H2O2-mediated Cross-linking between
Lactoperoxidase and Myoglobin
ELUCIDATION OF PROTEIN-PROTEIN RADICAL TRANSFER REACTIONS*
Olivier M.
Lardinois and
Paul R. Ortiz
de Montellano
From the Department of Pharmaceutical Chemistry, School of
Pharmacy, University of California, San Francisco, California
94143-0446
Received for publication, March 8, 2001, and in revised form, April 4, 2001
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ABSTRACT |
The
H2O2-dependent reaction of
lactoperoxidase (LPO) with sperm whale myoglobin (SwMb) or horse
myoglobin (HoMb) produces LPO-Mb cross-linked species, in
addition to LPO and SwMb homodimers. The HoMb products are a LPO(HoMb)
dimer and LPO(HoMb)2 trimer. Dityrosine cross-links are
shown by their fluorescence to be present in the oligomeric products.
Addition of H2O2 to myoglobin (Mb), followed by
catalase to quench excess H2O2 before the
addition of LPO, still yields LPO cross-linked products. LPO
oligomerization therefore requires radical transfer from Mb to LPO. In
contrast to native LPO, recombinant LPO undergoes little
self-dimerization in the absence of Mb but occurs normally in its
presence. Simultaneous addition of 3,5-dibromo-4-nitrosobenzenesulfonic
acid (DBNBS) and LPO to activated Mb produces a spin-trapped radical
electron paramagnetic resonance signal located primarily on LPO,
confirming the radical transfer. Mutation of Tyr-103 or Tyr-151 in SwMb
decreased cross-linking with LPO, but mutation of Tyr-146, Trp-7, or
Trp-14 did not. However, because DBNBS-trapped LPO radicals were
observed with all the mutants, DBNBS traps LPO radicals other than
those involved in protein oligomerization. The results clearly
establish that radical transfer occurs from Mb to LPO and suggest that
intermolecularly transferred radicals may reside on residues
other than those that are generated by intramolecular reactions.
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INTRODUCTION |
Amino acid centered radicals are now established as catalytic
intermediates in a variety of enzymes, including cytochrome c peroxidase (1), prostaglandin H synthase (2),
ribonucleotide reductase (3), pyruvate-formate lyase (4), and galactose oxidase (5). Protein radical formation not associated with the normal
function of the protein is also observed, as, for example, in the
reaction of Mb1 with
H2O2 (6-13). Both the catalytic and incidental
protein radicals are most commonly generated by a metal-catalyzed
reaction within the protein itself, but the formation of protein
radicals by reaction of the protein with an exogenous species such as
the hydroxyl radical is also known (14, 15). Protein radicals,
particularly those not intrinsic to the catalytic mechanism, can lead
to cross-linking of the protein, cleavage of the protein backbone, and
the formation of protein peroxy radicals and protein peroxides (16).
These radical reactions can be physiologically relevant, as in the
formation of a tyrosine-cross-linked protective shell by sea urchin
ovoperoxidase (17). In most situations, however, protein radicals
facilitate pathological or toxicological processes (18). Despite their importance, the factors and mechanisms that control the formation, localization, delocalization, and propagation of protein free radicals
remain obscure.
The reactions of sperm whale and horse heart MetMb with
H2O2 have served as a useful model for the
investigation of protein radicals. The early observation by EPR of a
protein radical in the reaction of MetMb with
H2O2 has been more precisely defined by recent
work (12, 19). Site-specific mutagenesis, in conjunction with protein
digestion, peptide sequencing, mass spectrometry, and EPR spectroscopy,
has established that MetMb cross-linking involves the formation of
Tyr-103 and Tyr-151 dityrosine cross-links (10, 12, 13). Tyr-146, the
third tyrosine in the protein, forms a radical, but it may not
participate in chemical reactions (13). Of the two tryptophan residues,
Trp-7 and Trp-14, only Trp-14 appears to be involved in the formation
of a protein-peroxy radical that can co-oxidize styrene (13, 19).
Evidence has also been obtained for the formation of a radical located
on Cys-110 (20), which is unique to human myoglobin, and on an
aliphatic MetMb amino acid residue (21). All of these protein radicals are associated with the formation of a ferryl (FeIV=O)
species in the reaction of MetMb with H2O2.
Recent work indicates that the reaction of MetMb with
H2O2 initially yields a transient Compound
I-like species, in which the ferryl is coupled to a porphyrin rather
than a protein radical (22, 23). It is therefore likely that the
protein radical is generated by subsequent rapid electron transfer from
the protein to the porphyrin radical. The residue that provides the
electron that directly quenches the porphyrin radical is not known, but
the resulting protein radical appears to transfer efficiently from one
residue to another over considerable distances. Thus, radical reactions
involving Tyr-103, Tyr-151, Trp-14, Cys-110 (human Mb only), and Lys-42
have been identified, and mutation of any one of these residues only
suppresses the reactions at that specific site. Furthermore, the
Tyr-151 radical is still formed, even though the closest Tyr-151 ring
carbon is 12 Å from the heme iron, when Tyr-103 and Tyr-146, which are
closer to the heme, have been mutated to phenylalanines. A relay
mechanism for dispersal of the free radical center is therefore not a
prerequisite for radical formation at a distal residue in MetMb.
