(Received for publication, June 20, 1995; and in revised form, August 14, 1995)
From the
We extended our investigation on the structure of the redox
centers of bd-type ubiquinol oxidase from Escherichia coli using cyanide as a monitoring probe. We found that addition of
cyanide to the air-oxidized O-bound enzyme caused
appearance of an infrared C-N stretching band at 2161
cm
and concomitant disappearance of the 647 nm
absorption band of the cytochrome d (Fe
)-O
species. Addition of
cyanide to the air-oxidized CO-bound enzyme also resulted in
disappearance of the 635 nm absorption band and the 1983.4
cm
C-O infrared band of the cytochrome d (Fe
)-CO species. The resulting species had
a derivative-shaped electron paramagnetic resonance signal at g = 3.15. Upon partial reduction with sodium dithionite, this
species was converted partly to a transient heme d (Fe
)-C=N species having an electron
paramagnetic resonance signal at g
= 2.96
and a C-N infrared band at 2138 cm
. These
observations suggest that the active site of the enzyme has a heme-heme
binuclear metal center distinct from that of the heme-copper terminal
oxidase and that the treatment of the air-oxidized enzyme with cyanide
resulted in a cyanide-bridging species with ``heme d(Fe
)-C=N-heme b
(Fe
)'' structure.
Escherichia coli has two terminal ubiquinol oxidases in
the aerobic electron transfer chain: bo-type ubiquinol
oxidase, which is expressed under high oxygen tension, and bd-type ubiquinol oxidase, which predominates under low oxygen
tension(1, 2) . These oxidases are structurally
unrelated, but both catalyze the two-electron oxidation of ubiquinol-8
and the four-electron reduction of dioxygen to produce
water(1, 2) . The E. coli bd-type ubiquinol
oxidase has been isolated and was found to consist of two polypeptide
chains: subunit I (58 kDa) and subunit II (43
kDa)(3, 4) . The cydAB genes coding for both
polypeptides have been cloned and sequenced(5) . Within this
enzyme, it has been claimed, there are three types of cytochrome heme
species based on the optical spectra: cytochrome d, cytochrome b, and cytochrome b
(6, 7) . Cytochrome d has a chlorin chromophore (heme D) (8) exhibiting a
characteristic absorption maximum at 628 nm in the fully reduced state
and is a primary binding site for exogenous
ligands(3, 4, 9) . The dioxygen molecule
forms a very stable adduct with ferrous cytochrome d showing
its Fe
-O
stretching vibration at
568 cm
(essentially identical to that of
myoglobin)(10) , and thus the enzyme in the air-oxidized state
is actually an oxygenated form. The cytochrome d moiety also
forms a remarkably stable oxoferryl (Fe
=O)
adduct(11) . Subunit I contains cytochrome b
that shows its
and
peaks at 562 and 532 nm,
respectively, in the reduced state (12) and most likely forms
the ubiquinol-8 binding site(13) . Cytochrome b
is an unusual b-type cytochrome. It
was suggested that the optical spectrum of this cytochrome is very
similar to that of high spin cytochrome c peroxidase, and its
and
bands are at 595 and 562 nm, respectively, in the
reduced minus oxidized difference spectrum(7) . Recent low
temperature CO photolysis experiments monitored by the infrared
C-O stretching absorption suggest that cytochrome d and
cytochrome b
form a binuclear
center(14, 15) .
In a previous study we analyzed
the purified bd-type ubiquinol oxidase by resonance Raman,
FT-IR, ()and EPR spectroscopies (16) . We found that
heme d-bound C-O and Fe
-CO
stretching frequencies were at 1980.7 and 471 cm
,
respectively, in the fully reduced state. These values differ
considerably from those of the heme-copper respiratory oxidases and the
oxygen-carrying hemoproteins, both groups possessing a His residue as
the heme axial
ligand(17, 18, 19, 20) . EPR
analyses on the air-oxidized O
-bound and
Na
S
O
-reduced nitric oxide-bound
forms revealed that there is no superhyperfine structure originating
from the heme axial
N ligand in the central resonance of
the nitric oxide EPR signals(16) . These results suggest
strongly that the heme d axial ligand of bd-type
ubiquinol oxidase is either a His residue in an anomalous condition or
some other residue making the molecular structure around the
oxygen-binding site different from those of the heme-copper respiratory
oxidases. Indeed, electron nuclear double resonance spectroscopy has
shown that the axial ligand to heme d is most probably not a
histidine or other strong nitrogenous ligand(21) . In the
present study we extended our investigation on the redox centers of the E. coli bd-type ubiquinol oxidase using cyanide as a
monitoring probe.
