differences can be noted. First, the [Fe-CN] stretching
vibration (
434 cm
) of the linear conformers of
catalases are considerably lower than that observed for the linear
conformer of HRP and other heme proteins that bind cyanide in an
essentially linear configuration (456 cm
)
(). In fact, this is the lowest reported
[Fe-CN] stretching frequency identified for a linear
heme protein cyanide adduct. Also the
[Fe-CN] modes of
the bent conformer of both BLC-CN and ANC-CN (about 350
cm
) are 10 cm
lower than that of
HRP-CN (360 cm
) and are virtually identical to that
of the bent conformer of the one-electron reduced sulfite reductase
(352 cm
) which also possesses a negatively charged
proximal ligand ( i.e. thiolate) (Han et al., 1989).
Second, the magnitudes of the observed isotope-dependent shifts of
stretching modes for linear conformers of the catalases are apparently
slightly smaller than that observed for the linear conformer of HRP
(substitution of
C
N
for
C
N
yields 4-6
cm
shifts for the catalases but an 8 cm
shift for HRP-CN). Third, we reported for HRP-CN that the
deprotonation of a single amino acid leads to downshifts of the
[Fe-CN] for both the linear conformer (attributed to the
direct effect of abolishing the hydrogen bond with bound cyanide) and
for the bent conformer (through the indirect alteration of the proximal
Fe-N
(His) bond strength) (Al-Mustafa and Kincaid,
1993). However, in BLC-CN only the
[Fe-CN] mode of the
linear conformer downshifts upon raising the pH while that of the bent
form remains unchanged.
[Fe-CN]
stretching vibration ( e.g. axial ligation, hydrogen bonding,
and the variation in the degree of tilting or bending). A major factor
that might contribute to the lowering of the
[Fe-CN] is
the unique distal-side structure of the heme active site in catalase.
The crystal structures of the three catalases (Murthy et al.,
1981; Vainshtein et al., 1986; Murshudov et al.,
1992; Fita and Rossman, 1985a) reveal that the essential distal
histidine (Margoliash et al., 1971) is almost parallel to the
heme plane. This orientation of the distal histidine is different from
the orientation found in cytochrome c-peroxidase (Finzel
et al., 1984) and that expected for HRP (Sakurada et
al., 1986). Thus, the steric and hydrogen bonding interactions
between the bound cyanide and the distal histidine in catalases are
expected to increase the tension on the Fe-C-N linkage, increasing the
off-axis distortion relative to that of HRP. This should lead to
further lowering of the
[Fe-CN] stretching frequency,
and to the observed decrease in the magnitude of the isotopic shift in
catalase relative to those observed for the cyanide adducts of HRP and
sulfite reductase (Al-Mustafa and Kincaid, 1993; Han et al.,
1989). Another distinctive feature of the catalase active site
structures is the coordination of a phenolate oxygen from an endogenous
tyrosine residue to the heme iron (Murthy et al., 1981;
Vainshtein et al., 1986; Murshudov et al., 1992). It
has been shown that increasing the basicity of the trans-axial ligand
strength in the cyanide-ligated FeOEP complexes leads to a
corresponding decrease in the
[Fe-CN] stretching
frequency (Uno et al., 1988). Similar behavior has been
detected for the CO adducts of model compounds, in studies which
demonstrate that the
[Fe-CO] frequency is highest when
the trans-axial ligand is very weak or absent (Kerr et al.,
1983). Recently, for the CO adducts of BLC, we reported (Hu and
Kincaid, 1992) the observation of the
[Fe-CO] and the
[C-O] at 542 and 1908 cm
,
respectively. These values are comparable to those reported for the CO
adducts of HRP (537 and 1904 cm
, respectively
(Evangelista-Kirkup et al., 1986) and 541 and 1906
cm
(Uno et al., 1987)). Furthermore, the
reported
[Fe-CO] and
[ C-O] values
correlate with those of other heme proteins and model compounds
containing a nitrogenous trans-axial ligand, indicating that the
-donating effect of the proximal phenolate oxygen in catalase is
comparable to that of the imidazole found in HRP and other heme
proteins (Yu et al., 1988; Li and Spiro, 1988). Therefore, the
difference in the proximal ligands between catalase and HRP is not
expected to contribute significantly to the lowering of the
[Fe-CN] frequencies of catalases.
(His) bond strength (Teroaka and Kitagawa, 1981;
Thanabal et al., 1988). The 4 cm
pH-induced
shift observed for the
[Fe-CN] mode of the bent
conformer of HRP-CN is attributable to a change in the trans-axial
ligand bond strength which is triggered by the deprotonation of the
distal histidine (Teroaka and Kitagawa, 1981). The lack of any
measurable shift in the
[Fe-CN] mode of the bent
conformer of BLC-CN implies that the distal- and the proximal-sides in
catalase function independently.
Table: Axial mode frequencies
(cm), band widths (FWHM in
cm
) and relative intensities
obtained by computer simulation of difference spectra of cyanoferric
HRP with different labeled cyanide isotopomers
Table: The comparison of
cyanide adducts of various heme proteins
, E. coli sulfite reductase in its oxidized
form; MetHb CTT, hemoglobin III from Chironomus thumni thumni;
LPO, lactoperoxidase; MPO, myeloperoxidase, CCP, cytochrome c
peroxidase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.