LPO, a mammalian peroxidase, catalyzes the physiologically relevant
oxidation of thiocyanide to antibacterial products, as well as the
oxidation of iodide, bromide, and conventional peroxidase substrates
such as ABTS and guaiacol (24). Its catalytic mechanism involves
reaction with H2O2 to form a Compound I species
with a FeIV=O ferryl species and a porphyrin radical
cation. This initial Compound I decays to a second Compound I species
in which the FeIV=O is paired with a protein radical (25,
26). We have recently shown that formation of the protein radical form
of the activated enzyme leads to dimerization of LPO through the
formation of a dityrosine cross-link involving residue Tyr-289 in each
of two enzyme molecules (27). Spin trapping/EPR evidence was also
obtained for the formation of protein radicals at sites other than the tyrosine involved in the cross-linking reaction (27). The roles of
these protein radicals in LPO function remain unclear, although a
previous study has argued that the protein radicals may be required for
certain catalytic functions (26), and autocatalytic covalent binding of
the prosthetic heme group to the LPO protein can be formulated as a
radical process (28). Although the evidence is more sparse than that
for MetMb, it appears that the protein radical undergoes some degree of
dispersion throughout the LPO structure, but it is not known whether
this dispersion is an equilibrium or unidirectional process.
As indicated by the work with MetMb and LPO, protein radicals
delocalize readily, even to distant sites within a protein, once they
are generated at a well-defined site, i.e. in the vicinity of the heme iron atom. In this study, we have investigated whether the
protein radicals thus generated can transfer from one protein to
another and whether this mechanism of radical propagation can produce
protein radicals in the second protein distinct from those formed by
radical generation within the protein itself.
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EXPERIMENTAL PROCEDURES |
Materials--
SwMb, HoMb, equine apomyoglobin, and LPO from
bovine milk were acquired from Sigma. The commercial LPO (80-150
units/mg protein; A412/A280 = 0.88-0.95)
shown to be homogeneous by SDS-PAGE was desalted over prepacked
Sephadex G-25 (PD-10) gel filtration cartridges before use. The
commercial SwMb and HoMb were further purified by chromatography on a
(2.6 × 100-cm) Sephacryl S-200 HR column (Amersham Pharmacia
Biotech). The column was equilibrated and run at 0.5 ml/min in 50 mM potassium phosphate buffer, pH 6.8. Prepacked Sephadex
G-25 (PD-10) gel filtration cartridges were purchased from Amersham
Pharmacia Biotech. All other chemicals were of analytical grade and
were purchased from Sigma or Roche Molecular Biochemicals.
Analytical Methods--
Absorption spectra were recorded on a
Cary 1E UV-visible spectrometer (Varian, Victoria, Australia).
Dityrosine content was measured with a PerkinElmer Life Sciences LS 50 B fluorimeter (Beaconsfield, United Kingdom). SDS-PAGE was done on a
Pharmacia LKB Phast System (Amersham Pharmacia Biotech). EPR
measurements were performed with an ER/200D EPR spectrometer from
Brucker, Inc. (Billerica, MA). Circular dichroism was measured with a
J-710 spectropolarimeter from Jasco (Tokyo, Japan).
Preparation and Expression of Site-directed Protein
Mutants--
The mutant SwMb proteins and wild-type recombinant rLPO
were expressed and purified as described previously (12, 13, 19, 28).
Recombinant SwMb proteins were oxidized to the met form using a slight
excess of K3Fe(CN)6 and then passed over a
PD-10 column eluted with 50 mM potassium phosphate buffer,
pH 6.8. The protein samples were then further purified by
chromatography on a (2.6 × 100-cm) Sephacryl S-200 HR column and
concentrated by ultrafiltration (Centricon YM 10; Amicon; molecular
weight cut off = 10,000) before use. The column was equilibrated
and run as described for the native protein (see above). The far
ultraviolet circular dichroism spectra of wild-type and mutant Mb
proteins at pH 6.8 were fully superimposable (data not shown). In all
of the SwMb tyrosine and tryptophan mutants examined, the UV-visible spectra observed were nearly identical to those of the native protein,
indicating that changes to the structure in the vicinity of the heme
iron were minimal (data not shown).
