(Received for publication, September 1, 1995; and in revised form, December 20, 1995)
From the
A heme d prosthetic group with the configuration of a cis-hydroxychlorin -spirolactone has been found in the
crystal structures of Penicillium vitale catalase and Escherichia coli catalase hydroperoxidase II (HPII). The
absolute stereochemistry of the two heme d chiral carbon atoms has been
shown to be identical. For both catalases the heme d is rotated 180
degrees about the axis defined by the
-
-meso carbon atoms,
with respect to the orientation found for heme b in beef liver
catalase. Only six residues in the heme pocket, preserved in P.
vitale and HPII, differ from those found in the bovine catalase.
In the crystal structure of the inactive N201H variant of HPII catalase
the prosthetic group remains as heme b, although its orientation is the
same as in the wild type enzyme. These structural results confirm the
observation that heme d is formed from protoheme in the interior of the
catalase molecule through a self-catalyzed reaction.
The vast majority of heme proteins contain iron protoporphyrin
IX (heme b) as the prosthetic group, but in recent years an increasing
number of naturally occurring porphyrins with different covalent
structures has been emerging(1) . Among those, chlorins and
isobacteriochlorins, which contain a partially saturated porphyrin
macrocycle, have received special attention. In particular, two
proteins isolated from Escherichia coli, the terminal oxidase
that predominates at low levels of oxygen (1) and the catalase
HPII(2) , have been shown to contain two structurally related
chlorins (Fig. 1) that have been generically termed heme d.
Spectroscopy data on the demetallized and esterified prosthetic groups
of both enzymes were consistent (3) with the presence of a
-spirolactone at the saturated pyrrole ring III of the macrocycle
in the C-6 position and a vicinal C-5 hydroxy group, although the
relative orientation of these two groups was shown to be different in
the two proteins(2) . In the terminal oxidase, the two
substituents have the more stable trans configuration (Fig. 1A, ii), while in the prosthetic group
of HPII they have the cis configuration (Fig. 1A, i). The absolute stereochemistry of
the two chiral centers of the HPII heme d has never been reported.
Figure 1:
A, structures of the cis- (i) and trans- (ii) heme d
hydroxy--spirolactones and the related cis- (iii) and trans- (iv) diol compounds.
The absolute configuration shown for the two chiral carbon atoms of i is the one obtained in this work from the crystal structure
of PVC and HPII catalase. The absolute configuration of the two trans-heme d derivatives related to the cytochrome d terminal oxidase (ii, iv) is at present unknown, and the
one shown in the figure is only for the purpose of comparison. B, heme b structure with the Protein Data Bank atomic
nomenclature used in the Introduction. The orientation of the heme
shown (defined by the relative positions of vinyl groups in pyrrolic
rings I and II) corresponds to the one found in beef liver catalase
when the heme is viewed from the distal
side.
Additional data on the cytochrome d terminal oxidase (4) suggested that the actual form of the prosthetic group
present in the interior of the enzyme was the corresponding trans-diol (Fig. 1A, iv), and that
lactonization occurred spontaneously during isolation of the heme. By
analogy with this system, and due to the facile formation of the
-lactone ring(3, 5) , it was also assumed that
the corresponding cis-diol (Fig. 1A, iii) was the actual species in the active site of
HPII(2, 6) . The enzymes responsible in E. coli for the conversion of protoheme into heme d have never been
identified, and this led Timkovich and Bondoc (1) to propose
that the formation of cis-heme d may be catalyzed by HPII
itself. According to this hypothesis, Loewen et al.(7) reported the conversion of the protoheme cofactor to
heme d in the presence of a source of hydrogen peroxide, using heme b
containing recombinant HPII.
