From the
Cytochrome bd has been purified from Azotobacter
vinelandii by a new simplified procedure. The heme and total iron
content has been measured, as has the number of high affinity CO and NO
binding sites. Spectral changes indicate high affinity binding of CO
and NO to heme d only, with a stoichiometry of 1 molecule of gas per 2
molecules of heme b or per 3 atoms of iron. The results clearly define
a stoichiometry of one heme d per complex. Low affinity binding of CO
and NO to heme b595 also occurs at higher ligand concentrations. EPR
heme-nitrosyl signals are seen with NO bound to both hemes b595 and d
but with no indication of spin exchange coupling. Exposure of the
air-oxidized complex to alkaline pH results in removal of molecular
oxygen from heme d and a change in line shape of the high spin region
of the EPR spectrum. Cyanide binds to both heme d and heme b595 in the
air-oxidized complex, displacing molecular oxygen from heme d. The rate
of cyanide binding to heme d as assessed by spectral changes at 650 nm
does not correlate with the rate of binding to heme b595 as assessed by
the loss of the high spin EPR signal. In addition, the cyanide binding
rate in the presence of reductant is only 3 times that of the rate of
binding to the air-oxidized enzyme, in contrast to the
copper-containing oxidases where strong redox cooperativity makes these
two rates differ by a factor of at least 10
The cytochrome bd terminal oxidase complex of Azotobacter vinelandii is a quinol oxidase required for
respiratory protection during aerotolerant nitrogen
fixation(1) . It shows structural (2) and genetic (1, 3) similarities to the Escherichia coli enzyme, although the apparent oxygen affinity of the A.
vinelandii complex is thought to be much lower(4) . The E. coli complex comprises two membrane-associated subunits of
molecular mass 58 and 43 kDa(5) . Similar properties are
reported for the A. vinelandii complex(6) . Cytochrome bd contains at least three redox-active centers: heme b558,
heme b595, and one or two d-type hemes. EPR signals indicate two major
high spin ferric heme signals attributed to b595 and heme d. A low spin
signal has been assigned to heme b558(7) . Coulometric and CO
binding studies on the E. coli enzyme (8) have
suggested the presence of 2 mol of heme d. In support of this
suggestion, there is evidence that the complex can bind simultaneously
to molecular oxygen and peroxide(9) , and nitrate appears to
bind to heme d with two distinct affinities(10) . However, the
presence of two hemes d per complex has been questioned on the basis of
EPR studies(7, 11) . CO binding studies on heme b595
have also given equivocal results, with optical measurements and
potentiometric titrations appearing to differ(12, 13) .
Unfortunately, the use of optical spectroscopy to assign the absorbance
bands in the Soret region is difficult, mainly because of band overlap
and weak absorption, but in the
The cytochrome bd complex is
of particular interest not only because of its role in respiratory
protection in A. vinelandii, where its enzyme activity is one
of the highest of all the terminal oxidases, but also because the
mechanism of oxygen reduction can be contrasted with that of the
copper-containing oxidases. These latter oxidases also reduce oxygen to
water in a binuclear heme/copper center with strong cooperativity
between the two redox metals. Several well defined intermediates have
been detected in this center(14) . The copper-containing
oxidases have the additional capability of proton
translocation(15) , which is thought to involve coordination
changes in or around the copper/heme binuclear
center(16, 17) . In comparison, little is known about
the reaction mechanism of the cytochrome bd oxidase, which
shares no primary sequence homology with the copper-containing oxidases
although mechanistic similarities exist. Oxygenated and peroxy states
have been detected (18, 19) as well as an oxoferryl heme d(20) .
