(Received for publication, April 3, 1995; and in revised form, June 22, 1995)
From the
The structures of the cyanide and triiodide complexes of Arthromyces ramosus peroxidase (ARP) at different pH values were investigated by x-ray crystallography in order to examine the behavior of the invariant residues of arginine (Arg-52) and distal histidine (His-56) during the enzyme reaction as well as to provide the structural basis of the active site of peroxidase. The models of the cyanide complexes at pH 7.5, 5.0, and 4.0, respectively, were refined to the R-factors of 17.8, 17.8, and 18.5% using 7.0-1.6-Å resolution data, and those of the triiodide complexes at pH 6.5 and 5.0 refined to 16.9 and 16.8% using 7.0-1.9-Å resolution data.
The structures of the cyanide
complexes at pH 7.5, 5.0, and 4.0 are identical within experimental
error. Cyanide ion bound to the heme in the bent conformation rather
than in the tilt conformation. Upon cyanide ion binding, the
N atom of His-56 moved toward the ion by rotation of
the imidazole ring around the C
-C
bond, but there was little conformational change in the remaining
residues. The distance between the N
atom of His-56 and
the nitrogen atom of the cyanide suggests the presence of a hydrogen
bond between them in the pH range investigated. In the triiodide
complexes, one of the two triiodides bound to ARP was located at the
distal side of the heme. When triiodide bound to ARP, unlike the
rearrangement of the distal arginine of cytochrome c peroxidase that occurs on formation of the fluoride complex or
compound I, the side chain of Arg-52 moved little. The conformation of
the side chain of His-56, however, changed markedly. Conformational
flexibility of His-56 appears to be a requisite for proton
translocation from one oxygen atom to the other of HOO
by acid-base catalysis to produce compound I. The iron atom in
each cyanide complex (low-spin ferric) is located in the heme plane,
whereas in each triiodide complex (high-spin ferric) the iron atom is
displaced from the plane about 0.2 Å toward the proximal side.
Peroxidases (donor: HO
oxidoreductase
(EC 1.11.1.7)) are a class of heme proteins which oxidize a wide
variety of organic and inorganic compounds by the use of hydrogen
peroxide. In addition to their physiological importance, they have
attracted attention because of useful applications which include assays
of biological substances (Allain et al., 1974; Akimoto et
al., 1990) and large scale use for deliginification and the
bleaching of dyes (Pedersen and Carlsen, 1994). The reaction generally
consists of the following steps (Reactions
I-IV).
Since the crystal structure of yeast cytochrome c peroxidase (CCP), ()a typical class I peroxidase
(Welinder, 1992), was first determined at 1.7-Å resolution
(Finzel et al., 1984), the mechanism of compound I formation,
the first step in the above equations, has been proposed for CCP. The
mechanism of compound I formation suggested is that the distal
histidine and arginine concertedly stabilize charge separation and
facilitate proton transfer from one oxygen to the other of the peroxide
as well as heterolytic cleavage of its O-O bond (Finzel et
al., 1984). To confirm the validity of this mechanism, mutant CCP
proteins have been prepared, and their three-dimensional structures and
spectroscopic and kinetic properties extensively studied. For example,
Erman et al.(1993) reported that the distal histidine is
critical for the rapid formation of compound I of CCP, and Vitello et al.(1993) reported that arginine is responsible for
substrate binding in the heme pocket and for stabilizing compound I.
The three-dimensional structures of a lignin peroxidase from Phanerochaete chrysosporium (LiP), a peroxidase from Arthromyces ramosus (ARP) and a manganase peroxidase from P. chrysosporium (all class II peroxidases) have been,
respectively, determined at 2.0-, 1.9-, and 2.06-Å resolutions
(Edwards et al., 1993; Piontek et al., 1993; Poulos et al., 1993; Kunishima et al., 1994; Sundaramoorthy et al., 1994)). ()Although the histidine and
arginine residues at the distal side of the heme are conserved in ARP
and LiP, structural comparison of these peroxidases with CCP has shown
that the conformations of the distal histidine residue (His-56 in ARP)
relative to the heme differ (Kunishima et al., 1994). In
addition, preliminary structural refinement of the triiodide complex of
ARP, used for phase determination by the isomorphous replacement method
(Kunishima et al., 1994), has shown that the behavior of the
distal arginine (Arg-52 in ARP) differs from that observed in CCP when
it forms a complex with fluoride (Edwards and Poulos, 1990) or when it
is converted to compound I
(Fülöp et al., 1994).
