(Received for publication, July 17, 1995; and in revised form, November 20, 1995)
From the
The identity of the non-extractable heme of mammalian
lactoperoxidase (LPO) has remained unsolved for over 40 years. Accepted
possibilities include a constrained heme b or an
8-thiomethylene-modified heme b. Recent studies of myeloperoxidase
(MPO) (Fenna, R., Zeng, J., and Davey, C.(1995) Arch. Biochem.
Biophys. 316, 653-656; Taylor, K. L., Strobel, F., Yue, K.
T., Ram, P., Pohl, J., Woods, A. S., and Kinkade, J. M., Jr.(1995) Arch. Biochem. Biophys. 316, 635-642) suggest possible
prosthetic group similarities between MPO and LPO. To address heme
identity for LPO, we used comparative magnetic circular dichroism (MCD)
spectroscopy of LPO versus myoglobin (Mb), horseradish
peroxidase (HRP), and MPO. MCD spectra of native
Fe-LPO and
Fe
-CN
-LPO are
10 nm red
shifted from analogous forms of Mb and HRP, including the formate-Mb
adduct. MCD spectra of native LPO and MPO are opposite in sign, and MCD
spectra of their cyanoadducts also differ. These data indicate the LPO
heme is distinct from heme b of Mb and HRP as well as from ``heme
m'' of MPO. From this work and literature analysis, we suggest
that the non-extractable ``heme l'' of LPO has the two vinyl
groups of heme b but lacks the 2-sulfonium-vinyl linkage of heme m. The
observed red shifts in LPO spectra may derive from ester linkages to
protein as for MPO. Strong spectral analogies between LPO and mammalian
peroxidases (e.g. from saliva, eosinophils, thyroid,
intestine) indicate similar prosthetic heme moieties.
Lactoperoxidase (LPO, ()EC 1.11.1.7) is a mammalian
peroxidase that is the product of exocrine gland secretion, e.g. into milk, saliva, and tears(1, 2, 3) .
Its normal physiological role appears to be as a bacteriocide,
converting thiocyanate to hypothiocyanate in an
H
O
-dependent reaction. The bacteriocide
function of LPO is also of primary importance for the closely related
salivary peroxidase (SPO)(4) . LPO has recently been found in
human colostrum and may be a useful criterion for distinguishing
between hormone-dependent and hormone-independent types of mammary
cancer(5) . Yet another functional role for LPO may be in the
degradation of catecholamines such as norepinephrine(6) .
Finally, soluble LPO is both a functional and a spectral model for
membrane-bound thyroid peroxidase (TPO)(3, 7) .
The series of mammalian peroxidases, including LPO, SPO, TPO, eosinophil peroxidase, intestinal peroxidase, and myeloperoxidase (MPO), have related protein primary structures ( (8) and (9) , and references therein) and prosthetic heme moieties that are not readily extracted by conventional approaches(1, 2, 3) . The proteins also display spectral signatures that are distinct from those of known heme groups (Table 1)(1, 2, 3) . As a consequence, the structural identity of the heme moieties of mammalian peroxidases has been a subject of considerable controversy.
Proposed structures for the prosthetic group of LPO are shown in Fig. 1. Heme b (Fig. 1A) is found in many common heme proteins. These include myoglobin (Mb) and hemoglobin, the P450 enzymes, catalases, and peroxidases from yeast, plant, and fungal sources (cytochrome c peroxidase, horseradish peroxidase (HRP), lignin peroxidase, and chloroperoxidase). Evidence for heme b (Structure A) as the heme of LPO came from Pronase digestion of the enzyme(17) . Sievers (17) proposed that the anomalous electronic absorption spectrum of LPO and its pyridine hemochrome derivative, relative to those of known heme b-containing proteins (see Table 1), arose from an unusually constrained active-site pocket surrounding the heme. The 8-thiomethylene-substituted derivative of iron PPIX (Fig. 1B) was proposed as the LPO heme by Clezy and colleagues (18) following treatment of LPO with 2-mercaptoethanol and urea. This hypothesis would explain the lack of ready extractibility for the LPO heme moiety but does not fully address observed spectral variations from heme b.
Figure 1: Proposed heme structures for lactoperoxidase. A, iron protoporphyrin IX (heme b)(17) ; B, 8-thiomethylene-substituted heme b(18) ; C, the m heme of myeloperoxidase(9, 19) .