The reconstituted system is capable of forming a transmembrane
electrochemical gradient, although without translocation of
protons(21) . At the oxygen reduction site, interaction between
the high spin heme groups, b595 and d, has been suggested by EPR
studies(7) . CO titrations (22) and Fourier transform
infrared spectroscopy (23) have led to the proposal of a
heme-heme binuclear center at the oxygen binding site. In the present
work, we have purified and characterized the complex from A.
vinelandii in order to quantitate the binding of various ligands
at the oxygen binding site. The results clearly show the presence of
one heme d per cytochrome bd complex. In addition, the results
of CO, NO, and CN
The thorough
characterization of the purified complex provides a consistent heme
stoichiometry from several independent methods (µM heme b
from pyridine hemochrome spectra, µM iron from atomic
absorption, µM high affinity binding of CO and NO) and can
now be used to provide a reliable extinction coefficient in the
Cyanide has been shown to induce a large
red shift in the Soret peak of air-oxidized complex of E.
coli, and some of this change (up to one third) is reported to
take place before any significant development of the trough at 648
nm(35) . On the basis of MCD evidence, Krasnoselskaya et al.(35) conclude that up to two-thirds of the air-oxidized
complex purifies in the oxygenated state, which prevents fast cyanide
binding to either heme b595 or heme d. The present results show that
the addition of substrate to the enzyme increases the rate of cyanide
binding to heme d, consistent with a faster removal of molecular oxygen
from this heme under turnover conditions. What is interesting to note
is that cyanide binding in the presence of substrate is only
approximately 3 times faster than under non-turnover conditions. This
contrasts with mitochondrial cytochrome c oxidase, where there
is strong redox interaction between the redox centers and the rates of
binding of cyanide to the oxidized and partially reduced enzyme differ
by a factor of 10
Full displacement of molecular
oxygen occurs with both cyanide binding and exposure to alkaline pH in
the air-oxidized complex. However, after incubation with cyanide for
the same time period as used for measuring the optical changes, the
loss of spin intensity in the high spin region of the EPR spectrum is
less than one-third. We therefore suggest that in the air-oxidized
complex, heme b595 is predominantly responsible for the high spin
signals and that most heme d in air-oxidized enzyme is in the
oxygenated form. Resonance Raman studies have shown that the oxygenated
form of cytochrome d is very similar to
oxyhemoglobin(38) . Indeed, removal of oxygen from the
air-oxidized complex by argon treatment produces a partially reduced
state(9) . This would then indicate an EPR silent Fe(II) ground
state for the oxygenated heme d. Previous quantitations vary in the
amount of the oxygenated form
present(9, 35, 39) , but most estimates give
values between 70-100%. The present results with hydroxide and
cyanide treatment indicate that some of the remaining heme d is ferric
and contributes between 20-30% to the spin intensity at g
= 6. (This should not be confused with the EPR signals from
oxidized preparations under anaerobic conditions, where almost 100% of
the heme d has been shown to be ferric high spin and axial(7) .)
With cyanide treatment, a new low spin signal appears with the
formation of the cyanide complex of ferric heme d. It is interesting to
note that the low spin signal around 2.55, assigned to heme
d(11) , disappears on cyanide ligation, indicating that a small
amount of low spin ferric heme d is present in the air-oxidized
preparation. A more complicated picture arises for hydroxide treatment.
The formation of a low spin ferric heme-hydroxide complex might be
expected to give rise to an easily detectable EPR signal (S =
See ``Experimental Procedures'' for details of
purification stages.
Analysis and conditions of measurement were as
described under ``Experimental Procedures.'' The number of
determinations is given in parentheses.
We thank Dr. S. Hill (Nitrogen Fixation Laboratory,
University of Sussex) for growth of A. vinelandii cultures and
Dr. Steve Rigby for helpful discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
. The results do
not support the idea of the presence of two strongly interacting hemes
in a binuclear center.
-band region three heme types have
been distinguished(8) .
binding argue against the presence
of a strongly interacting binuclear center in the enzyme.
Purification of the Cytochrome bd Complex
The
cytochrome bd-overexpressing A. vinelandii strain MK8
was grown essentially as described by Kelly et al.(1) .
All steps were carried out at 4 °C. Membranes from approximately 50
g, wet weight, of cells were prepared (1) and suspended to a
protein concentration of 10 mg/ml in 25 mM Tris/HCl, pH 8.0,
containing 10 mM EDTA and 0.5 M KCl. The membranes
were pelleted at 100,000 g for 1 h and resuspended in
0.1 M potassium phosphate buffer, pH 7.2 (10 mg of
protein/ml). Sodium cholate was added from a 10% (w/v) stock solution
to give a final concentration of 2% (w/v), and the mixture was left
stirring overnight. After centrifugation at 45,000
g for 20 min, the cytochrome bd-containing pellet was
washed twice in phosphate buffer alone to remove the remaining cholate.