Figure 1:
Visible
absorption spectral change upon addition of cyanide to the air-oxidized
O-bound enzyme. The difference spectrum was calculated as
the cyanide-treated (22 h after the addition of 5 mM cyanide )
minus the air-oxidized O
-bound enzyme. Sample concentration
was 0.218 mM (0.436 mM in heme B
concentration).
Figure 2:
FT-IR spectra of bd-type
ubiquinol oxidase in the C-O and C-N stretching vibration
region. The air-oxidized O-bound enzyme was exposed first
to carbon monoxide (
C
O) atmosphere (A); then cyanide (
C
N; 5
mM) was added anaerobically to the air-oxidized CO-exposed
enzyme, (B, taken 40 min after the addition of cyanide). Then
Na
S
O
was added anaerobically to the
air-oxidized CO-exposed cyanide-treated enzyme, and the development of
the 1980.7 cm
band and decay of the 2161
cm
band were recorded (C, D, and E; taken just after, 100 min after, and 16 h after the
addition of Na
S
O
, respectively).
Sample concentration was 0.60 mM (1.21 mM in heme B
concentration). The band at 2093 cm
is due
to free H
C
N.
Cyanide
also caused disappearance of ferric low spin signals at g = 2.46, g
=
2.32 (Fig. 3, A and B), and g
= 1.83 (not shown) at 15 K. Those were assigned to a minor
form of ferric cytochrome d(9) . Concomitantly, a
derivative-shaped EPR signal at g = 3.15 developed (Fig. 3B) together with another low spin signal at g = 2.82. Prolonged incubation of the sample at 4
°C in the dark caused an increase in intensity of these EPR signals (Fig. 3C). The EPR signal at g = 3.15
is distinct from the g = 3.3 signal previously assigned
as ferric low spin cytochrome b
(32, 33, 34) ,
although both signals overlapped.
Figure 3:
Effects of cyanide addition on the EPR
spectra of bd-type ubiquinol oxidase. Shown are: A,
the air-oxidized O-bound state; B, 10 min after
addition of cyanide (5 mM); C, 1 week after addition
of cyanide (5 mM); and D, just after addition of
sodium dithionite to C. The conditions were: temperature, 8 K;
modulation frequency, 5 Gauss; power, 5 mW; sample concentration, 0.179
mM (0.358 mM in heme B
concentration).
Anaerobic addition of
NaS
O
to this cyanide-pretreated
form caused a rapid disappearance of the high spin signals together
with the low spin signal at g = 2.82. The
derivative-shaped EPR signal at g = 3.15 persisted and
a transient EPR species with g
= 2.96
appeared (Fig. 3D). The g
=
2.96 signal can be ascribed to a cyanide adduct of cytochrome d(Fe
)(32, 35) . Prolonged
incubation of the Na
S
O
-treated
sample on ice removed the derivative-shaped EPR signal and the
transient EPR signal completely.
On the other hand, the g = 6 high spin rhombic EPR signal diminished
appreciably upon prolonged incubation with cyanide, whereas the axial
high spin signal changed little during the incubation. Although the
assignment for the g = 6 high spin axial signal ()is still controversial, there seems to be a consensus that
the g = 6 high spin rhombic signal is due to cytochrome b
(32, 33, 39) . From
these pieces of evidence we conclude that the cyanide-bridging
structure at the active site is a kind of ``heme d(Fe
)-C=N-heme b
(Fe
).''