Unexpectedly preliminary ligand binding experiment showed that most
ligands bound hardly to the heme besides triiodide and cyanide. Because
the behavior of these residues may be affected by the pH, we examined
how the structure of ARP changes when it binds ligand at different pH
values.
Heme proteins that form complexes with ligands have been investigated extensively to elucidate the nature of the heme iron and structure-property relationships, such as the spin state versus the displacement of the iron atom from the heme plane (Perutz, 1970). For peroxidases, although the cyanide bound forms of CCP, LiP, horseradish peroxidase, and Coprinus cinereus peroxidase have been characterized by NMR (Satterlee and Erman, 1991; de Ropp et al., 1991; Banci et al., 1991; Thanabal et al., 1988; Veitch et al., 1994), crystallographic studies of these forms are lacking; only the structures of the inhibitor-bound forms of CCP are known (Edwards and Poulos, 1990). In addition, different modes of ligation to the heme have been reported; e.g. in CCP cyanide binds to the heme iron in a tilted configuration (Edwards and Poulos, 1990), whereas in metmyoglobin it binds to its heme iron in a bent configuration (Neya et al., 1993). This difference might be ascribed to the protein environment of the heme, but it is likely that for some heme proteins unambiguous results could not be obtained because of the lack of detailed three-dimensional structures. In particular, in the case that ligand sizes are small, a high resolution x-ray crystal structure is required to detect any subtle structural change and to define the mode of ligand binding.
We present here detailed structural features of the heme and its environment as deduced from high resolution x-ray analysis of the cyanide and triiodide complexes and show the behavior of the distal histidine and arginine residues when ARP binds to cyanide and triiodide.
Complex formation of ARP with triiodide was
monitored by adding 3 mM KI solution stepwise to 6
µM ARP solution containing 1 mM KI. The
equilibria of triiodide and ARP were examined at pH 6.0, 7.5, and 9.0,
the buffers used being 50 mM sodium succinate for pH 6.0, 50
mM Tris-HCl for pH 7.5, and 50 mM sodium borate for
pH 9.0. The ionic strength of the reaction mixture was adjusted to 0.1 M with sodium nitrate. 3 mM KI
solution
was prepared by diluting 30 mM KI
stock solution
(prepared by adding iodine to about 8 eq molar excess KI) with 100
mM KI solution.
Preparation of the fluoride complex was
attempted by adding sodium fluoride to the ARP solution at pH 5.5. But
the absorption spectrum did not change significantly even when a 30 eq
molar excess of sodium fluoride was added. ()
The triiodide-derivative crystals were prepared by
soaking native ARP crystals for 2 h in mother liquor consisting of 33%
saturated ammonium sulfate, 20 mM sodium succinate, pH 6.0,
and 2 mM KI. The derivative crystals were
isomorphous with the native ones. For preparation of the derivative, it
was essential to lower the pH. The crystal soaked at pH 7.5 showed
little change in x-ray diffraction intensity.
Initially for each cyanide
complex, the solvent molecules around the heme were omitted, and the
model was refined by XPLOR using the diffraction data with F>2 in the 7.0-1.6-Å
resolution range. A 2F
- F
map clearly located the cyanide ion at
the sixth coordination position of the heme iron. When the positions of
the peaks in the 2F
- F
and F
- F
maps satisfied the
geometries of the hydrogen bond with the protein and/or another water
molecule, they were included in the subsequent refinement. The final
refinement was done including the solvent molecules as well as the
cyanide ion.
For the triiodide complexes, a difference Fourier map
with the coefficient of (F - F
) exp(i
)
at 2.2-Å resolution located two clearly resolved triiodide ions
near the heme, where F
and F
are the structure factors of the
triiodide complex and the native crystal, respectively. An F
- F
difference Fourier using the refined derivative phases
(
) was then used to position the
water molecules. Final refinement used the diffraction data with F>2
in the 7.0-1.9-Å
resolution range. Results are given in Table 2. The mean
coordinate error for each model is estimated to be about 0.2 Å
(Luzatti, 1952).
Figure 1: Absorption spectra of the native ARP (solid line), the cyanide (dot-dash line), and triiodide (dashed line) complexes. The spectrum of the native ARP is identical to that of C. cinereus peroxidase (Morita et al., 1988).
Triiodide binding to ARP results in a shift
of from 405 to 402 with an isosbestic point at 414
nm. The spectrum of the triiodide complex is apparently the high-spin
type. The ARP spectra with 1 eq triiodide added to the solution at pH
7.5 and 9.0 were similar to the spectra at pH 6.0 (data not shown).