The pellet was suspended in 0.1 M phosphate buffer, pH 7.2,
(15 mg of protein/ml), and lauryl maltoside was added to a final
concentration of 1.5% (w/v). The suspension was stirred for 1 h before
being centrifuged at 45,000
g for 15 min. Ammonium
sulfate was added to the supernatant to give 55% saturation. The
mixture was left for 30 min and centrifuged at 45,000
g for 15 min. The red pellet was discarded. Raising the ammonium
sulfate concentration to 80% and adjusting the pH to 7.2 with dilute
ammonium hydroxide precipitates the oxidase complex. The floating
pellet was dissolved in 0.1 M phosphate buffer, pH 7.2
(10-15 mg of protein/ml) and stored under liquid nitrogen.
Chemicals and Assays
The protein concentration of
vesicle suspensions and membrane protein preparations was measured by a
modified Lowry method(24) . Ubiquinone-1 was a kind gift from
Dr. R. Cammack (King's College London). Before use, it was
chromatographed on Merck silica gel 60 TLC plates, using
chloroform:hexane:diethylether 10:10:1 as solvent. The ubiquinone-1 was
then dissolved in ethanol. The assay for oxidase activity was based on
that described by Kita et al.(25) using a Clark-type
oxygen electrode at 25 °C containing 1 ml of 0.1 M potassium phosphate, pH 7.9, plus ubiquinone-1 and DTT(
)at the concentrations indicated. The reaction was
started by adding 5-10 µg of cytochrome bd that had
been pre-incubated on ice for 5 min in 10 µl of 0.1 M phosphate buffer, pH 7.9, containing 1 mM phospholipid (L-
-phosphatidylcholine from Soybean, Type IV-S, Sigma).
Optical spectra were recorded on a Varian Cary 210 spectrophotometer at
room temperature, using a spectral bandwidth of 1 nm and a scan rate of
2 nm s
. The spectra were stored in digitalized form
for treatment. SDS-polyacrylamide gel electrophoresis was performed in
a minigel unit (10
7.5 cm) essentially as described by Laemmli (26) with the exception that the sample buffer contained 6%
(w/v) SDS and 8 M urea in order to prevent aggregation of the
oxidase complex. The samples were loaded onto the gel without prior
incubation. Gels were stained with Coomassie Brilliant Blue (2% in 45%
(v/v) methanol, 10% (v/v) acetic acid) and destained in the same
solvent without dye. Pyridine hemochrome spectra were determined by
mixing 0.75 ml of diluted cytochrome preparation with 175 µl of
pyridine and 75 µl of 1 M NaOH. The dithionite-reduced
minus ferricyanide oxidized difference spectrum was immediately
recorded. The b-type hemes were quantified using an extinction
coefficient of 24 mM
cm
(556 nm minus 540 nm)(27) . Continuous wave EPR
measurements were recorded on a Bruker ESP300 spectrometer fitted with
a TE103 rectangular cavity and a Hewlett-Packard microwave frequency
counter 5350B and an Oxford instruments liquid helium flow cryostat
ESR900. Spectra were base line-corrected by subtraction of a cavity
spectrum of water/buffer under identical conditions. The iron and
copper content of the samples was determined by atomic absorption
spectroscopy using a Varian AA-1475 spectrometer.
Carbon Monoxide and Nitric Oxide Binding
Titrations
Nitric oxide was generated by reaction of 2 M sulfuric acid with sodium nitrite under nitrogen atmosphere and
washed by passage through 1 M sodium hydroxide and water.
Saturated solutions of carbon monoxide or nitric oxide were prepared by
bubbling the respective gas through ice-cold water for 10 min. The
concentration of the gas in solution was determined by titration of a
myoglobin standard. Aliquots of the stock solution were added
anaerobically to the reduced cytochrome preparation at room
temperature. After equilibration (2-3 min), the spectrum was
recorded over the range 400-700 nm. The end point of a titration
was determined by bubbling the gas through the suspension at room
temperature to obtain a saturated sample.