Activity and Concentration
Measurements--
Peroxidase activities were determined with ABTS as
the reducing substrate (29). Formation of the ABTS radical cation was monitored at 414 nm (
ABTS·, 414 = 36 mM
1 cm
1; assay conditions: 100 µM ABTS, 100 µM
H2O2, and 50 mM sodium acetate, pH
4.5). Enzyme concentrations were estimated by measuring the absorbance
of the heme Soret band using the following extinction coefficients:
LPO, 412 = 112.3 mM
1
cm
1 (30), SwMb
MetMb, 409 = 157 mM
1 cm
1 (31), and HoMb
MetMb, 409 = 188 mM
1
cm
1 (31).
SDS-PAGE of Protein Samples--
Cross-linking experiments were
carried out at 25 °C for 5 min. The reactions were terminated by
addition of the SDS-PAGE sample buffer. Samples were allowed to
stand in SDS for 10 min and heated at 100 °C for 2 min before
loading onto the gels, which were developed and then stained with
Coomassie Blue.
Cationic Ion-exchange Chromatography--
A stock solution of 72 µM LPO and 741 µM HoMb was prepared in 50 mM potassium phosphate buffer, pH 6.8. The LPO-HoMb stock solution was incubated with H2O2 at a final
concentration of 1 mM for 5 min at 25 °C. Excess
H2O2 was then consumed by incubation with
catalase (1.25 units/ml) for 15 min. The resulting solution was applied
to a 2.6 × 20-cm SP Sepharose Fast Flow column (Amersham Pharmacia Biotech) equilibrated with 50 mM potassium
phosphate, pH 6.8. The column was washed with 50 mM
potassium phosphate buffer (pH 6.8) and then eluted using the stepwise
procedure described in the text (0-450 mM NaCl). The
fractions corresponding to LPO-HoMb heteromers (as assessed by SDS-PAGE
analysis) were pooled in two lots. Each lot was desalted and
concentrated by ultrafiltration (Centricon YM 30; Amicon; molecular
weight cut off = 30,000). The concentrated samples contained about
4 mg total protein/ml and were stored at 4 °C for subsequent analysis.
Enzymatic Digestion of the LPO-HoMb Heteromers--
A 100-µl
sample of each fraction of heteromers separated by cation-exchange
chromatography was taken for analysis. The samples were diluted to a
volume of 2 ml with 8 M urea in 50 mM Tris-HCl, pH 8, containing 2 mM CaCl2 before they were
concentrated down to 100 µl by diafiltration (Centricon YM 30;
Amicon). After adding 20 µl of 50 mM dithiothreitol, the
samples were incubated at 60 °C for 60 min. The resulting mixtures
were then diluted to 380 µl with 50 mM Tris-HCl, pH 8, containing 2 mM CaCl2 and treated with Pronase
(1:10, w/w) for 24 h and leucine aminopeptidase (1:25, w/w)
for 12 h at 37 °C. The hydrolyzed samples were frozen and stored at
70 °C for subsequent analysis.
Reverse Phase HPLC Analysis of the LPO-HoMb Heteromers--
A
dityrosine standard or LPO-Mb hydrolysate samples were injected onto a
Vydac 218TP54, 4.6 × 250-mm, C18 reverse phase HPLC column. The
column was eluted with a linear gradient rising from 100% solvent A to
10% solvent B in 45 min (solvent A, 0.1% trifluoroacetic acid in
water; solvent B, 0.085% trifluoroacetic acid in acetonitrile). A
rapid gradient to 90% solvent B and then back to 100% solvent A was
then run to regenerate the column. The eluent was monitored with a
UV-visible detector set at 280 nm. Fluorescent compounds were detected
with a PerkinElmer Life Sciences 650-10S fluorescence spectrophotometer (
ex = 280 nm,
em = 410 nm) coupled in-line with the HPLC system.
Preparation of the Dityrosine Standard--
Dityrosine was
prepared using the enzymatic method of Amadò et al.
(32) and purified by reverse phase HPLC as described previously
(27).
Spin Trapping Experiments--
EPR measurements were performed
with an ER/200D EPR spectrometer operating at 9.80 GHz with a TM
cavity. X-Band, first derivative absorption spectra were obtained with
the following settings: microwave power, 25 mW; center field, 3480 gauss; time constant, 100 ms; sweep time, 50 s; modulation, 0.32 millitesla at a frequency of 100 kHz; and total sweep width, 125 gauss.
Spectra were taken at 18 °C to 22 °C. The magnetic field range
and center were estimated by comparing the EPR spectrum from a
LPO/H2O2 reaction mixture with that of the
stable nitroso compound potassium nitrosodisulfonate. The potassium
nitrosodisulfonate splitting was taken to be 13.091 gauss, and the
center peak was taken to correspond to a g of 2.0056 (33). The reactions were all performed in 50 mM potassium
phosphate solution (pH 6.8) containing 200 µM
diethylenetriaminepentaacetic acid to inhibit possible catalysis by
trace transition metals.