For a number of fungal catalases, the
presence of heme prosthetic groups with altered covalent structure has
been inferred from their electronic spectra. The optical spectra of
catalases from Penicillium vitale(8) and Neurospora crassa(9) have been suggested to arise
from a chlorin-like structure. In other organisms, e.g. Penicillium
chrysogenum(10) , the electronic spectrum of the ferric
enzyme resembles that of a chlorin derivative ( = 405, 590, 720 nm), although the authors do not mention
this peculiarity in their report. To our knowledge, no relationship
with the bacterial heme d structure had been proposed for the
prosthetic group of these fungal catalases.
Catalase (hydrogen
peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6) is present in
virtually all aerobic organisms where it dissociates hydrogen peroxide
into molecular oxygen and water. The most common form of the enzyme is
a homotetramer with one porphyrinic prosthetic group per subunit. The
three-dimensional structures of five of these heme-containing catalases
have been reported: P. vitale (PVC) ()(Protein Data
Bank code 4CAT; polyglycine coordinates from a structure determination
at 2.0-Å resolution, see below)(11) , beef liver (BLC)
(Protein Data Bank codes 7CAT and 8CAT; both structures at 2.5-Å
resolution) (12) Micrococcus lysodeikticus (MLC) (13) (at 1.5Å resolution), HPII catalase from E. coli(6) (Protein Data Bank code not yet available) and Proteus mirabilis (PMC) ((14) ; Protein Data Bank
codes 1CAE and 1CAF, at 2.2- and 2.8-Å resolution, respectively).
PVC and HPII catalases contain a C-terminal domain of about 150
residues, with a ``flavodoxin-like'' topology that is absent
in BLC, MLC, and PMC. These smaller catalases, lacking a
``nucleotide binding'' domain, can bind a molecule of
NADPH(14) , while no nucleotides have been found to bind to PVC
or HPII. The three-dimensional structure of native PVC was reported at
2.0-Å resolution with an agreement R factor of 31.4% for
a partial, x-ray, sequence(11) . The resulting electron density
map was not sufficient, despite of the resolution, to precise the
chemical modifications present in the heme prosthetic group and
suggested by the spectroscopic data. For HPII the 2.8-Å
resolution structure (R factor 20.1%) supported the presence
of a modified heme in the enzyme, although its conformation could not
be defined(6) .
In this work we describe the configuration
of the heme d group now determined in the crystal structures of PVC at
1.8-Å resolution for both the native enzyme and the complex with
3-amino-1,2,4-triazole (with R factors of 16.7 and 14.8%,
respectively) and in the structure of the HPII catalase with azide, at
2.2-Å resolution (R factor 18.1%). Comparison of the
heme environment in PVC and HPII catalase with the mammalian BLC
reveals common peculiarities of the heme d pockets. The configuration
of the heme b group found in the crystal structure, at 2.2-Å
resolution (R factor 18.5%), of the essentially inactive N201H
variant of HPII(7) , in which the distal Asn residue has been replaced by a histidine, is also considered.
The heme d group characterized in the active sites of the
refined crystal structures of both PVC and HPII catalase possesses the
structure of the cis-hydroxy -spirolactone (Fig. 1A, i), as can be clearly inferred from
the electron density maps (Fig. 2). The absolute configuration
of the two
-carbon atoms of the macrocycle bearing the hydroxy and
the spirolactone substituents is S and R, respectively. In the two
enzymes, the electron density of the methyl and vinyl side chains of
the pyrrole rings I and II indicates that the orientation of the heme d
is mostly inverted (see below) with respect to that found for the heme
b group in BLC(12) , while the electron density for the
-spirolactone and the hydroxy heme substituents clearly appears in
the pyrrole ring opposite to the essential distal histidine, Fig. 3. Therefore, the heme d prosthetic group of PVC and HPII
catalase is mainly the result of modification of the pyrrole ring III,
in agreement with the biochemical data available for HPII(2) .
Figure 2:
Stereo
views of (F - F
) omit maps of the heme groups of PVC (A), HPII (B), and the N201H variant (C).