Purification of the Cytochrome bd
Complex
Cytochrome bd has been purified previously from E. coli(5, 25) , Klebsiella
pneumoniae(13) , Photobacterium
phosphoreum(28) , and A. vinelandii(6) .
The experimental conditions for the first three organisms cannot be
directly applied to A. vinelandii, mainly because of the
instability of the complex in the detergents used. We have developed a
simple and fast procedure for purification of the complex from the
overexpressing mutant strain MK8 of A. vinelandii based on
ammonium sulfate precipitation. This was briefly described by
Jünemann and Wrigglesworth(29) , and a full
characterization of the preparation is given here. summarizes the results of a representative preparation of
the cytochrome bd complex. The initial wash in KCl removes
flavin and b/c-type cytochromes as judged from the
optical spectra (results not shown). The crucial step appears to be the
extraction in lauryl maltoside, where the oxidase is solubilized
selectively. Any extra contaminating b-type cytochrome is
precipitated by the subsequent ammonium sulfate treatment which was
also shown, by EPR, to remove iron-sulfur complexes. An SDS gel of the
final preparation (Fig. 1) shows the presence of two main bands
indicative of polypeptides with molecular masses of 50 and 31 kDa.
These values can be compared with values of 50.1 and 35 kDa as
calculated from the nucleic acid sequences of the two subunits of
cytochrome bd from A. vinelandii. (It should be noted
that the values of 59.7 and 42 kDa quoted in the literature for this
calculation (30) are higher because no allowance for water
removal on peptide bond formation was made.) Components with other
molecular weights comprised less than 12% on the basis of densitometry
scans of Coomassie-stained gels. A pyridine hemochrome spectrum of the
purified complex (not shown) revealed only b-type (556 nm) and d-type
(608 nm) hemes to be present. Quantitation of the hemes by this method
depends on the extinction coefficient for the pyridine hemochromes. No
reliable value has been found for d-type heme but an early value of
20.7 mM cm
for b-type
heme (41) has been remeasured as 24.0 mM
cm
(27) . Using this latter figure, the
present preparation contains 10.3 nmol of heme b/mg of protein (). The iron content determined by atomic absorption
spectroscopy was found to be 17.6 nmol/mg of protein (),
which is 1.7 times the amount of the total b-type heme content
determined by the pyridine hemochrome method. As expected, no copper
was detected in the final preparation. The final preparation oxidized
ubiquinol-1 (ubiquinone-1 plus DTT) with specific activity similar to
that of the preparation described by Kolonay et
al.(6) . The activity with alternative substrates was
lower, being 230 nmol of O
consumed/s/mg of protein using
duroquinol (250 µM plus 1 mM DTT) and 127 nmol
O
consumed/s/mg of protein with N,N,N`,N`-tetramethyl-p-phenylenediamine
(1 mM plus 5 mM ascorbate).
Figure 1:
SDS-polyacrylamide gel of purified
cytochrome bd complex from A. vinelandii. Gel
dimensions: 7.5 10
0.075 cm, with loadings of 2 and 4
µg of protein.
Spectroscopic Properties
The absolute spectra of
air-oxidized and dithionite-reduced complex are shown in Fig. 2together with the difference spectrum. The main features
are similar to those of the cytochrome bd complexes purified
from P. phosphoreum(28) , E. coli(9) ,
and K. pneumoniae(13) . The samples as prepared
contained a high proportion of the stable oxygenated species with a
peak in the air-oxidized spectrum at 646 nm (trough in the difference
spectrum at 650 nm). An indicative measure of the purity of the complex
is the ratio of peak intensities in the reduced minus air-oxidized
spectra at 560-580 nm and 630-650 nm, which approaches 1
for the purer preparations. The slight shoulder around 550 nm is not
due to c-type cytochrome but has been ascribed to the
-band of heme b595 in E. coli(21, 31) .