When ammonium sulfate, however, was present in the solution, the
binding of triiodide to ARP was inhibited at a pH higher than 7.0.
Figure 2:
(2F - F
) map of the cyanide complex (pH 5.0) at 1.6-Å resolution.
The heme in a protein binds to
a cyanide with the C-N bond being often tilted from the normal of the
heme plane, where two ways of binding are possible: tilt and bent
conformations (Deatherage et al., 1976). As deviation from the
spherical shape of the electron density is small due to the short C-N
distance of the cyanide (1.15 Å), it was difficult to define its
orientation precisely even by 1.6-Å resolution analysis. From Table 3there appears to be no clear correlation between the bent
angles and pH values. The cyanide is tilted slightly from the normal of
the heme plane (the mean angle of the FeC(CN) vector
to the heme plane is 87°), and the bending of
Fe
C-N is significant (the mean angle of
Fe
C-N is 159°). Therefore the bent configuration
is more likely for the cyanide complex of ARP. Edwards and
Poulos(1990), however, reported that the cyanide model fit slightly
better to the electron density in the tilted configuration on the basis
of the x-ray crystal structure analysis of the cyanide bound complex of
CCP at 1.85-Å resolution.
Figure 3: Superposition of the model of the cyanide complex (blue) on that of the native ARP (white). The model of the native ARP is at pH 5.5 (Footnote 4) and that of the cyanide complex is at pH 5.0.
NMR studies on cyanide-bound
horseradish peroxidase, CCP, LiP, and C. cinereus peroxidase enabled assignment of the signal of the hydrogen atom
bonded to each N of the distal histidine in the pH
range of 7 and 6 (Thanabal et al., 1988; Satterlee and Erman,
1991; de Ropp et al., 1991; Veitch et al., 1994). Our
present crystallographic and NMR results show that it is reasonable
that His-56 is the hydrogen donor in the hydrogen bond in the
cyanide-bound ARP, which is why its N
is protonated in
the pH range investigated. The N
atom of His-56 must be
protonated because the hydrogen bond network of N
(His-56)
C=O (Asn-93) and NH
(Asn-93)
C=O (Glu-87) is retained in the
cyanide complexes. The hydrogen bond between the N
atom
of His-56 and the cyanide even at pH 7.5 is noteworthy because the
N
atom of the distal histidine is usually deprotonated
at pH 7.5, the pK
value of the free
histidine being about 6. We conclude that the pK
of the hydrogen-bonded network of
CN
H
-His-56 in the
cyanide complex is much higher than that of the distal histidine in the
native enzyme. Upon cyanide binding, a few of the water molecules are
rearranged to form a hydrogen bond network, the cyanide pushing Wat-674
away by 1.6 Å and Wat-676 by 1.8 Å (Fig. 3).
Figure 4:
Difference Fourier maps for the triiodide
complex. a, the Fourier coefficient was (F - F
) exp(i
), where F
, F
, and
, respectively,
are the observed structure factors of the complex and the native
crystals and the phase angle of the native crystal at pH 7.5. An
carbon backbone and the active site residues (Arg-52 and His-56) of ARP
are superimposed on the map. The minimum contour level is
±3.5
. b, close-up view of the internal triiodide
ion. The coefficient was (F
- F
)
exp(i
), where F
and
were calculated from the atomic parameters of all the atoms
except those of the ion.
The
geometry of the triiodide bound heme is given in Table 4. For
convenience, iodine atoms of the internal site are named I,
I
, and I
in the order of their proximity to the
heme iron. I
is placed above the heme iron, and the
triiodide is nearly parallel to the heme plane. The interaction between
the heme iron and the triiodide appears to be weak as judged by the
little spectral change on triiodide binding.
Figure 5: Superposition of the active site residues of the triiodide complex (brown) on those of the native ARP (white).
In contrast to
Arg-52 in ARP, a small but significant conformational change of His-56
was induced by triiodide binding. The imidazole ring of His-56 rotated
along the C-C
and
C
-C
bonds by 8 and 4°,
respectively, resulting in the maximum positional shift of 0.73 Å
at N
. This movement must be due to the large van der
Waals radius of the iodine atom; the distance between I
in
the triiodide complex and N
(His-56) is only 2.85
Å, less than the sum of their van der Waals radii, if the
conformation of His-56 is unchanged.