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RESULTS |
H2O2-mediated Cross-linking between
LPO and MetMb--
Fig. 1 shows the profile
of mixtures of LPO and MetMb on a SDS-polyacrylamide gel after
incubation with H2O2. Two types of Mb, HoMb and
SwMb, were utilized. As already known (12, 13, 27),
H2O2 causes the covalent oligomerization of LPO
(Fig. 1, A and B, lane 2) and SwMb (Fig.
1B, lane 4) but no more than a barely detectable trace on
the gel with HoMb (Fig. 1A, lane 4). When the HoMb-LPO
mixtures were exposed to H2O2, two new bands occurred at positions corresponding to Mr 97,000 and Mr 127,000 (Fig. 1A, lane
6), suggesting the formation of two types of heteromers, a dimer
(LPO-Mb) and a trimer (LPO(Mb)2), respectively. Traces of
two or three closely spaced bands of Mr
~200,000, likely due to (LPO)2 homodimers and
possibly also due to (LPO)2-Mb heterotrimers, were also
present. One additional band was obtained with a molecular weight of
~153,000 using SwMb in place of HoMb (Fig. 1B, lane 6) suggesting the formation of a LPO(Mb)3
heterotetramer. These dimeric and oligomeric entities could not be
attributed to S-S bond formation because (a) the protein
samples were treated with 2-mercaptoethanol before electrophoresis, and
(b) there are no cysteine residues in SwMb or HoMb.
Inclusion of HoMb in the LPO/H2O2 system both
decreased LPO homodimer and homotrimer formation and increased LPO-HoMb
hetero-oligomerization in a concentration-dependent manner
(Fig. 2). This suggests that HoMb
competes with the two residues on the LPO surface involved in
homotrimer formation. Heteromer formation increases to a maximum using
reaction mixtures containing 1:40:40 stoichiometric concentrations of
LPO, Mb, and H2O2, respectively (data not
shown). No heteromer was observed by SDS-PAGE in incubations of
apo-HoMb, LPO, and H2O2 (data not shown).
Significant hetero-oligomerization only occurred when ferric HoMb or
ferric SwMb was incubated with H2O2 in the
presence of LPO.

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Fig. 1.
A, SDS-PAGE analysis of the
cross-linking between HoMb and LPO. Lane 1, LPO; lane
2, LPO + H2O2; lane 3, HoMb;
lane 4, HoMb + H2O2; lanes
5 and 6, HoMb + LPO + H2O2.
B, SDS-PAGE analysis of the cross-linking between SwMb and
LPO. Lane 1, LPO; lane 2, LPO + H2O2; lane 3, SwMb; lane
4, SwMb + H2O2; lanes 5 and
6, SwMb + LPO + H2O2. The
following concentrations were used: [H2O2] = 312 µM, [LPO] = 10 µM, [HoMb] = 375 µM, [SwMb] = 375 µM, and
[polyacrylamide] = 20% (lanes 1-5) and 7.5% (lane
6).
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Fig. 2.
Cross-linking between HoMb and LPO: effect of
the amount of HoMb added. Lane 1, LPO; lane
2, LPO + H2O2; lanes 3-6, HoMb + LPO + H2O2. The following concentrations were
used: [H2O2] = 125 µM;
[LPO] = 10 µM; [HoMb] = 47 µM
(lane 3), 94 µM (lane 4), 188 µM (lane 5), and 375 µM
(lane 6); and [polyacrylamide] = 7.5%.
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Isolation and Characterization of the LPO-HoMb Heteromers--
The
LPO-HoMb heteromers were separated by chromatography on a cationic
exchange column (SP Sepharose Fast Flow). A typical elution pattern
(Fig. 3A) indicates the
presence of three major protein components (peaks 1-3) and several
minor components. One of the minor components is the LPO homodimer,
which elutes at ~170 ml (Fig. 3A). The fraction
corresponding to the earliest eluting major peak (peak 1) contained
only HoMb and was not investigated further. The fractions corresponding
to peaks 2 and 3 were pooled individually and desalted and concentrated
by ultrafiltration. The molecular weights and the purities of the
resulting solutions were determined on a SDS-polyacrylamide gel (Fig.
3B). As shown, peak 2 yielded two bands with apparent
molecular weights of 108,000 and 130,000, which are consistent
with the LPO-HoMb heterodimer and the LPO(HoMb)2
heterotrimer, respectively. Peak 3 also yielded two bands, one of which
was of higher molecular weight than that of native LPO and corresponded
to the LPO-HoMb heterodimer at Mr
97,000.

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Fig. 3.