The EPR spectrum of the air-oxidized purified complex is shown in Fig. 3(uppertrace). Simulation of the spectrum
in the high spin region suggests the presence of both axial and rhombic
components. A prominent high spin axial component at g = 6
comprises over two-thirds of the signal and, in the E. coli complex, has been assigned to heme d(7, 11) . The
remaining high spin rhombic features at g = 5.5 and g =
6.3 have been assigned to heme b595. Features associated with ferric
low spin heme can be seen at g = 3.4 and around g = 2.55.
The g = 3.4 signal has been shown in E. coli to titrate
with an E
of 226 mV and has been assigned to heme
b558(7) . Signals around g = 2.5 in the E. coli enzyme have been resolved into a combination of three different
components including a rhombic form with g
and g
features at 2.61 and 2.48(11) . The intensity of the
signals was found to vary from preparation to preparation, but in redox
titrations they all titrated with the same redox midpoint potential and
have been ascribed to low spin ferric heme d(11) . We ascribe
the signal around g = 2 in the present preparation to a small
amount of free radical with a signal close to the g
component of the high spin axial signal. A small amount of
nonspecifically bound iron can be seen at g = 4.3, consistent
with the iron content measured by atomic absorption being slightly
higher than 1.5 times the total b-type heme, assuming a b/d heme ratio
of 2:1.
Figure 2:
Absorption spectra of purified cytochrome bd complex from A. vinelandii. Samples contained 3.1
µM oxidase complex in 0.1 mM potassium phosphate,
pH 7.2. Air-oxidized (solidline), dithionite-reduced (dashedline), and difference spectra (dottedline) are shown.
Figure 3:
EPR spectra of the purified cytochrome bd complex from A. vinelandii. The air-oxidized
samples (26 µM) were suspended in potassium HEPES, pH 7 (uppertrace) and in Tris-HCl, pH 9 (1-min exposure) (lowertrace). Conditions of measurement were as
follows: temperature, 5 K; microwave frequency, 9.35 GHz; microwave
power, 20 mW; modulation amplitude, 10 G; modulation frequency, 100
kHz; gain, 2 10
; time constant, 163 ms. The spectra
are base line-corrected by subtraction of the spectrum of buffer
measured under the same conditions. Simulated spectra (program by Neese
(45)) are shown as dottedlines. The parameters used
in the simulations were as follows: rhombic component, g
,
g
, and g
at 6.20, 5.47, and 2.00, respectively,
with linewidths 16.0 G, 19.5 G, and 100 G, and axial component,
g
, g
, and g
at 5.915, 5.88, and
2.00, respectively, with linewidths 9.5 G, 40 G, and 100 G. See
``Results'' for the relative contributions of each component
to the final spectra.
Hydroxide Effects on the Air-oxidized Cytochrome bd
Oxidase
Fig. 3(lowertrace) shows the
effect of short time exposure at alkaline pH on the EPR spectrum of the
air-oxidized bd complex. The main change is a decrease in the
high spin axial component and an increase in the rhombic signal.
Changes in the optical spectrum at 646 nm (not shown) indicated that
deoxygenation of heme d occurred at alkaline pH. Simulation of the EPR
spectra (Fig. 3, dottedlines), assuming no
pH-dependent change in the zero field-splitting parameters for the
axial and rhombic components, indicates that the total intensity of the
high spin components drops by approximately one-third at alkaline pH.
However, most of this drop is due to a loss of the axial component. The
relative amount of the rhombic species approximately doubled (from 28
to 74%), corresponding to an absolute increase by a factor of 1.8. It
would appear that a pH-dependent removal of molecular oxygen from
oxygenated heme d is associated with a change in the line shape of the
high spin signal in the EPR spectrum. No significant changes were
detected in the low spin region. In particular, no signals indicative
of the formation of any low spin ferric complex with hydroxide (S = ) could be identified. The EPR changes were fully
reversible on resuspending the sample back in pH 7 buffer after
ultrafiltration through an Amicon YM10 membrane.
