(Received for publication, December 1, 1995; and in revised form, February 15, 1996)
From the
The diheme cytochrome c peroxidase from Paracoccus
denitrificans was modified with the histidine-specific reagent
diethyl pyrocarbonate. At low excess of reagent, 1 mol of histidine was
modified in the oxidized enzyme, and modification was associated with
loss of the ability to form the active state. With time, the
modification reversed, and the ability to form the active state was
recovered. The agreement between the spectrophotometric measurement of
histidine modification and radioactive incorporation using a
radiolabeled reagent indicated little modification of other amino
acids. However, the reversal of histidine modification observed
spectrophotometrically was not matched by loss of radioactivity, and we
propose a slow transfer of the ethoxyformyl group to an unidentified
amino acid. The presence of CN bound to the active
peroxidatic site of the enzyme led to complete protection of the
essential histidine from modification.
Limited subtilisin treatment of the native enzyme followed by tryptic digest of the C-terminal fragment (residues 251-338) showed that radioactivity was located in a peptide containing a single histidine at position 275. We propose that this conserved residue, in a highly conserved region, is central to the function of the active mixed-valence state.
Bacterial cytochrome c peroxidases (CCPs) ()are diheme enzymes that oxidize monoheme Class I
cytochromes c and reduce hydrogen peroxide to water. The most
extensively characterized example is from Pseudomonas aeruginosa and contains a high potential, electron-transferring heme and a
low potential, peroxidatic heme(1, 2) . In the
oxidized state, the enzyme is inactive(1, 3) ; the
high potential heme is partially high spin, and the low potential heme
is low spin and inaccessible to ligands(2) . Reduction of the
high potential heme causes the low potential heme to become high spin
and enables binding of hydrogen peroxide(4, 5) . We
have studied the enzyme from Paracoccus denitrificans and have
found very similar characteristics(6, 7) , although a
distinctive difference is the Ca
requirement in P. denitrificans CCP for the low to high spin transition at
the peroxidatic heme(8) .
The amino acid sequence of P. aeruginosa CCP suggested that the molecule was constructed in two domains, each having the general appearance of a monoheme Class I cytochrome c(9) . This family of cytochromes is characterized by a heme attachment site and proximal histidine ligand near the N terminus and a distal methionine ligand near the C terminus. Ellfolk et al.(9) proposed that the C-terminal domain followed this pattern and acted as the high potential electron-transferring site. The N-terminal domain appeared to lack an appropriately placed methionine, and Ellfolk et al. proposed that the sixth iron ligand of this domain was a histidine donated from the C-terminal domain and conferring a low redox potential. In the mixed-valence form of the enzyme, this histidine would be released from the iron, the heme would become high spin, and hydrogen peroxide could enter the active site.
The amino acid sequence of P. aeruginosa CCP has been revised on the basis of the gene sequence determined
by Ridout et al.(10) , and the key histidine in the
proposal of Ellfolk et al.(9) is His-261. We have
determined the amino acid sequence of P. denitrificans CCP, ()which shows 61% identity to the P. aeruginosa enzyme. The histidine in P. denitrificans CCP
corresponding to His-261 in P. aeruginosa CCP is at position
275.
During the preparation of this paper, the crystallographic structure of the oxidized form of the P. aeruginosa enzyme was determined (11) . In contrast to the original proposal of Ellfolk et al.(9) , the ligand to the proposed N-terminal peroxidatic heme is, in fact, His-71 (position 85 in P. denitrificans CCP numbering), and His-261 is situated at the interface of the two heme domains.
This paper describes the modification of P. denitrificans CCP with the histidine-selective reagent diethyl pyrocarbonate. We show that modification of His-275 in the oxidized form of the enzyme completely abolishes the ability of the enzyme to form the active mixed-valence state.