The orientation of the
imidazole ring of the distal histidine in ARP differs from the
orientations in CCP and LiP (Table 5). They are constrained by
the hydrogen bond between the N atom and the O=C
of asparagine (Asn-93 in ARP) (Kunishima et al., 1994). The
rotations caused by triiodide binding occurred in a direction such that
the conformation of His-56 in ARP was close to the conformations in CCP
and LiP, but their amounts were not large compared to the difference
between the tortion angles in these peroxidases.
Finzel et al.(1984) speculated on the coordination of hydroperoxide
to CCP, and on the basis of the structure of the fluoride-bound CCP,
Edwards and Poulos(1990) suggested that O1 becomes hydrogen-bonded to
both NH and N
H
of
the distal arginine (Arg-48; identical to Arg-52 in ARP) by movement of
its side chain during compound I formation. One of the functional roles
of the distal arginine is neutralization of the negative charge of
fluoride (as well as HOO
), and in the case of CCP,
fluoride binding to the heme was achieved by movement of its side
chain. We obtained no evidence that ARP forms a complex with fluoride
ion. In the case of ARP the assumed position of the fluoride and that
of the N
of Arg-52 are too far apart to interact. A
possible explanation for the inability of ARP to form a stable fluoride
complex is the lack of interaction, because the side chain of Arg-52 of
ARP is fixed.
Figure 6:
Proposed mechanism of compound I formation
of ARP. According to Finzel et al.(1984) the reaction consists
of three steps. In step 1 hydrogen peroxide binds to the active site by
substituting for two water molecules, producing a Michaelis-Menten ES complex. In step 2 the proton attached to the oxygen atom
that interacts with the heme iron translocates to the N of the distal histidine, producing the transitional complex I. In
step 3 the proton attached to the distal histidine translocates to the
other oxygen atom of hydroperoxide (transitional complex II), and
heterolytic cleavage of the O-O bond occurs concomitantly, generating
compound I and one water molecule.
The proposed model of
the HO
-enzyme complex is shown in Fig. 7. Hydrogen peroxide would displace two water molecules in
the active site; Wat-674 which is the closest one to the heme iron and
is hydrogen-bonded to the N
of His-56, and Wat-675
which is hydrogen-bonded to the N
of Arg-52 and
Wat-674. It is possible to place hydrogen peroxide on the distal side
of the heme in such a way that one oxygen atom is on the heme iron and
the other is hydrogen bonded to Arg-52 without conformational change in
the Arg-52 side chain. If the H-O-O-H torsion angle in hydrogen
peroxide is assumed to be orthogonal, a stable conformer, the hydrogen
bond of N
-H (Arg-52)
O1 will direct
the O2-H bond toward the N
of His-56 because the
N
of Arg-52 bears hydrogen and is a donor of the
hydrogen bond. Such geometry of the hydrogen peroxide relative to the
active site of ARP appears to be favorable for the subsequent proton
abstraction from O2 to the N
of His-56.
Figure 7:
Proposed structure of the
HO
-bound form of ARP. The distances between the
hydrogen peroxide and ARP in this model are
O1
N
(Arg-52); 2.9 Å,
O2
Fe; 2.8 Å, H(O2)
N
(His-56); 2.4 Å.
Directing
the N-H of His-56 toward O1 for proton translocation is
achieved by rotations around the C
-C
and C
-C
bonds. The directions of
these rotations are the same as when triiodide binds to ARP, but the
amount is larger. If these bonds were rotated so as to place
H-N
(His-56) on the O1 atom with the remaining residues
unchanged, the geometries of the hydrogen bond between N
of His-56 and O
of Asn-93 would become
unfavorable. To achieve a tetrahedral arrangement around O1, i.e. to interact the O1 and His-56-N
, which is
favorable for the subsequent proton translocation from
His-56N
to O1, another type of additional movement such
as the shift of hydroperoxide and/or the dynamic behavior of the
protein molecule should be considered. (
)We could not build
a reasonable model of the transitional complex II for ARP assuming that
the conformation of the distal histidine is retained as in the native
enzyme.
In conclusion, the structures of the native and triiodide-bound form of ARP suggest that the mechanism of compound I formation of ARP is similar to that of CCP as proposed by Finzel et al.(1984). The behavior of the active site residues during compound I formation for ARP differs, however, from that for CCP. Our computer modeling study suggests that Arg-52 maintains the conformation of hydrogen peroxide favorable for compound I formation without side chain movement and that conformational flexibility of His-56 is necessary for proton translocation from one oxygen atom to the other of the hydroperoxide.
The atomic parameters (codes 1ARU, 1ARV, 1ARW, 1ARX, and 1ARY) have been deposited in the Protein Data Bank, Chemistry Department, Brookhaven National Laboratory, Upton, NY.