CO Binding to Reduced Cytochrome bd
The CO reduced
minus reduced difference spectra of the purified complex at low (3
µM) and saturating (>0.8 mM) CO concentrations
are shown in Fig. 4. Binding of CO induces changes in the
-band spectrum of reduced heme d. Titration of these changes (644
nm minus 623 nm) shows saturation at low (stoichiometric) amounts of CO (Fig. 5A), giving an average of 5.1 ± 0.4 (n = 4) nmol of CO bound/mg of protein, equivalent to 1 CO
bound/complex (). The difference spectrum in the Soret
region for these titration experiments is more complex (Fig. 4).
A clear trough can be seen at 443 nm, but also present is a double
trough between 415 and 426 nm. At higher CO concentrations, no further
changes occur in the heme d spectrum at 644 nm minus 623 nm, but a
deepening of the trough at 426 nm occurs together with smaller changes
at 560 nm and 590-600 nm. We attribute these changes to CO
binding with low affinity to heme b595.
Figure 4:
CO reduced minus reduced difference
spectrum at low (3 µM) (A) and high
(approximately 1 mM) (B) CO concentrations. The
samples were reduced by dithionite. Other conditions of measurement
were as in Fig. 2.
Figure 5:
CO and NO binding titration to purified
preparations of the reduced cytochrome bd complex.
Experimental details were as described under ``Experimental
Procedures''. A, CO binding as monitored by following
using 0.44 mg ml complex the spectral changes in the
-band region of oxygenated heme d
(
A
). B, NO binding using
A
with 0.86 mg ml
complex.
NO Interaction with the Reduced Cytochrome bd
Complex
The optical difference spectrum of NO interaction with
dithionite-reduced cytochrome bd is shown in Fig. 6. A
titration of the redshift of the heme d -band on NO binding (653
nm minus 627 nm in the difference spectrum) results in 5.5 ± 0.4
nmol of NO bound/mg of protein (Fig. 5B). This high
affinity binding is associated with changes in the Soret region of the
optical spectrum at 443 nm and around 425 nm. On increasing the NO
concentration to higher than stoichiometric amounts, features develop
at 437, 560, and 595-600 nm, which we attribute to low affinity
binding to heme b595. Binding of NO to reduced cytochrome bd also induces a heme-nitrosyl EPR spectrum (Fig. 7, tracea) with some similarities to that reported for the NO
complex of the
-subunit of hemoglobin(42) . The nature of
this spectrum changes as the NO concentration is increased from
stoichiometric amounts to higher levels (Fig. 7, traceb) consistent with NO binding to a separate heme site.
Subtraction of the two spectra from each other (Fig. 7, uppertrace) shows the low affinity species (appearing at high
NO concentrations) to have strong rhombic symmetry similar to that seen
in NO derivatives of several ferrous hemeproteins (42) including the
-subunit of hemoglobin(43) . A well resolved hyperfine
structure was not obvious under the conditions used for either the low
NO affinity or high NO affinity spectra. Samples for EPR were taken
alongside the optical titrations, at low and high ligand
concentrations. When quantified against a copper/EDTA standard, the
spin concentration at stoichiometric amounts of NO was 5.2 nmol/mg of
protein, increasing to 10 nmol/mg of protein at high NO concentrations.
We conclude that there are two NO binding sites of equal concentration
but different affinities. The spectral evidence indicates that these
are heme d and heme b595, respectively. No obvious evidence for strong
spin coupling between these hemes can be seen. This contrasts with the
cytochrome c oxidases, where the nitrosyl signal from
NO-ligated heme a
disappears when NO binds to the close by
Cu
(32) .
Figure 6:
The optical spectrum of NO interaction
with dithionite-reduced cytochrome bd complex (0.86 mg
ml) in 0.1 M potassium HEPES, pH 7. A, 7.5 µM NO concentration; B, 1-min
bubbled NO through the sample.
Figure 7:
EPR spectra of NO-treated
dithionite-reduced cytochrome bd complex. NO and cytochrome bd concentrations were as in Fig. 6. EPR conditions were as in
Fig. 3 except the temperature was 77 K, and the microwave power was 6.3
mW.