Figure 3:
Modification of a single histidine from P. denitrificans CCP abolishes catalytic activity and the
ability of the enzyme to form the mixed-valence high spin state. a, loss of ability to form the characteristic high spin 380 nm
band on modification of a single histidine. CCP (4 µM) in
5 mM Hepes, pH 7.5, was modified for 20 min with DEPC at the
following concentrations: 0, 1.5, 2.8, 5.6, 8.4, 12.2, 16.4, and 24.6
µM. The extent of modification for each DEPC concentration
was calculated to be 0, 0.12, 0.27, 0.46, 0.58, 0.68, 0.86, and 1.00
mol of histidine modified per mol of protein, respectively. The ability
of each sample to form the mixed-valence high spin state was tested by
difference spectroscopy as described under ``Experimental
Procedures.'' Each difference spectrum with decreasing
A
correlates to the increased extent of
modification as listed above. b, loss of ability to form the
380 nm band in the mixed-valence enzyme and loss of catalytic activity
as histidine modification increases. The percent high spin of each
sample was calculated from the changes at 380 nm in a.
Aliquots of each sample were assayed, and activity was determined (see
``Experimental Procedures'').
Figure 7:
Identification of the
Arg-251-Met-338 peptide as the site of modification. CCP (20
µM) in 10 mM Hepes, pH 7.5, was modified to an
extent of 0.9 mol of histidine modified per mol of protein with
[C]DEPC (100 µM). The modified
sample was desalted through a small Sephadex G-25 column (12
1
cm), equilibrated in 10 mM Hepes, pH 7.5, before being
digested with subtilisin. To CCP (4-20 µM) in 10
mM Hepes, pH 7.5, were added 1 mM ascorbate, 10
µM diamino durol, and 1 mM CaCl
.
Subtilisin (Carlsberg) was added to give a substrate/enzyme ratio of
500:1 (w/w). After 60 min at 0 °C, subtilisin was inhibited by
1 mM phenylmethanesulfonyl fluoride, 10 mM EGTA. The
digest was made to 1% in SDS and loaded onto a Pharmacia Superdex 75
column equilibrated in 20 mM Tris, pH 7.3, 100 mM NaCl. The eluate was monitored at 280 nm, and the resultant two
main peaks were collected. The protein composition of the two peaks was
determined by Coomassie Blue staining after SDS electrophoresis on a
15% polyacrylamide gel. The remainder (two-thirds) of the two peaks was
used for radioactive counting. The distribution of label between the
two fractions is shown in the histogram. The contribution of labeled
undigested protein to the fraction containing the Glu-1-Thr-250
peptide was deducted to give a corrected dpm. Lane 1, M
standards; lane 2, undigested CCP; lane 3, subtilisin digest; lane 4, peak 1 from the
Superdex G-25 column; lane 5, peak
2.
Mass spectrometry was
performed using a BioQ triple quadrupole mass spectrometer equipped
with a pneumatically assisted electrospray source (Fisons, Altrincham,
United Kingdom) operating in positive ion mode. The resolution of the
mass spectrometer allows for isotopic resolution of peptides under M
3000. By averaging the mass of elemental
isotopes according to their natural distribution and thus determining
the average mass of individual residues, the theoretical M
values of peptides over 3000 were calculated.
Figure 1:
UV difference spectra of the
modification of imidazole, P. denitrificans CCP, and yeast CCP
with diethyl pyrocarbonate. A: panel i, P.
denitrificans CCP (3.5 µM) in 5 mM Hepes, pH
7.5, was modified with DEPC to a final concentration of 20
µM. After 2 min, a modified CCP minus unmodified CCP
difference spectrum was taken and showed 0.57 mol of histidine modified
per mol of protein, based on = 3.2
mM
cm
for
ethoxyformylhistidine. Panel ii, the reaction of DEPC with P. denitrificans CCP was complete within 20 min and showed 1.0
mol of histidine modified per mol of protein. B: 5 mM imidazole in 5 mM Hepes, pH 7.5, was incubated with 4
µM DEPC. The difference spectrum obtained at 2 min showed
3.8 µM ethoxyformylimidazole (
= 3.0 mM
cm
). The spectrum did not change in the
subsequent 20 min. C: yeast CCP (3.8 µM) in 5
mM Hepes, pH 7.5, was modified with DEPC to a final
concentration of 20 µM. After 15 min, a difference
spectrum was taken and showed 1.1 mol of histidine modified per mol of
protein. The wavelengths of individual peak maxima are indicated on the
respective spectra.