The structures of the prosthetic groups modeled inside the density are
also shown. The proximal tyrosine ligand (not shown) resides in the lower part of the drawing (see Fig. 3), and
the pyrrolic rings closer to the viewer are rings II and III. Ring III
is modified in both PVC and native HPII but not in the N201H variant
(see ``Results''). D, detail of the heme d group of PVC. The quality of the electron density allows us to
distinguish the relative positions of the methyl carbon and the
hydroxylic oxygen atoms.
Figure 3:
Van der Waals representation of the heme
d. The proximal tyrosine ligand (Tyr in PVC) is shown to
emphasize the orientation toward the proximal side of the ring III
hydroxylic and lactonic oxygens, which can interact with the aromatic
ring of the tyrosine. The modified ring is far from the essential
histidine (His
in PVC).
In all heme-containing catalases whose structures have been
reported, the heme prosthetic groups are well buried inside the
tetramer, about 20 Å from the nearest molecular
surface(6, 11, 12, 13, 14) .
Therefore, the presence of heme d and the 180° rotation, with
respect to BLC, of the prosthetic group in PVC and HPII can be assumed
to be accommodated by peculiarities that are probably common to the
heme pockets of both proteins. While the pyrrolic rings III and IV of
heme b are symmetric with respect to a 180° rotation about the axis
defined by the -
-meso carbon atoms, the ring I and ring II
methyl and vinyl substituents exchange positions. Thus the observed
preference in the orientation in which heme b binds to a particular
heme protein is determined by the different contacts that these
substituents make with the polypeptide chain. For PVC and HPII, the
presence of a
-spirolactone ring and an additional hydroxy group
makes the heme d more asymmetric. Nevertheless, the N201H variant of
HPII, which possesses an unmodified heme b group, exhibits the same
heme orientational preference as the wild type enzyme and opposite to
the one found in BLC. It is therefore logical to assume that the
different contacts of the methyl and vinyl groups in one heme
orientation or in the reverse are the main determinant of the
orientation of the heme inside the pocket. Heme-contacting residues
that apparently govern the heme orientation in PVC (Ile
,
Val
, Pro
, and Leu
) are
preserved in HPII (Ile
, Ile
,
Pro
, and Leu
) and show marked differences
from the structurally equivalent residues in BLC (Met
,
Ser
, Leu
, and Met
,
respectively). However, none of the substitutions appears to introduce
by itself a strong steric hindrance, and therefore, individually, they
would not provide a definitive explanation for the observed heme
inversion. It is also possible that other differences could be at play
during the initial binding of the heme groups to catalase.
The
interactions of the modified pyrrolic ring in the heme d group are
represented for both PVC and HPII in Fig. 4. A hydrogen bond is
formed between the hydroxy group (OND) of heme d, acting as the
hydrogen donor, and the O- oxygen of a serine residue (sequence
number 349 in PVC and 414 in HPII). This serine is in turn
hydrogen-bonded to the carboxylate oxygen of an aspartic residue
(sequence number 53 in PVC and 118 in HPII) from another subunit
related by a molecular dyad axis(6, 11) . These
interactions must contribute to the stabilization of the heme d with
the hydroxyl oxygen pointing toward the proximal side. In BLC (12) and MLC (13) the equivalent residue is an alanine
(residue 356 in BLC). However, in PMC (14) a serine residue is
maintained in this position, although heme d has not been reported. O2D
is hydrogen-bonded to a water molecule (Fig. 4), which in turn
is bound to the unmodified propionic group. O2D also interacts with the
NE2 atom of a glutamine residue (residue 354 in PVC) that is present in
most catalase sequences but is replaced by histidine in BLC (residue
361). In PVC, but not in the HPII model, O2D can also form a hydrogen
bond with the guanidinium group of Arg
. On the distal
side, the modified pyrrolic ring forms almost exclusively hydrophobic
interactions with the side chains of Ala
and Val
in PVC (Ile
and Val
, respectively, in
HPII). The existence of these interactions further confirms the
orientation of the heme d ring III oxygens toward the proximal side (Fig. 4). In both enzymes, the heme d oxygens O1D and OND are
close to the aromatic ring of the tyrosine axial ligand, with several
interatomic distances shorter than 3.5 Å (Fig. 3). These
polar interactions (23) could alter the electronic distribution
in the aromatic ring, influencing the catalytic properties of heme d in
catalases.
