(Received for publication, November 27, 1995; and in revised form, February 2, 1996)
From the
The heme axial ligands of bd-type ubiquinol oxidase of Escherichia coli were studied by EPR and optical
spectroscopies using nitric oxide (NO) as a monitoring probe. We found
that NO bound to ferrous heme d of the air-oxidized and fully
reduced enzymes with very high affinity and to ferrous heme b of the fully reduced enzyme with low affinity.
EPR spectrum of the
NO complex of the reduced enzyme
exhibited an axially symmetric signal with g-values at g
= 2.041 and g
= 1.993 and a clear triplet of triplet (or a triplet of
doublet for the
NO complex) superhyperfine structure
originating from a nitrogenous proximal ligand trans to NO was
observed. This EPR species was assigned to the ferrous heme d-NO complex. This suggests that the proximal axial ligand of
heme d is a histidine residue in an anomalous condition or
other nitrogenous amino acid residue. Furthermore, the EPR line shape
of the ferrous heme d-NO was slightly influenced by the
oxidation state of the heme b
. This indicates
that heme d exists in close proximity to heme b
forming a binuclear center. Another axially
symmetric EPR signal with g-values at g
= 2.108 and g
= 2.020
appeared after prolonged incubation of the reduced enzyme with NO and
was attributed to the ferrous heme b
-NO complex.
The bd-type ubiquinol oxidase is an alternative
terminal oxidase in the aerobic respiratory chain of Escherichia
coli and is expressed predominantly under low oxygen
pressure(1) . This enzyme catalyzes the oxidation of
ubiquinol-8 and the reduction of molecular oxygen to water and forms a
proton electrochemical gradient across the cytoplasmic membrane via
scalar proteolytic reactions(2) . It is composed of two
subunits (I and II)(3, 4) . Based on optical
spectroscopic properties, the enzyme contains two hemes B and one heme
D associated with cytochrome b, cytochrome b
, and cytochrome d, respectively (5) . Cytochrome d has a chlorine chromophore, heme
D(6, 7, 8) , and is a primary exogenous
ligand binding site(9) . In the air-oxidized condition, heme d is actually in a reduced state and coordinates a molecular
oxygen(10) , as an Fe
-O
diamagnetic EPR-inactive state. Heme b
is
claimed as a six-coordinated ferric low-spin heme component of the
oxidase which has been shown to be contained within subunit I (11, 12) and has been implicated in the oxidation of
the substrate, ubiquinol-8, in the cytoplasmic membrane. Heme b
is a ferric high-spin
center(13, 14) . EPR-active ferric heme b
and heme b
exhibit an
intense g = 6.0 axial high-spin signal and, overlapped
with this, rhombic high-spin signals (5, 13, 14) . Additional two minor ferric
low-spin species at g
= 3.3 and g
= 2.5 were also reported
previously(5, 13, 15) . However, the
assignment for these EPR signals is still controversial. In our
previous study, we have reported that anaerobic addition of nitric
oxide (NO) to the air-oxidized enzyme caused an exchange of ferrous
heme d-bound O
with NO leading to an appearance of
ferrous heme d-NO EPR signal around g = 2
region without eliminating the ferric high-spin signals. From a rough
estimation of spin contents of these EPR-active species, we concluded
that the ferrous heme-NO EPR signal and the ferric high-spin signals
each corresponds to about 1 mol of heme/mol of the enzyme, and, thus,
the bd-type ubiquinol oxidase contains only 1 mol each of
cytochrome b and cytochrome d as the redox
components(16) . However, our recent results for the heme B and
metal contents analyses of the purified bd-type ubiquinol
oxidase indicated that there were two hemes B and one heme D in the
oxidase(5) . This result is distinctly incompatible with our
previous EPR results. In addition, recent metal and heme content
analyses as well as ligand binding titration experiments of bd-type ubiquinol oxidase from Azotobacter vinelandii indicated the stoichiometry of two B-type hemes and one heme D per
molecule(17) . Moreover, they reported that both CO and NO bind
with high affinity to the ferrous heme d at a stoichiometry of
1 mol of ligand/mol of enzyme and with low affinity to the reduced heme b
(17) . Our previous EPR analyses on the
NO-bound forms of the air-oxidized enzyme revealed that there is no
superhyperfine structure originating from the heme axial
N
ligand trans to NO in the central resonance of the heme d-NO EPR signals(16) . A recent electron nuclear
double resonance study suggested that the proximal ligand to heme d is not a histidine residue or other strong nitrogenous
ligand(18) . In the present study, we have extended our EPR
investigation on the NO complex of bd-type ubiquinol oxidase
from E. coli in order to clarify the stereochemical and
electronic structure of heme d as well as heme b
active center. The present results clearly
show the presence of a triplet of triplet superhyperfine structure in
the EPR spectra originating from heme d nitrogenous proximal
ligand trans to NO.