Most recently, Fenna and colleagues (9) and Taylor et al.(19) have determined the structure of the ``heme m'' prosthetic group of native MPO (Fig. 1C). These studies clearly identified the prosthetic group as an iron porphyrin, resolving a long standing controversy where unusual spectral properties of MPO were suggested to derive from an (atypical) iron chlorin prosthetic moiety (for example, (13) and references therein). Fenna et al.(9) suggested that close similarities in the protein sequences of MPO and LPO made it possible that LPO could have heme-protein linkages and a heme structure related to that of MPO.
Each of the three proposals for the native heme moiety of LPO (Fig. 1) has had its supporters. Evidence for the identity of the LPO heme has come from a variety of different experimental approaches. These include resonance Raman(20, 21) , NMR(22, 23) , and EPR (24) spectroscopy, as well as other types of biochemical analyses(25, 26) .
Two additional factors of importance with respect to the heme of
native LPO are 1) the nature and identity of the 5th axial ligand to
the heme moiety and 2) the presence or absence of a 6th ligand. The 5th
ligand, donated by the protein, is known to have a considerable effect
on observed spectral properties as well as on functional
characteristics. The classical example is Fe-CO-P450
(Soret band at
450 nm) in contrast with Fe
-CO-Mb
(Soret at
420 nm), where the observed differences derive from an
axial S-Cys
ligand in the case of P450 and an
axial N-His ligand in the case of Mb (e.g.(27) ). The S-Cys
5th ligand of
P450s has an effect on all of the spectral properties of these enzymes
relative to the analogous forms of other heme
proteins(28, 29, 30, 31, 32) .
Yet other examples are the N-His
5th ligand of
HRP, cytochrome c peroxidase, and presumably other peroxidases (33) and the O-Tyr
5th ligand of
catalases ((34) , and references therein). The 5th ligand to
the heme of LPO is either a histidine (35) or the deprotonated
N-His
common to many peroxidases (Refs. 22, 24,
36, and 37, and references therein). The 6th ligand of LPO has
variously been reported to be an unusual formate (35) or
H
O(20, 21) . Some assignment
inconsistencies exist such as a reported 5-coordinate
structure(37, 38) , whereas the body of other evidence
indicates that native LPO is
6-coordinate(20, 21, 39) .
In this work,
we have addressed the heme moiety of LPO with respect to the structural
proposals in Fig. 1. Our approach is comparative spectral
analysis of LPO and the analogous forms of: 1) Mb and HRP, both of
which have heme b and, respectively, either N-His or
N-His 5th ligands; and 2) MPO, which has the
novel heme m and N-His
ligation(9, 19) . These proteins were examined
in both their native and in the Fe
-CN
forms, using MCD and electronic absorption spectroscopy. MCD
spectroscopy is able to distinguish easily between 5- and 6-coordinate
ferric heme systems (16, 27, 34) and is
sensitive to heme-bound water(16, 34) . Thus we
directly compare LPO properties with those of heme systems proposed to
have identical prosthetic heme moieties, identical axial ligation, or
identity in both heme group and axial ligands.
Our data demonstrate
obvious spectral differences between the hemes of LPO and Mb or HRP,
and between the hemes of LPO and MPO, in both native and
Fe-CN
forms. Furthermore, the
spectral differences do not derive from axial ligands, as shown by
comparison of native LPO and Mb-formate. Thus, we suggest that the LPO
heme (called here heme l) is distinct from either heme b or heme m,
although it has similar peripheral substituents to each. Given the high
degree of spectral and protein sequence agreement between mammalian
peroxidases (8, 9, 19) , we suggest that the
heme l of LPO, or a very closely related heme, is likely to also be the
heme of TPO, SPO, eosinophil peroxidase, and other mammalian
peroxidases.
Bovine LPO was a kind gift from Prof. Harold M. Goff,
Department of Chemistry, University of Iowa. The enzyme was isolated
and purified as described
previously(36, 40, 41, 42, 43, 44) .
Enzyme concentration was determined using the published value:
(mM) at 412 nm = 114(45, 46) . Wild
type, recombinant human Mb was prepared as described
previously(16) . HRP, Sigma type XII (highly purified Sigma
type VI), was used as purchased.
All samples were examined in a 100
mM potassium phosphate buffer, pH 7.0. The cyanide adducts
were prepared by addition of a 50-200-fold excess of a cyanide
stock solution to a serum-stoppered cuvette (10 µl of a 0.5 M stock). The formate adduct of Mb was prepared by addition of
a stock solution to a final concentration of 0.1 M(27) .