Figure 4: Stereo views of the environment of the modified pyrrolic ring III of heme d in PVC (A) and HPII (b) and of heme b in the N201H variant (C). Hydrogen bonds are represented by dotted lines, and the lengths are indicated (see ``Results'').
The structure of the heme b-containing N201H variant of
HPII allows us to analyze the changes introduced in the protein when
heme d is present in the interior of the catalase molecule. As
indicated above, the orientation of the heme is also reversed with
respect to BLC in the structure of the N201H variant. Heme inversion is
therefore previous and perhaps unrelated to heme d formation. The main
structural changes between the wild type enzyme, containing heme d, and
the N201H variant are in the side chains of residues Gln and Ile
(Fig. 4). In the N201H structure,
the negative charge of the ring III propionic group is neutralized by
two salt bridges with the guanidinium group of Arg
(Fig. 4C). In wild type HPII, this guanidinium
group interacts with the OE1 oxygen of Gln
, whose side
chain experiences an important rearrangement (Fig. 4B).
The torsion angle
of residue Ile
,
situated in the distal side above the modified pyrrolic ring, changes
from -56° in N201H to 168° in the native structure,
probably to avoid contacts with the heme d carbon atoms CMD and CAD
that project outside the heme plane. In PVC the equivalent residue is
an alanine, and no reorientation is required.
Neither extra solvent
molecules nor cavities have been detected in the two heme d-containing
catalases in the vicinity of the modified pyrrolic ring. In fact, no
important volume changes between the heme b and heme d structures are
expected, since the increment in volume due to the presence of an extra
hydroxy group in the modified heme d is, at least in part, balanced by
the formation of the covalent bond between O1D and C3D to give the
cyclic -lactone.
The present crystallographic study of PVC and HPII catalase
active sites has shown that the major orientation of the prosthetic
group in these two enzymes differs by a 180° rotation about the
-
-meso carbon axis with respect to that found for the heme b
group in BLC. However, there is also evidence of heme rotational
disorder in the pocket of these two catalases, a phenomenon that has
been reported for other heme proteins like myoglobin (24) and
cytochrome b
(25) , from NMR studies. The
protein residues responsible for the observed preferences in the
orientation in which the heme is bound and the possible functional role
for the existence of heme rotational disorder in those proteins are
subjects of continuing debate.
The chemical nature of the heme d in
PVC and HPII catalase has been determined, and the presence of a
hydroxy group and a -spirolactone ring with a relative cis configuration in the active form of the prosthetic group has been
confirmed. The absolute configuration of the two carbon atoms of the
modified pyrrolic ring III of the macrocycle has also been determined.
The heme d prosthetic group, which is formally the result of the
cyclization of the cis-diol (Fig. 1A), has
both hydroxy groups directed toward the proximal side of the enzyme,
while it is generally accepted that the oxygen chemistry occurs on the
distal side of the prosthetic group(7) . This apparently
contradictory observation raises an interesting question regarding the
biosynthesis of the cis-heme d, which will have to be taken
into consideration by any mechanism that tries to explain the formation
of this derivative.
The electron density indicates that in both PVC and HPII catalase, the heme modification occurs only in the pyrrolic ring opposite to the essential distal histidine. Thus the presence of a small percentage of rotationally inverted heme in the active sites of both PVC and HPII catalase implies the existence of some ring IV modified heme in the interior of both. Experiments performed with the extracted prosthetic group of HPII (7) showed the presence of a small fraction of an unidentified product, chromatographically distinct from the ring III modified heme d, which could correspond to this second heme d-type derivative.