The nature of
the EPR signal at g = 2.82 is not clear because the
intensity of this signal was preparation-dependent. This may be due to
a minor form of heme d(Fe)-CN derived
from the cyanide-bridging species in which the bridging structure is
partially destroyed upon freezing. This scenario is consistent with the
observation of only one C-N stretching infrared band (i.e. the 2161 cm
band) at room temperature. However,
other possibilities (e.g. heme b
(Fe
)-CN or heme b
(Fe
)-NC species) cannot
be completely excluded.
The addition of
NaS
O
to the preformed cyanide
complex produced a new, but transient, low spin heme species having
with the g
= 2.96 EPR signal (Fig. 3D) and the 2138 cm
cyanide
infrared band (Fig. 2). This species is assignable to heme d(Fe
)-CN
unambiguously(32, 35) . The observed spectral changes
are most likely due to a breakage of the cyanide-bridging structure
caused by the reduction of cytochrome b
heme
itself or by the conformational change around the binuclear center upon
reduction of cytochrome b
heme. Such influence
of the redox states of other metal centers on the heme d has
been observed for the bound C-O stretching frequencies, i.e. the C-O band appears at 1980.7 cm
in the
fully reduced state, whereas it is at 1983.4 cm
in
the partially reduced state (Fig. 2)(16) . These
spectral characteristics are indicative of the heme-heme interactions
and have some similarities to those of the heme-copper
oxidase(22, 37, 40) .
The lower g value (2.96 for the partially reduced state and
2.82 for the air-oxidized state) and the higher C-N stretching
frequency (2138 cm
) of the heme d(Fe
)-CN moiety compared with those of
corresponding species of the heme-copper oxidase (g
= 3.24 (41) and 2123 cm
(
)for the E. coli bo-type ubiquinol oxidase and g
= 3.58 (42) and 2132
cm
(37) for bovine cytochrome c oxidase) might be ascribed, however, mainly to the chlorin
macrocycle of heme d. It was reported that cyanide adducts of
ferric hemoproteins containing chlorin macrocycle exhibited a g
value as low as 2.6(43) . It is also
known that presence of electron-withdrawing groups (such as hydroxyl
groups) on iron porphyrin macrocycle leads to an increase of the bound
C-N stretching vibration(31) .
We should consider, at this
point, a possible structure of the heme-heme binuclear center to obtain
detailed insight into the dioxygen reduction mechanism of bd-type quinol oxidase. In the cyanide-bridging structure, the
C atom of the cyanide is likely bound to the Fe atom of heme d with the Fe-C bond being perpendicular to the heme plane
(with the Fe-C and C=N bond lengths as 1.9 and 1.1
Å, respectively(38) ). The
Fe(b)-NC distance and
Fe(b
)-N-C angle would vary markedly
with the relative distance and orientation of the two metal centers (38) . The orientations of heme d and heme b
have been determined by EPR studies on
oriented multilayer preparations of cytoplasmic membrane fragments (44) . Both ferric high and low spin heme d are
oriented with their heme planes perpendicular to the membrane plane.
Ferric high spin heme b
is oriented with its
heme plane at approximately 55 ° to the membrane plane. Thus, heme d and heme b
planes are facing each
other with 35 ° orientation. Assuming the
Fe(b
)-N bond to be perpendicular to the
heme plane with the bond length of 2.2 Å(38) , the
distance of the two metal center was calculated as 5.1 Å. In this
case, peripheral group(s) of the two hemes must be in a close contact
each other, the access of an amino acid residue(s) into the ligand
binding pocket (i.e. the space between the two metal centers)
must be highly restricted, and the ligand-binding pocket itself would
be very hydrophobic. The hydrophobic nature of the ligand-binding
pocket is consistent with the unusual stability of the oxygenated
species as well as the oxoferryl (Fe
=O)
adduct(10, 11) . This might be one of the reasons why bd-type ubiquinol oxidase is more resistant to anionic
inhibitors such as cyanide and azide than bo-type ubiquinol
oxidase(2, 3) .