The difference spectrum of the modified P. denitrificans CCP displays an initial gain of absorbance at 245 nm (Fig. 1A, panel i), followed by slow formation of a trough between 265 and 300 nm (panel ii). Modification of free imidazole generated the ethoxyformylimidazole derivative, which gave a peak maximum at 233 nm, but no corresponding changes at higher wavelengths (Fig. 1B). As will be discussed below, the loss of ability to form the active state of P. denitrificans CCP is correlated with absorbance gain at 245 nm and not with the presence of the 265-300 nm features.
The modified
yeast CCP difference spectrum (Fig. 1C) is very similar
to the modified P. denitrificans CCP spectrum. For both
proteins, a DEPC/protein ratio of 20 µM:4 µM resulted in modification of 1 mol of histidine/mol of
protein. This was calculated using the extinction coefficient of
ethoxyformylhistidine at 245 nm of 3.2 mM
cm
(16) . A possible source of error
for this calculation is the effect of spectral features near the peak
at 245 nm. For example, the trough at 265-300 nm in the modified
difference spectra of bacterial and yeast CCPs may have a
``pulling down'' effect on the 245 nm peak. An independent
check on the extent of modification was made by measuring the
incorporation of [
C]DEPC. These experiments have
determined that, in general, there is good agreement between the two
methods of quantitation, with radioactive labeling giving slightly less
(
10%) than spectroscopic measurements. This indicates first that
A
is a reliable measurement of histidine
modification and second that there is little uptake into amino acids
other than histidine.
Fig. 2a shows the extent of
modification of P. denitrificans CCP as DEPC concentration was
raised. At each DEPC concentration, the modification was allowed to
proceed until the absorbance maximum at 245 nm was obtained (20
min). The biphasic character of the plot of DEPC demonstrates the
existence of an easily modifiable histidine. Eventually, at high DEPC
concentrations, an apparent maximum of four histidines are modified
(data not shown). We expected a maximum of three histidines to be
available for modification since the remaining three are assumed to be
coordinated to heme (two as proximal ligands and one as a distal ligand
for the peroxidatic heme). The discrepancy that we see in our measured
maximum value of four may be due to bis-modification of
histidine(22) . A similar biphasic pattern was obtained by
following the extent of modification at a single high DEPC
concentration (data not shown). A rigorous interpretation of the
results was complicated by the fact that the final extent of
modification is the result of the rate of modification, the rate of
ethoxyformylhistidine reversal, and the rate of hydrolysis of the
reagent. Fig. 2b compares the extent of modification at
pH 6 and 7.5, and it indicates that much higher DEPC concentrations are
required at pH 6 to achieve modification of 1 mol of histidine, a
result consistent with the requirement that histidine must be
unprotonated to be modified(23, 24) . We chose pH 7.5
for subsequent studies.
Figure 2:
Extent of modification of P.
denitrificans CCP with increasing concentrations of DEPC. a, P. denitrificans CCP (4 µM) in 5
mM Hepes, pH 7.5, was treated with increasing concentrations
of DEPC, and the reactions were allowed to proceed to completion at
each concentration. The moles histidine modified were calculated using
= 3.2 mM
cm
. At a concentration of 8 mM DEPC,
an apparent maximum of four histidines were modified (data not shown on
figure). The boxed area indicates a subset of points used in b. b, shown is a comparison of histidine modification
of CCP at pH 6 and 7.5. Modification of CCP (4 µM) at pH 6
was carried out in 5 mM Mes.
Fig. 3a demonstrates the loss of ability to form the high spin 380 nm band in the mixed-valence enzyme after modification of histidine in the oxidized state. These data are plotted in Fig. 3b along with loss of catalytic activity upon modification of histidine. Thus, modification of a single histidine in P. denitrificans CCP abolishes both the ability to form the high spin state and activity. Although a single histidine was also readily modified in yeast CCP at similarly low DEPC/protein ratios, modification did not cause loss of activity.