A, separation of LPO-HoMb heteromers by
chromatography on a cationic exchange column. Elution profile before (- - -) and after (____) incubation with 1.3 equivalents of H2O2. The following
concentrations were used: [LPO] = 72 µM, [HoMb] = 741 µM, [catalase] = 0.4 nM, and
[H2O2] = 1 mM. B,
SDS-PAGE analysis (7.5%) of the main fractions isolated on the
cationic exchange column. The sample were reduced by 2-mercaptoethanol
and boiled before electrophoresis. Lane 1, LPO; lane
2, LPO+ H2O2; lane 3, peak 2;
lane 4, peak 3. The following concentrations were used:
[LPO] = 10 µM, and [H2O2] = 30 µM.
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The reactions of some hemoproteins with H2O2
are known to result in oligomerization of the proteins due to the
formation of tyrosine-tyrosine cross-links (10, 34, 35). The dityrosine cross-links can be detected by their characteristic ultraviolet fluorescence (36, 37). To evaluate the presence of dityrosine cross-links between LPO and Mb, the fractions corresponding to peaks 2 and 3 were pooled individually, desalted, and concentrated by
ultrafiltration as described above. They were then digested with
Pronase and leucine aminopeptidase. Analysis of the hydrolysates of the
two peaks by reverse phase HPLC (Fig. 4,
A and B) showed in both cases a product eluting
at 21 min with a strong fluorescence at 410 nm (excitation, 280 nm).
The identity of this fluorescent compound was confirmed by
demonstrating co-migration with authentic dityrosine on reverse phase
HPLC (Fig. 4C). The fluorescence excitation and emission
maxima of the fluorescent compounds were virtually identical to those
of authentic, purified dityrosine at acid, neutral, and alkaline pH
(data not shown), providing strong additional evidence that LPO-Mb
hetero-oligomerization was due to the formation of tyrosine-tyrosine
cross-links between the two proteins.

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Fig. 4.
Reverse phase HPLC analysis of the
proteolytic hydrolysate of peak 2 (A) and peak 3 (B) from the cationic exchange column and of a
dityrosine standard (C). The pooled fractions of
peak 2 (95-115 ml) and peak 3 (132.5-147.5 ml) were desalted,
concentrated by ultrafiltration, and subjected to proteolytic
hydrolysis and analysis by reverse phase HPLC as described under
"Experimental Procedures." The fluorescence of the column effluent
at 410 nm (excitation, 280 nm) is shown.
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Protein-Protein Radical Transfer Reactions--
The possible
occurrence of a radical transfer from Mb to LPO in the
Mb/H2O2/LPO system was investigated by
sequentially adding catalase and then LPO to the
Mb/H2O2 reaction mixture. Catalase and LPO were
added 10 and 20 s after H2O2,
respectively, to allow time for the catalase to remove unreacted
H2O2 and thus to ensure that cross-linking was
not due to LPO radicals formed by direct reaction with
H2O2. As can be seen (compare Fig.
5 with Fig. 1), adding catalase before
LPO had little effect on the type of cross-linked products that were
formed. In particular, the dimeric and trimeric LPO-Mb forms were
observable despite the addition of catalase to the HoMb incubation
(Fig. 5A, lanes 1 and 6). Similar results were
obtained with SwMb (Fig. 5B, lanes 1 and 6),
except that additional bands are present due to the simultaneous
formation of SwMb homodimers. Control experiments performed in the
absence of Mb showed that (a) when catalase was omitted,
monomeric LPO was partially converted to dimeric and trimeric products
(Fig. 5, A and B, lane 5) and
(b) addition of catalase 10 s before the addition of
LPO prevented oligomerization of the enzyme (Fig. 5,
A and B, lane 4). Thus, under the
conditions used, catalase is effective in removing unreacted
H2O2 before the addition of LPO. Thus, the
oligomerization of LPO observed in the presence of Mb in this
experimental protocol is the result of a radical transfer from Mb to
LPO.

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Fig. 5.
A, cross-linking between HoMb and LPO:
effect of time-delayed injection of catalase and LPO. Lanes
1 and 6, complete system with HoMb,
H2O2, catalase, and LPO; lane 2, as
described in lane 1, except that
H2O2 was not added; lane 3, as
described in lane 1, except that LPO was not added;
lane 4, as described in lane 1, except that HoMb
was not added; lane 5, as described in lane 1, except that HoMb and catalase were not added. B,
cross-linking between SwMb and LPO: effect of time-delayed injection of
catalase and LPO. Lanes 1 and 6, complete system
with SwMb, H2O2, catalase, and LPO; lane
2, as described in lane 1, except that
H2O2 was not added; lane 3, as
described in lane 1, except that LPO was not added;
lane 4, as described in lane 1, except that SwMb
was not added; lane 5, as described in lane 1, except that SwMb and catalase were not added. Catalase and LPO were
added to the incubation 10 and 20 s, respectively, after the
addition of H2O2, regardless of whether Mb was
present or not. The following concentrations were used: [LPO] = 11 µM, [HoMb] = 350 µM, [SwMb] = 350 µM, [catalase] = 0.3 µM,
[H2O2] = 500 µM, and
[polyacrylamide] = 20% (lanes 1-5) and 7.5% (lane
6).