The three protein samples and their various
adducts were examined by electronic absorption spectroscopy in either
5-mm or 1-cm quartz Suprasil cuvettes using Varian-Cary 219, Hitachi
U-2000, or Hitachi U-3200 spectrophotometers. MCD spectra were obtained
on a Jasco J-720 spectropolarimeter fitted with a 1.5-tesla (15 kG)
electromagnet in the sample compartment with the magnetic field
direction parallel to the direction of light propagation. Following
collection of the MCD/CD data, the samples were re-examined using
electronic absorption spectroscopy. ()In no case was an
absorption spectral change greater than
0.5% observed.
The
MCD/CD instrument was calibrated daily for intensity with ammonium d-10-camphor sulfonate (Jasco) and was calibrated bimonthly
with neodymium glass for wavelength. The MCD/CD data for each sample
were obtained using the following experimental conditions: 1 nm
bandwidth, 25 scan accumulation, 200 nm/min scan rate, 0.5 nm
resolution, and 13.9 kG (1.39 tesla) magnetic field. Final MCD data
were corrected for natural CD and for the buffer blanks using the Jasco
software and were then corrected for concentration, light path, and
magnetic field. The data presented in the figures have been smoothed
using the Jasco software. Samples were maintained at 14 °C
during data collection by use of a custom-made flow-through cell holder
and dedicated water bath(16, 32, 34) .
Final MCD data are presented in units of
(moles/liter
cm
tesla)
, where
(deg
cm
dmol
tesla
)
= 3300
.
The basic hypothesis on which this work rests is that
structural identity results in spectral identity. Even relatively small
structural changes can induce significant spectral effects. MCD
spectroscopy is well known as a useful probe in the structural analysis
of biological heme systems (e.g.(27) and references
therein). One particular advantage is the low sample concentrations
required relative to other biophysical methods. A second advantage lies
in the accuracy of results, with general agreement between MCD and
x-ray methods (e.g.(16) and (34) ). For
example, MCD spectroscopy indicated heme-bound HO for
ferric forms of His-64
Gln and His-64
Gly engineered
recombinant mutant Mbs (pH 5.6); these data were shown to concur with
results from x-ray analysis (16) .
The sensitivity
of MCD spectroscopy to heme-bound H
O has also been useful
in resolution of a controversy with regard to water occupancy in
functional states of mammalian, fungal, and bacterial
catalases(34) .
In this work, we use direct spectral
comparison for evaluation of heme b and heme m as potential prosthetic
groups for LPO. If native LPO has a heme b prosthetic group, its
spectra should be similar to those of either Mb or HRP, depending on
the 5th (axial) ligand (N-His or N-His,
respectively) to the LPO heme group and its coordination state
(6-coordinate, high spin or 5-coordinate, high spin, respectively). If
ferric LPO has heme b with a carboxylate 6th ligand (35) causing the observed red shifting of the electronic
absorption spectrum (Table 1), then one would expect the formate
adduct of Mb to display a close spectral similarity.
Yet another
data set addressing the heme moiety of native LPO comes from
preparation and analysis of the low spin, 6-coordinate
Fe-CN
adducts of LPO, Mb, HRP, and
MPO. With all the proteins having CN
as 6th ligand
and N-His or N-His
as 5th ligand, only
differences in the respective heme moieties could explain any observed
spectral variation. Therefore, we would expect
Fe
-CN
-LPO to display close spectral
similarity to Fe
-CN
-Mb or -HRP if
the LPO moiety is heme b or to
Fe
-CN
-MPO if the LPO heme moiety is
heme m.
In the
electronic absorption spectra, the high spin marker band of native LPO
is at 626 nm in comparison with 633 nm for human Mb (high spin,
6-coordinate) and 643 nm for HRP (high spin, 5-coordinate). The Soret
band of LPO is at 412 nm in contrast to 409.5 nm for Mb and 403 nm for
HRP. Upon formation of the Fe-CN
adducts, the spectra of all the proteins are altered, the high
spin marker band disappears from the visible region, and the Soret
bands are red shifted. For HRP and Mb the spectral features of the
cyanide adducts are not identical although they are very closely
similar to one another. This is as expected given identical heme
moieties and identical 6th ligands, with the only difference between
the Fe
-CN
adducts of HRP and Mb
being their respective 5th ligands. In contrast, the absorption
spectrum of Fe
-CN
-LPO is markedly
red shifted from the cyano adducts of HRP and Mb. Again, in the case of
the fluoro adducts, we see a close similarity in the spectral
properties of Fe
-F
-HRP and -Mb,
with the spectral features of the LPO complex being distinctly
different. Considering the identity of the 6th ligands for these three
proteins and the common assumption that LPO has a 5th ligand identical
to either Mb or HRP, these data do not appear to support a common heme
moiety for the three proteins.