Anaerobic addition of nitric oxide (NO) to the air-oxidized enzyme caused an exchange of
ferrous heme d-bound O
with NO leading to an
appearance of axially symmetric heme d-bound NO EPR signals at g
= 2.041 and g
= 1.993 at 77 K as shown in Fig. 1A, upper trace. The addition of NO to the air-oxidized enzyme did
not eliminate the ferric high-spin signals, neither the axial nor the
rhombic signal, at 15 K as reported previously(16) . If NO
binds to the ferric high-spin cytochromes b, their nitrosyl
complexes will become EPR-inactive due to spin-pairing. Thus, it is
concluded that NO does not bind to the ferric high-spin cytochromes b species. The weak ferric low-spin signals at g = 2.46, 2.32, and 1.83 disappeared completely upon the
treatment with NO indicating a formation of the EPR-inactive ferric
heme d-NO complex. The spectral pattern of the ferrous heme d-NO complex was quite different from those of usual ferrous
hemoprotein-NO complexes. This indicates an unusual coordination
structure at the (5th ligand)-heme-NO moiety in cytochrome d.
Figure 1:
EPR spectra of
the purified NO complexes of bd-type ubiquinol oxidase at 77
K. The EPR spectrum of the air-oxidized enzyme in the presence of
nitric oxide (NO) under anaerobic condition (A).
The EPR spectra of the nitrite (Na
NO
)-treated (B) and Na
NO
-treated (C)
enzymes within a few minutes after reduction by solid sodium dithionite
under anaerobic condition. The upper and the lower traces indicate the 1st and the 2nd derivative EPR spectra, respectively.
Conditions of measurements are as follows: microwave frequency, 9.23
GHz; incident microwave power, 5 milliwatts; 100-kHz field modulation
width, 0.2 millitesla (mT).
A careful observation revealed a hyperfine structure in the g region of the second derivative spectrum
of the ferrous heme d-bound NO EPR signals, which might be
ascribed to a superhyperfine interaction with another
N
nucleus trans to NO (lower trace in Fig. 1A). In order to clarify whether this is really a
superhyperfine splitting due to
N nucleus trans to NO or not, the EPR spectrum of the
NO complex was
compared with that of the
NO complex. Within a few minutes
after addition of solid sodium dithionite to the nitrite
(Na
NO
or
Na
NO
)-treated enzyme, axially symmetric NO EPR
signals similar but much more intense than that of the NO-bound form of
the air-oxidized enzyme appeared. The increment in the signal intensity
of the nitrite-treated dithionite-reduced enzyme indicated that the
minor ferric low-spin heme d in the air-oxidized enzyme was
also converted to the ferrous heme d-NO species. In addition,
a clear triplet of triplet (for the
NO complex) or a
triplet of doublet (for the
NO complex) superhyperfine
splitting appeared in the g
region as
shown in Fig. 1, B and C (the upper and lower traces in Fig. 1indicated 1st and 2nd
derivative EPR spectra, respectively). The principal g-values
were estimated to be g
= 2.041 and g
= 1.993, and the superhyperfine
constants were
A
1.5 mT,
A
1.4 mT,
a
= 0.67 mT for the
NO complex and
A
2.1 mT,
A
1.9 mT,
a
= 0.67 mT for
the
NO complex, respectively. The triplet of triplet
and/or triplet of doublet splittings in the g
signal can reasonably be ascribed to the superhyperfine
interaction with another
N nucleus trans to NO.
This result is inconsistent with electron nuclear double resonance
studies in which the proximal axial ligand to heme d was
concluded to be not a histidine residue or other nitrogenous
ligand(18) . However, the electron nuclear double resonance
study was done for the air-oxidized form assuming that the axial
high-spin signal (g = 5.92, 2.0) was derived from heme d(18) and, therefore, not compatible with the
conditions of the present study.
It has been reported that there are
three invariant histidine residues (His-19 and His-186 in subunit I and
His-56 in subunit II) in bd-type ubiquinol oxidases from E. coli(20, 21) and A.
vinelandii(22, 23) . A site-directed mutagenesis
study on bd-type ubiquinol oxidase from E. coli enzyme showed that only two His residues appeared to function as
heme axial ligands(24) . In subunit I, His-186 is most likely a
heme axial ligand to cytochrome b, and His-19 is
likely an axial ligand to either cytochrome b
or
cytochrome d(24) . Although it is uncertain at this
moment whether His-19 is the proximal axial ligand of cytochrome d or not, our present EPR results strongly suggest that the proximal
axial ligand of cytochrome d is a histidine residue in an
anomalous condition or other nitrogenous amino acid residue. The
appearance of the clear superhyperfine splitting in the nitrite-treated
dithionite-reduced enzyme (but not so obvious in the NO-complex of the
air-oxidized enzyme) indicates that the coordination structure around
the axial ligand binding site of heme d may be altered
slightly upon reduction of the other redox center (i.e. heme b
) or direct electrochemical interaction between
oxidized heme b
and heme d-NO may be
changed. This observation is consistent with the notion that heme d exists in close proximity to heme b
, which
might form a binuclear center(5, 25) .