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Essentially the same result was obtained using wild-type rLPO in place
of native LPO (Fig. 6). rLPO displayed an
enzymatic activity that was 50% lower than that of the native protein
(data not shown) and, in unexpected contrast to the native form,
exhibited little self-dimerization upon treatment with
H2O2 (Fig. 6, lane 5). Nevertheless,
~50% of the monomeric rLPO migrates in the location of LPO-Mb
heterodimers and LPO homodimers when treated with
H2O2 in the presence of HoMb (Fig. 6,
lanes 1 and 6).

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Fig. 6.
Cross-linking between HoMb and rLPO.
Lanes 1 and 6, complete system with HoMb,
H2O2, catalase, and rLPO; lane 2, as
described in lane 1, except that
H2O2 was not added; lane 3, as
described in lane 1, except that rLPO was not added;
lane 4, as described in lane 1, except that HoMb
was not added; lane 5, as described in lane 1, except that HoMb and catalase were not added. Catalase and LPO were
added to the incubation 10 and 20 s, respectively, after the
addition of H2O2, regardless of whether Mb was
present or not. The following concentrations were used: [rLPO] = 9.5 µM, [HoMb] = 350 µM, [catalase] = 0.3 µM, [H2O2] = 500 µM, and [polyacrylamide] = 20% (lanes 1-5)
and 7.5% (lane 6).
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An intense ESR spectrum characteristic of an immobilized nitroxide was
detected when DBNBS was simultaneously added with LPO after
preincubation of Mb/H2O2 with catalase to
remove excess H2O2 (Fig.
7A). No signal could be
detected in the absence of either SwMb (Fig. 7B) or
H2O2 (Fig. 7C), indicating that
formation of the EPR signal requires the presence of this protein and
is not due to radicals formed by the direct reaction of LPO with H2O2. A much weaker signal was observed with
the SwMb/H2O2/DBNBS system in the absence of
LPO (Fig. 7D). This signal, which is reproducibly less than
30% of the intensity of the signal from the complete system, is
believed to originate from direct reaction of
H2O2-activated SwMb with DBNBS. Identical
behavior and signals were observed when HoMb was used in place of SwMb
(data not shown).

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Fig. 7.
ESR spectra obtained from a mixture of SwMb,
H2O2, catalase, and LPO in the presence of
DBNBS. A, complete system with SwMb,
H2O2, catalase, LPO, and DBNBS; B,
as described in A, except that SwMb was not added;
C, as described in A, except that
H2O2 was not added; D, as described
in A, except that LPO was not added. Catalase and LPO were
added to the incubation 10 and 20 s, respectively, after the
addition of H2O2, regardless of whether Mb was
present or not. DBNBS was added simultaneously with LPO. The following
concentrations were used: [LPO] = 290 µM, [SwMb] = 2.3 mM, [catalase] = 0.15 µM,
[H2O2] = 3.6 mM, and [DBNBS] = 18 mM. The instrumental parameters were as follows:
modulation amplitude, 1G; time constant, 100 ms; receiver gain, 5 × 105; modulation frequency, 100 kHz; microwave frequency,
9.80 GHz; and microwave power, 25 mW.
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Involvement of Specific Mb Residues--
To identify the residues
involved in cross-linking and in radical transfer reactions,
site-directed mutant Mb proteins in which the tyrosine or tryptophan
residues were replaced by phenylalanines were used. The results of
cross-linking experiments carried out with mixtures of native LPO and
recombinant Mb proteins are shown in Fig.
8A. The double integrated
areas of the ESR spectra obtained when DBNBS was added simultaneously
with the LPO to the reaction mixture are shown in Fig. 8B.
The areas of the corresponding background signals obtained in the
absence of LPO are also shown. As before, catalase and LPO were added
10 and 20 s, respectively, after H2O2 to
remove unreacted H2O2 and to ensure that the
cross-links and/or ESR signals were not due to radicals formed by
direct reaction of LPO with H2O2. Replacement
of Tyr-103 or Tyr-151 significantly inhibited cross-linking of the SwMb
with LPO, and the triple tyrosine mutant gave no detectable
cross-linking (Fig. 8A). This implicates these two tyrosine
residues in the hetero-oligomerization process. In contrast, removal of
Tyr-146, Trp-7, or Trp-14 had little or no effect. Spin trapping
experiments showed the presence of radical species capable of reaction
with DBNBS in the incubations of native LPO with all the recombinant Mb
proteins that were investigated (Fig. 8B). Maximal ESR
signal intensities were obtained with the single Y103F and Y151F
mutants as well as with the triple tyrosine mutant (Fig.