A further spectroscopic indicator of the active enzyme is
the Ca-dependent appearance of a g
= 2.89 signal in the EPR spectrum of the mixed-valence
enzyme, which can be equated with the high spin peroxidatic heme at
room temperature (Fig. 4a). Because of the high protein
concentrations needed for EPR spectroscopy, endogenous Ca
allows partial appearance of this g
= 2.89 signal in the spectrum of the mixed-valence without
added Ca
. The enzyme modified at a single histidine
cannot undergo the transition to the active form (Fig. 4b) and is mostly trapped in a mixed-valence low
spin form with g
= 3.00.
Figure 4:
EPR spectroscopy of modified and native P. denitrificans CCPs. a, EPR spectra of native CCP
in oxidized, mixed-valence, and mixed-valence + Ca states. Nine ml of CCP (4 µM) in 5 mM Hepes, pH 7.5, were concentrated to 180 µl, and spectra were
recorded from this 200 µM sample. b, EPR spectra
of modified CCP in oxidized, mixed-valence, and mixed-valence +
Ca
states. To 9 ml of CCP (4 µM) in 5
mM Hepes, pH 7.5, was added DEPC to a final concentration of
20 µM, which resulted in 1.1 mol of histidine modified per
mol of protein. The solution was concentrated, and spectra were
recorded as described for the native sample. Experimental conditions
were as follows: temperature, 7.5 K; microwave frequency, 9.65 GHz;
microwave power, 2 milliwatts; modulation, 1 millitesla; and gain, 1.6
10
. g values for selected resonances are
shown.
Our difference spectrum due to modification at pH 7.5 showed the presence of a shallow trough between 265 and 300 nm (Fig. 1A, panel ii). We wanted to be certain that this was not an indicator of modification of aromatic acids such as tyrosine or tryptophan, which may affect the activity of the protein. A difference spectrum (Fig. 1A, panel i) obtained after modification for 2 min showed no trough between 265 and 300 nm, and yet the histidine modified correlated well with the proportion of protein unable to form the high spin state (data not shown). Also, during reversal of modification, the 245 nm peak is gradually lost, but the 265-300 nm trough remains (Fig. 5a). Again, the loss of histidine modification correlates well with the recovery of the ability to form the active high spin state (Fig. 5b). The reversal of modification does not follow a simple exponential; in 5 mM Hepes, pH 7.5, 50% of the label is lost in 6 h. Taken together, these results indicate that the appearance of the 280 nm trough is not associated with changes in the activity of the enzyme and that those changes we observed are due to histidine modification.
Figure 5: Loss of ethoxyformylhistidine with time correlates with regain of active enzyme. a, difference spectra of modified minus unmodified CCP were taken at different times after DEPC addition. CCP (12 µM) in 5 mM Hepes, pH 7.5, was modified with DEPC to a final concentration of 45 µM. The absorbance at 245 nm was maximal at 20 min (1.0 mol of histidine modified per mol) and fell thereafter due to slow hydrolysis of ethoxyformylhistidine. Times shown are hours after this maximal extent of modification. b, the loss of ethoxyformylhistidine after a given time was obtained from measurements at 245 nm and correlated with the extent of the ability to form the high spin state as described in the legend of Fig. 3.
Figure 6:
Relative susceptibility to DEPC
modification of the different forms of P. denitrificans CCP.
The different redox and spin states of CCP (4 µM in 5
mM Hepes, pH 7.5) were formed as described under
``Experimental Procedures.'' The cyano-mixed-valence enzyme
was formed by the addition of 100 µM NaCN to the
mixed-valence high spin state. DEPC was added to each form of CCP until
the enzyme's ability to form the high spin state was abolished.