Figure 2:
EPR spectra of the NO-bound fully reduced bd-type ubiquinol oxidase from E. coli at 77 K. The
EPR spectrum of the fully reduced enzyme after prolonged incubation
with NO (B). The EPR spectrum of the nitrite-treated
dithionite-reduced enzyme within a few minutes after addition of solid
sodium dithionite is duplicated for comparison (A). The enzyme
concentrations of A and B are the same. Spectrum
C is obtained by subtraction of the two spectra from each other (B - f A; f means
subtraction factor; here f = 1). Conditions of
measurements are as in Fig. 1.
Figure 3: Changes in spin content associated with the formation of the NO-bound reduced enzyme. The nitrite-treated dithionite-reduced enzyme under anaerobic condition was allowed to stand on ice, and EPR spectra were measured at 3 min, 30 min, 1.5 h, 3.5 h, 5.5 h, and 6 h + NO at 77 K. The spin content of the ferrous-heme-NO was estimated by double integration of the EPR spectrum. The NO complexes of the air-oxidized (dotted line) and the fully reduced (bold line) enzymes are illustrated for comparison. The comparative spin content 1 denoted 1 mol of heme/mol of enzyme, which was determined from the ferrous NO complex of sperm whale myoglobin. The inset illustrates the formation of the NO-bound reduced enzyme versus incubation time.
Figure 4: Optical spectra of purified bd-type ubiquinol oxidase from E. coli. Solid thin line, air-oxidized form; dotted line, NO-bound air-oxidized form; solid bold line, fully reduced form; and dashed line, NO-bound fully reduced form.
In our previous study on bd-type ubiquinol oxidase, we proposed that this enzyme contains only 1 mol each of the cytochrome b and the cytochrome d as the redox components (16) . However, we estimated the spin contents of the ferrous heme-NO and the ferric high-spin species by double integration method at 15 K without considering the effects of temperature-dependent relaxation time on the shape and the intensity of the EPR signals, thus leading to an incorrect conclusion.
A consistent heme stoichiometry that bd-type ubiquinol oxidase contains two hemes B and one heme D
was supported by pyridine hemochrome spectra of the purified enzyme in
the present study. As the ferrous pyridine hemochrome of heme D was
reported to be unstable(6, 28) , the spectrum was
measured in aqueous alkaline pyridine solution immediately after
reduction with solid sodium dithionite. The spectrum of freshly
prepared pyridine hemochrome of bd-type ubiquinol oxidase is
shown in Fig. 5. Subtracting a pyridine hemochrome spectrum of
heme B (obtained from 2 equimolar sperm whale myoglobin) from the
pyridine hemochrome spectrum of bd-type ubiquinol oxidase, a
reliable spectrum of the pyridine hemochrome of heme D was obtained as
shown in Fig. 5. For the peak of cytochrome d in
the reduced minus air-oxidized difference spectra (inset in Fig. 5), a molar extinction coefficient for bd-type
ubiquinol oxidase has been reported(5) . Using our recent molar
extinction coefficient of 27.9 mM
cm
for the redox pair 628 nm (for reduced
state) minus 651 nm (for air-oxidized state)(5) , a reliable
extinction coefficient at 608 nm of the pyridine hemochrome of heme D
was estimated to be 23.4 mM
cm
.
Figure 5: Pyridine hemochrome spectrum of bd-type ubiquinol oxidase (solid thin line) and the deconvoluted spectra of heme B (dashed line) and heme D (solid bold line), respectively. The pyridine hemochrome spectrum of bd-type ubiquinol oxidase was recorded immediately after reduction with solid sodium dithionite. The inset illustrates the difference spectrum of the fully reduced minus air-oxidized enzymes.
In conclusion, bd-type
ubiquinol oxidase from E. coli is a cytochrome bbd-type enzyme as reported
previously(5, 13, 14, 17) . Optical
and EPR results indicate that NO binds to the reduced heme d with very high affinity and to the fully reduced heme b with low affinity. Two kinds of EPR signals
correspond to both NO complexes of fully reduced cytochrome d and b
with 1:1 spin contents. A clear
superhyperfine interaction with
N nucleus trans to NO in the EPR spectrum of the NO complex of cytochrome d indicates that the proximal axial ligand of heme d is a
histidine residue or other nitrogenous amino acid residue. Further
detailed studies are desired to reveal the precise structure of the
active center of this enzyme.