8B). These intensities are approximately double the intensities seen with the other Mb mutants or with the wild-type enzyme. This may result from an increased efficiency of radical transfer from Mb to LPO caused by blocking the tyrosine radical to
dityrosine decay pathway.

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Fig. 8.
A, cross-linking between recombinant
SwMb and native LPO. Lane 1, complete system with SwMb
wild-type, H2O2, catalase, and LPO; lane
2, as described in lane 1, but with Y103F mutant in
place of wild-type; lane 3, as described in lane
1, but with Y146F mutant in place of wild-type; lane 4, as described in lane 1, but with Y151F mutant in place of
wild-type; lane 5, as described in lane 1, but
with Y103F/Y146F/Y151F mutant in place of wild-type; lane 6, as described in lane 1, but with W7F mutant in place of
wild-type; lane 7, as described in lane 1, but
with W14F mutant in place of wild-type; lane 8, as described
in lane 1, but with W7F/W14F mutant in place of wild-type.
The following concentrations were used: [LPO] = 11 µM,
[recombinant SwMb] = 350 µM each, [catalase] = 0.3 µM, [H2O2] = 500 µM, and [polyacrylamide] = 20%. Catalase and LPO were
added to the incubation 10 and 20 s, respectively, after the
addition of H2O2, regardless of whether Mb was
present or not. B, intensity of the ESR signal when DBNBS
was added 10 s after the addition of catalase in the
above-mentioned reaction mixtures. , complete system with
recombinant Mb, H2O2, catalase, LPO, and DBNBS;
, system with recombinant Mb, H2O2,
catalase, and DBNBS, but no LPO, for comparison. The following
concentrations were used: [LPO] = 290 µM, [recombinant
SwMb] = 1.80 mM each, [catalase] = 0.15 µM, and [H2O2] = 3.6 mM. ESR parameters were the same as those described in the
Fig. 6 legend.
|
|
 |
DISCUSSION |
The formation of SwMb homodimers and the analogous formation of
LPO homodimers on reaction of these hemoproteins with
H2O2 were established by earlier studies (27,
38). However, the formation of hetero-oligomeric species involving the
cross-linking of LPO to one, two, or possibly more Mb molecules (Fig.
1) is a new observation. As found earlier for the Mb and LPO
homodimers, fluorescence spectroscopy indicates that the
hetero-oligomeric LPO-Mb species, like the Mb and LPO homodimers, are
held together by dityrosine cross-links (Fig. 3). Interestingly, LPO-Mb
oligomers are formed with both SwMb and HoMb, even though HoMb does not self-dimerize because it lacks the Tyr-151 residue that is absolutely required for homodimer formation (Fig. 1). Earlier studies established that Tyr-151 was necessary for SwMb homodimerization, although the
principal dityrosine cross-link was formed between Tyr-151 of one
molecule and Tyr-103 of the other molecule (10). Tyr-146, which is
buried in the protein, is not involved in dimerization reactions, but
Tyr-151 can cross-link to itself. The dityrosine link between SwMb and
LPO could therefore involve Tyr-151 or Tyr-103 of Mb. Because HoMb
lacks Tyr-151, its dityrosine cross-link to LPO must be through
Tyr-103. This inference is strongly supported by the finding that
(a) mutation of either Tyr-103 or Tyr-151 to a phenylalanine
in SwMb greatly decreases oligomerization and (b) mutation
of both Tyr-103 and Tyr-151 suppresses oligomerization, whereas
mutation of Tyr-146, Trp-7, or Trp-14 has no effect on the reaction
(Fig. 8).
The LPO tyrosine residue linked to the tyrosine in Mb is more difficult
to define. One possibility is that the link involves Tyr-289, which is
the residue that is also involved in LPO self-dimerization (27). The
fact that LPO dimer formation is attenuated in the presence of SwMb or
HoMb supports this interpretation, but the evidence is inconclusive
because trapping of a LPO radical at another site if the LPO radical is
able to exchange among several sites on the protein would still
diminish the formation of LPO dimers. Furthermore, the formation
of LPO(HoMb)2 trimers involving dityrosine bonds virtually
requires that at least two tyrosines in LPO must be able to cross-link
to Mb to generate the HoMb-LPO-HoMb structure. In the case of SwMb,
formation of the LPO(HoMb)2 trimer does not absolutely
require the involvement of two LPO tyrosine residues because SwMb has
two tyrosines that can undergo cross-linking reactions. It is therefore
theoretically possible that the trimer has the structure LPO-SwMb-SwMb,
so that there is only one bond to the LPO unit. However, the formation
of a LPO(HoMb)2 trimer indicates that at least two
tyrosines in LPO are available for cross-linking to HoMb.