The DEPC/CCP ratio required to abolish the ability to form the high
spin state is noted next to each form of the enzyme. *, at a ratio of
330 DEPC:1 CCP, the cyano-mixed-valence enzyme retained 90% of its
ability to form the high spin state. In a separate experiment, samples
of CCP (4 µM) in 5 mM Hepes, pH 7.5, in the
oxidized and mixed-valence high spin states were treated with
[C]DEPC to final concentrations of 20 µM (5 DEPC:1 CCP) and 1.2 mM (300 DEPC:1 CCP). After 20 min,
these two solutions were desalted on Sephadex G-25 columns equilibrated
in 5 mM Hepes, pH 7.5, and 200-µl aliquots of the desalted
material were added to 4 ml of scintillation fluid. Moles of
[
C]ethoxyformylhistidine/mole of enzyme were
calculated as described under ``Experimental
Procedures.''
Mass spectrometry established that the
larger fragment (M 27,950.9) corresponded to the
N-terminal 250 amino acids containing both heme groups (M
predicted from the amino acid sequence of
27,950.93). The smaller fragment (M
9581.38)
accounted for the remaining non-heme C-terminal 87 amino acids (M
predicted from the amino acid sequence of
9579.87). Thus, subtilisin cleavage was limited to the peptide bond
between Thr-250 and Arg-251.
Subtilisin digestion of the
mixed-valence holoenzyme that had been radiolabeled in the oxidized
state to the extent of 1 mol of histidine/mol gave the same peptide
pattern as shown in Fig. 7. A ratio of 2.2:1 for radioactive
disintegrations in the Arg-251-Met-338 peptide relative to the
Glu-1-Thr-250 peptide was obtained, and if the contribution of
undigested protein to the Glu-1-Thr-250 fraction was allowed for,
this ratio became 2.9:1. This ratio is a strong indication that the
single essential histidine resides in the Arg-251-Met-338
fragment. However, the close correlation between modification of a
single histidine and loss of activity shown in Fig. 3had led us
to expect an even cleaner pattern of label distribution. We found that
a [C]DEPC-labeled protein sample left for 24 h
after modification had completely lost its ethoxyformylhistidine based
on spectroscopic measurements at 245 nm, yet had retained almost all
radioactivity. Cleavage and separation of the two subtilisin fragments
yielded an Arg-251-Met-338/Glu-1-Thr-250 radiolabel ratio
of 1:5. Therefore, over the 24 h, label was transferred from a specific
site on the Arg-251-Met-338 peptide to the Glu-1-Thr-250
peptide. We propose that a partial transfer of this type is the reason
for the ratio of 2.9:1 (rather than higher) that we observed for
analysis completed within
2 h.
As the Arg-251-Met-338
peptide contains both His-275 and His-322, unequivocal identification
of the essential histidine in CCP required isolation of a fragment
containing a single histidine. Therefore, the Arg-251-Met-338
peptide was subdigested with trypsin, generating six smaller fragments
(labeled A-F) (Fig. 8) that were isolated by reverse-phase
HPLC and identified by mass spectrometry. M values
were within 0.5 of those predicted from the amino acid sequence.
His-275 and His-322 were located in peptides E and F, respectively.
Figure 8:
Identification of His-275 as the
radiolabeled amino acid. The unmodified purified Arg-251-Met-338
peptide was prepared as described in the legend of Fig. 7.
Trypsin was added to give a substrate/enzyme ratio of 50:1 (w/w). After
10 min at 23 °C, trypsin was inhibited with 0.5 mM phenylmethanesulfonyl fluoride. The resultant peptides were
separated by HPLC as described under ``Experimental
Procedures'' and gave rise to the 214 nm profile labeled Unmodified. Six peptides were collected (A-F)
and identified by mass spectrometry (M values in parentheses). CCP modified to an extent of 0.9 mol of
histidine modified per mol of protein with
[
C]DEPC was cleaved with subtilisin, and the
resultant radiolabeled peptide (Arg-251-Met-338) was purified as
described in the legend of Fig. 7. The digestion of this peptide
with trypsin yielded the HPLC profile labeled Modified. The
unmodified peptide is labeled E1, while the modified peptide
is labeled E2. The distribution of radiolabel was determined
by radioactive counting and is shown in the histogram below each
peak.