The identification of a heme b group in the crystal structure of the inactive N201H variant of HPII agrees with the hypothesis (1, 7) that heme b is not converted in vivo into heme d by a specialized hydroxylating system and subsequently incorporated into PVC or HPII catalase. Rather, the heme b chromophore binds to the active site of the enzyme, where it is converted into the heme d derivative during the initial cycles of the catalytic turnover.
The high structural similarity of the heme binding pocket among catalases from different prokaryotic and eukaryotic sources does not imply the presence of the same prosthetic group in all of them. The changes necessary to accommodate the modified heme d chromophore in the interior of the pocket are relatively small. Through the analysis of several catalase sequences we have observed that most of the residues that participate in the stabilization of the heme d are also present in other catalases not containing this prosthetic group. It is therefore unclear whether the presence of heme d is related only to those residues in the pocket or if some more distant effects also have a significant contribution.
What is the functional role of heme d in catalases? At present this question does not have a definite answer, and one can only speculate. PVC and HPII catalase exhibit, apart from the different heme d prosthetic group, other structural and functional peculiarities that are not shared by other well characterized catalases, which may or may not be related to the presence of heme d in the active site of these two enzymes. In particular, the presence of the extra C-terminal domain and heme d may be correlated, as they are both attributes of PVC and HPII.
BLC isolated from natural sources always contains a relatively large amount of bile pigments from the oxidative degradation of the heme prosthetic group in the interior of the enzyme(26) . In contrast, PVC and HPII catalase contain almost exclusively heme d or, in certain cases, a small fraction of unmodified heme b. Heme d may be more resistant to the oxidative damage that heme b experiences during the catalytic turnover in BLC.
The catalytic cycle of a normally operating catalase involves two states of the enzyme, the resting Fe(III) state and the so-called compound I, which is the result of the reaction with one molecule of peroxide and is oxidized 2 equivalents above the resting enzyme. Compound I reacts with another molecule of hydrogen peroxide to give molecular oxygen and water, returning to the resting enzyme and so completing the catalytic cycle. However, under certain conditions, compound I of some catalases like BLC can be reduced by one electron to give compound II, which possesses an intermediate oxidation state between that of compound I and the resting enzyme. In turn, compound II can react with another molecule of peroxide to give compound III, a catalytically inactive intermediate. It is believed that the function of the bound nucleotide in BLC is to protect the enzyme from the formation of compound III, thus avoiding the inactivation of the enzyme (27) . Interestingly, PVC and HPII catalase that possess the extra C-terminal domain with a ``flavodoxin-like'' topology lack the ability to bind NADPH. Nevertheless, HPII catalase compound I shows an extraordinary resistance to reaction with one electron donors to give compound II(28) . According to the currently accepted electronic structure of the porphyrin macrocycle(29) , the partial saturation of one of the pyrrole rings does not destroy the aromatic character of a chlorin-like derivative. One consequence of this is that the Fe(III)/Fe(II) reduction potentials of d-type hemes fall within the normal range observed for heme b derivatives(1) . However, one cannot exclude the possibility that in the interior of the protein a substantial difference exists in the reduction potentials of the higher oxidation states involved in the catalytic turnover of catalase, which could explain the observed resistance of HPII catalase to form compound II. Other authors have noted the propensity of hydroxychlorin derivatives to form hydrogen bonds and have suggested that this property may contribute to their biological function by helping to anchor them within the interior of the heme protein(30) . Finally, if none of these correlations can be proved to be true, one can still consider the presence of heme d in PVC, HPII catalase, and very likely catalases from other sources as the equivalent to a neutral mutation in the primary sequence of an enzyme. In this hypothesis, heme d would be a ``mutated'' prosthetic group, whose modification would not affect the function of catalase. However, the fact that the structure of the heme d chromophore is well preserved in evolutionary distant organisms, like in the prokaryotic HPII catalase and the eukaryotic PVC, seems to argue against this possibility.