For MPO, the recent elucidation of the novel heme m structure (Fig. 1C) (9, 19) permits analysis of the observed features in the electronic absorption spectra. Presumably the atypical spectral properties of MPO are derived from both the unusual 2-substituent and the 1,5-ester connections to the protein. One would also expect the lowered symmetry of heme m, arising from constraints placed on the porphyrin macrocycle by tethering to the protein at the 1-, 2-, and 5-positions of the heme, to affect structural and consequently spectral properties. As reported by Fenna et al.(9) , the high degree of sequence homology between mammalian peroxidases (LPO, TPO, SPO, and intestinal peroxidase(7, 10) ) does not include the Met residue necessary to form the novel 2-substituent link. However, because the sequence homology between MPO and LPO includes the Glu and Asp residues needed for the 1- and 5-ester linkages, Fenna and colleagues (9) suggested that the heme of LPO might be at least partially analogous to that of MPO.
Figure 2: MCD spectra of native LPO and Mb-formate. Solid line, native LPO (20.7 µM); dotted line, Mb-formate (9.73 µM Mb, 0.1 M formate). Experimental conditions are as described under ``Materials and Methods.''
Figure 3:
MCD spectra of ferric-cyanide adducts of
LPO, Mb, HRP, and MPO. Solid line, LPO (15 µM,
200-fold CN); dotted line, Mb (8.70 -fold
µM, 50
CN
); dashed
line, HRP (18.4 µM, 100
CN
). Experimental conditions are as described under
``Materials and Methods.'' The inset is the
ferric-cyano adduct of MPO, adapted from (12) .
The first interesting
observation is that the MCD spectra of the
Fe-CN
adducts of Mb and HRP, while
very similar in shape, intensity, and band position, are not identical.
There is a small blue shift for the HRP complex relative to the Mb
complex, concurring with the slight blue shift of the absorption
spectral bands of the HRP-cyanide adduct relative to the Mb-cyanide
adduct. Given the clear-cut identity of the heme moieties as well as
the 5th and 6th ligands for cyano-HRP and cyano-Mb, it is intriguing to
speculate that the spectral differences noted here are derived from the
key structural difference between the two proteins, the protonation
state of the 5th ligand. New results for our laboratory, supported by
x-ray structural analysis(32) , reveal the notable sensitivity
of CD spectroscopy to the presence/absence of a hydrogen bond to
heme-bound H
O. We have also shown that MCD spectroscopy is
sensitive not only to the presence of axially bound H
O but
also to the presence of hydrogen bonding between the heme-bound water
and a distal residue, once again supported by x-ray structural analysis (16) .
This work is the first report of MCD
sensitivity to a hydrogen bond interaction at the 5th ligand of a heme
protein.
The MCD spectrum for the
Fe-CN
adduct of LPO, while similar
in overall shape to the analogous spectra of Mb and HRP, is red shifted
by
8-10 nm for all features. These include, for example, the
Soret peak of LPO at 423.5 nm (413.5 nm for HRP, 415.5 nm for Mb), the
crossover (MCD zero point) of LPO at 431 nm (421 and 423 nm for HRP and
Mb), and the Soret trough of LPO at 438.5 nm (428.5 and 430 nm for HRP
and Mb). These data indicate that the heme of LPO is not identical with
the heme b moiety of HRP and Mb. Furthermore, they indicate that the
LPO heme group differs from heme b by the presence of one or more
electron-withdrawing substituents in addition to the two vinyl moieties
of heme b (Fig. 1A).
The MCD spectrum of
Fe-CN
-LPO is also distinct from
that of Fe
-CN
-MPO (Fig. 3, inset)(12) . The latter has a resolved positive band
slightly below 400 nm, a stronger positive peak at
430 nm, a
crossover at
440 nm, and a trough at
447 nm. Given the newly
reported structure of heme m of MPO (Fig. 1C), the
peripheral substituents of this heme must play a dominant role in the
MCD spectrum, because MPO has also been shown to have a histidine 5th
ligand(9) . It is evident that the
Fe
-CN
adduct of LPO is distinct
from that of Fe
-CN
adducts of
either heme b or heme m systems.