The essential histidine of CCP was radiolabeled with
[C]DEPC; the modified Arg-251-Met-338
peptide was isolated and digested; and the resultant tryptic peptides
were separated as described for the native protein. The HPLC profile
for the modified tryptic digest displayed an extra peak E2, the
appearance of which correlated with the diminution of the original peak
E1. Almost all the label was contained in peptide E2 (Fig. 8).
Thus, we conclude that peptide E contains the essential histidine,
which, when ethoxyformylated, increases the retention time of the
peptide. This was confirmed by obtaining an elution profile that
matched that of the unmodified protein after reversal of the
modification in 0.2 M ammonia solution (data not shown). Also,
the M
of peptide E2 was determined to be 2472.62
by mass spectrometry. This corresponds well with the theoretical mass
of 2400.12 for peptide E (Asn-266-Lys-287) plus 72 for an
ethoxyformyl group. Thus, His-275 is the single histidine modified at
low DEPC/CCP ratios that is essential for activity.
Modification of a single most reactive histidine is associated with loss of activity (Fig. 3), and activity is regained as the modification slowly hydrolyzes (Fig. 5). In yeast CCP, 1 mol of histidine is also readily modifiable at low concentrations of DEPC, but in this case, modification was not associated with loss of activity (data not shown). This is consistent with the results of Bosshard et al.(27) , who found that three histidines could be modified with no loss of activity. Similarly, in horseradish peroxidase, two histidines were modified by DEPC, but it is the more slowly modified residue that is essential(26) .
Purification of the C-terminal fragment by molecular exclusion chromatography followed by tryptic digestion and peptide separation by reverse-phase HPLC allowed localization of radioactive labeling in the tryptic peptide containing His-275 within 4 h of modification. Over a longer time scale (e.g. 24 h), the radioactive label was retained on the protein, but attached to the N-terminal region (Glu-1-Thr-250). We are unaware of a precedent for such an intramolecular transfer of an ethoxyformyl group, but we propose that it is consistent with attack on ethoxyformylhistidine 275 by a lysine that is adjacent in the folded protein, but resides in the N-terminal sequence (Glu-1-Thr-250). Ethoxyformyllysine is not susceptible to nucleophilic attack(15) . This proposal will be tested by identification of the labeled residue in the Glu-1-Thr-250 peptide.
In the crystallographic structure of the P. aeruginosa enzyme(11) , His-261, the counterpart of His-275, is placed at the interface between the N-terminal domain (proposed to contain the peroxidatic heme) and the C-terminal domain (containing the electron-transferring heme). Fig. 7of (11) shows the histidine as part of a hydrogen-bonded network involving the juxtaposed heme propionates of both heme groups. Our finding that His-275 is the most easily modified histidine in the oxidized state would not have been consistent with the original role as heme ligand proposed by Ellfolk et al.(9) , but it is compatible with the position determined by x-ray crystallography.
Fulop et al.(11) proposed that this histidine may
form part of an electron transfer route between the two heme groups.
Our finding that its modification abolishes activity would be
consistent with disruption of this electron transfer route. However,
Fulop et al. realized that proposals concerning the active
mixed-valence enzyme but based on the oxidized form must be regarded
with skepticism. There are none of the likely catalytic residues near
the proposed peroxidatic heme that one would expect by analogy with
eukaryotic active sites(29, 30) , and histidine 71
must leave the heme before a hydrogen peroxide-binding site can be
formed. It is therefore entirely possible that major conformational
changes take place during formation of the active mixed-valence enzyme.
Indeed, one possibility is that the conserved region containing
Arg-265, His-275, and Trp-280 moves to form the distal side of a
restructured peroxidatic site. Although this sequence is not
-helical in the x-ray structure of the oxidized form (11) , the consensus of the four prediction methods used
revealed some
-helix in this region. For example, the method of
Gibrat et al.(18) predicts
-helix between
Asp-255 and Ala-268 in the P. denitrificans CCP sequence. In
response to the criticism that such major conformational changes would
be likely to be slow, it must be realized that the oxidized form that
has been crystallized is almost certainly not part of the catalytic
cycle and represents a dead-end conformation into which the protein
relaxes to protect the catalytic site in the absence of reductant.