Dityrosine cross-links are formed by the coupling of two tyrosine
radicals in a radical recombination process, hence radicals must be
present on both tyrosine residues for efficient coupling. To determine
whether a protein radical present on a Mb molecule can be transferred
to a tyrosine residue in LPO, Mb was exposed to a limited excess of
H2O2, and catalase was then added to quench the
remaining H2O2 before LPO was added to scavenge
the excess peroxide. Under conditions in which the excess
H2O2 is removed efficiently, cross-linking of
LPO is still observed. This finding requires transfer of the Mb radical
to LPO, followed by condensation with an Mb radical (Fig. 5). The
radical transfer could involve specific residues, for example, transfer
of the radical from Tyr-151 to the recipient site on LPO, or could
result from a more general and undefined process that does not require
the interaction of specific Mb residues with receptor residues in LPO.
The finding that mutation of Tyr-103 and/or Tyr-151, but not Tyr-146,
Trp-7, or Trp-14, inhibits or suppresses oligomerization clearly
establishes that the electron transfer is not mandatorily mediated by
Tyr-146, Trp-7, or Trp-14, although it is not possible to determine
whether it is mediated by Tyr-103 or Tyr-151 because these two residues are required to form the dityrosine link. However, the finding that the
trapping of LPO radicals by DBNBS is unimpaired when the triple
tyrosine mutant of SwMb is used as the radical donor (Fig. 8) and is
observed even in the absence of oligomerization indicates that radical
transfer occurs in the absence of any of the residues that have been
demonstrated to act as radical sites in Mb. Thus, although dimerization
requires the participation of specific tyrosines in Mb, the radical
transfer to LPO does not.
One very interesting finding is that rLPO is much less susceptible to
self-dimerization than the native protein in the absence of Mb, but the
two proteins form equal amounts of homodimers in the
presence of Mb. Previous work with the heterologously expressed protein
has shown that the primary difference between rLPO and LPO is that the
heme group is covalently bound to the protein in the native enzyme, but
only partially so in the recombinant protein (28). This difference,
which is consistent with the finding that the heme-protein
cross-linking reaction is autocatalytic (28), readily explains our
finding that the initial activity of the recombinant protein is half
that of the native enzyme. However, the lack of self-dimerization in
the absence of Mb is puzzling because the spectroscopic evidence
suggests that the secondary and tertiary structures of the recombinant
protein are unperturbed. The finding that self-dimerization does not
occur in the absence of Mb but occurs normally in its presence suggests that the radical obtained by transfer from Mb to rLPO may be located at
a different site from that at which it is located when generated by
direct reaction of rLPO with H2O2.
The present results establish that (a) the HoMb and SwMb
protein radicals can be transferred to LPO, (b) none of the
residues known to bear unpaired electron density in SwMb is
specifically required for the radical transfer, and (c) the
radical transferred to rLPO can support dimerization with Mb, even
though the radicals generated by direct reaction of rLPO with
H2O2 only very poorly yield the usual rLPO
homodimer. This demonstration that radical transfer occurs between Mb
and LPO complements recent reports that incubation of Mb with
H2O2 in the presence of serum albumin produces
EPR-detectable serum albumin-derived radicals through a radical
transfer mechanism (39-41). However, it was concluded from the serum
albumin experiments that a peroxy radical centered on Trp-14 was
responsible for electron abstraction from, and therefore radical
transfer to, serum albumin (40). Our finding that both the W7F and W14F
SwMb mutant are fully able to transfer a radical to LPO clearly shows
that neither tryptophan is essential for this reaction. It is possible
that the Trp-14-peroxy radical participates in the reaction when
present, but it is not essential for the radical transfer. The radical
transfer thus appears to occur directly from the amino acid radicals in
Mb. This finding is more closely mimicked by the finding that free
tyrosine facilitates the oxidation of serum albumin by horseradish
peroxidase (41), a reaction that presumably occurs by direct radical
transfer from the free tyrosine radical to the protein.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM32488.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: School of Pharmacy,
University of California, San Francisco, CA 94143-0446. Tel.: 415-476-2903; Fax: 415-502-4728; E-mail: ortiz@cgl.ucsf.edu.
Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M102084200
 |
ABBREVIATIONS |
The abbreviations used are:
Mb, myoglobin;
MetMb, metmyoglobin;
HoMb, horse myoglobin;
SwMb, sperm whale
myoglobin;
heme, iron protoporphyrin IX regardless of the oxidation and
ligation states;
LPO, lactoperoxidase;
rLPO, recombinant LPO;
DBNBS, 3,5-dibromo-4-nitrosobenzenesulfonic acid;
EPR, electron paramagnetic
resonance;
ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid;
SDS-PAGE, SDS-polyacrylamide gel electrophoresis;
HPLC, high pressure
liquid chromatography.
 |
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