Figure 4: MCD spectra of native ferric LPO, Mb, HRP, and MPO. Solid line, LPO (20.7 µM); dotted line, human Mb (10.9 µM); dashed line, HRP (19.93 µM). Experimental conditions are as described under ``Materials and Methods.'' The inset is the native ferric form of MPO, adapted from (12) .
For the native heme proteins, it is the MCD spectrum of MPO (Fig. 4, inset) that is anomalous. Note that its pattern of bands is reversed relative to those of LPO and Mb, with the most intense positive band at low energy and the most intense negative band at high energy. As previously discussed(11, 12) , this reversed MCD pattern is the expected one for an iron chlorin or a formyl-substituted porphyrin. Given that neither of these is consistent with the new reports of the heme m structure of MPO (see Fig. 1C)(9, 19) , it appears that this pattern is also representative of a very low symmetry heme with electron-withdrawing substituent(s). We suggest that three points of attachment between the heme and the protein may have a strong spectral affect, in analogy with cytochrome c, for which the two points of heme attachment to the protein lower the symmetry and affect the resonance Raman data(49) .
Evaluation of the native ferric heme proteins therefore indicates that the heme moiety of LPO is distinct not only from the heme b moiety of Mb and HRP but also from the heme m group of MPO. The LPO heme is likely to have additional substituent(s) that are electron withdrawing and/or symmetry lowering (beyond the two vinyl groups of heme b), although less so than occurs for MPO where the peripheral substituents result in a total reversal of the MCD pattern.
From sequence information discussed by Fenna et
al.(9) , it appears that LPO is sufficiently related to
MPO to result in retention of the two ester linkages to the heme (the
2-linkage is not possible). Given this information and NMR data
demonstrating close similarity between MPO and LPO(22) , ()a structural hypothesis for the heme moiety of
lactoperoxidase becomes possible.
The data presented in this work eliminate all three of the
structures in Fig. 1as the heme moiety of LPO. Iron PPIX (Fig. 1A) is eliminated by obvious spectral differences
between native and cyano-forms of LPO in comparison with those of Mb
and/or HRP. Previous suggestions of a formate-ligated heme b are also
eliminated. Spectral differences between LPO and its various ligand
complexes and the analogous Mb/HRP species are on the order of
8-10 nm. We suggest that this spectral variance is too large
to be accounted for by the simple modification of heme b that produces
the thioethylene-modified heme b (Fig. 1B). The novel
heme m group of MPO (Fig. 1C) can also be eliminated as
the prosthetic heme of LPO simply on the basis of clear-cut differences
between the MCD data shown.
Our proposal for the structure of the prosthetic group of LPO is shown in Fig. 5. We suggest that the LPO heme moiety, which we call heme l, has vinyl moieties at the 2- and 4-positions as is the case for heme b. In addition, the heme l of LPO has ester groups at positions 1 and 5 like heme m of MPO. This agrees with NMR data for MPO and LPO where spectral similarities between the two mammalian peroxidases were observed(22) . It is also consistent with reported sequence similarities between the two proteins(8, 9) . The two substituents should be sufficient to induce the observed red shifting of the MCD data and to prevent ready removal of the LPO heme without causing the complete spectral changes observed for MPO. Finally, there is the intriguing evidence from Ikeda-Saito and colleagues(24) , where the spectra of MPO following chemical modification (photoreduction) were surprisingly similar to those of LPO.
Figure 5: New structure proposal for the heme l moiety of LPO.
Mammalian peroxidases are fundamentally distinct from peroxidases from yeast (cytochrome c peroxidase), plant (horseradish and turnip peroxidases), or fungal (lignin and fungal peroxidases) species. The key difference is apparently simple; mammalian heme groups are non-extractable whereas yeast, plant, and fungal peroxidases all have extractable heme b prosthetic groups(1, 2, 3) . In many cases failure of mammalian heme groups to respond to conventional chemical treatment has been attributed to factors such as unusual or constrained active-site environments that retain the heme as is the case for catalase ((34) , and references therein). However, increasing evidence indicates that the origin of this non-extractability for the heme groups of the mammalian peroxidases rests in the important evolutionary distinction of covalent attachment of the heme group to the protein.