The Interaction of Cytochrome bd with
Cyanide
Previous studies using membrane particles from E.
coli and A. vinelandii have shown that the rate of
cyanide binding to cytochrome bd oxidase is more rapid at
higher rates of respiratory activity(33, 34) . In the
present case, binding of cyanide to the purified complex under turnover
conditions, with ubiquinol as substrate, occurs approximately 3 times
faster (second order rate constant of 2.6 M sec
) than the rate of binding of cyanide to
the air-oxidized complex, as assessed by spectral changes at 650 nm (Fig. 8). From Fig. 9it can be seen that the final species
formed is a ferric heme d-cyanide complex. A loss of oxygenated heme d
occurs in the air-oxidized enzyme (Fig. 9A), and cyanide
prevents the formation of ferrous heme d when added in the presence of
reductant (Fig. 9B). These results suggest that the
difference between the rates of cyanide binding to the complex in the
absence and presence of respiratory substrates is due to the faster
removal of molecular oxygen from heme d under turnover conditions
and/or a higher steady-state level of the actual CN
binding form of heme d. Krasnoselskaya et al.(35) have suggested concerted binding of cyanide to ferric
hemes d and b595, possibly as a bridging ligand. If this does occur in
the air-oxidized enzyme, the present results show that cyanide binding
to ferric heme b595 is not strong enough to prevent its reduction, in
contrast to the cyanide-bound heme d species, which remains ferric in
the presence of reductant. In the EPR spectrum (Fig. 10), there
is a drop in both the axial and rhombic components of the g = 6
signal when cyanide binds to the air-oxidized cytochrome bd complex. The intensity falls by approximately 30% after incubation
with 10 mM cyanide for 1 h and continues to fall with longer
incubation. It is clear that the full effect of cyanide on the high
spin signal is much slower than the binding of cyanide to heme d as
assessed by the optical changes at 650 nm. On the other hand, 1 h of
incubation with cyanide completely removes the g = 2.55 signal,
and a new signal appears at g = 2.97 (Fig. 10). Kauffman et al.(44) report a value of g = 2.99 for the
position of the heme d ferric cyanide signal in membrane preparations
from A. vinelandii incubated with 5 mM cyanide. A
smaller feature (at g = 3.23) was also noted by these workers.
In the present case, small but significant perturbations can also be
seen in the spectrum around the g = 2.97 signal in the
cyanide-treated sample. When heme b595 is reduced these are lost
together with the g = 3.4 signal from b558. The g = 2.97
signal remains, confirming the findings of the optical spectra (Fig. 9) that the low spin ferric cyanide complex of heme d is
not reduced by ubiquinol.
Figure 8:
Rate of
cyanide binding to oxygenated heme d in the absence () and
presence (
) of respiratory substrates. Conditions of measurement
were as follows: cytochrome bd (2.1 µM) in
potassium phosphate (0.1 M), pH 7.2, ubiquinone-1 (250
µM), and DTT (3 mM).
Figure 9:
Optical difference spectra of cyanide
treated air-oxidized cytochrome bd complex. Concentrations
were as in Fig. 8. Samples were incubated with cyanide for 30 min. A, cyanide-treated minus air-oxidized complex; B,
cyanide-treated complex in the presence of ubiquinone-1 (250
µM) and DTT (3 mM) minus air oxidized complex; C, control sample, reduced (ubiquinone-1 and DTT for 30 min)
minus air-oxidized complex.
Figure 10:
EPR spectra of cyanide binding to
purified cytochrome bd oxidase. Conditions of measurement were
as in Fig. 3. Other conditions were as in Fig.
9.
Purification of the Cytochrome bd Complex
The
first successful purification of cytochrome bd from A.
vinelandii was reported by Kolonay et al.(6) , who
used DEAE-Sepharose chromatography in the presence of dodecyl
maltoside. The present procedure provides a lower cost alternative
based on ammonium sulfate precipitation, which gives a final
preparation of comparable yield, purity, and activity. The final
concentration of b-type heme shown in is 10.3 nmol/mg of
protein. If a molecular mass of 85,100 Da is used for the molecular
mass of the complex from A. vinelandii, as derived from the
nucleic acid sequences for subunits I and II, then a theoretical value
can be calculated for the concentration of total heme b/mg of purified
complex. This turns out to be approximately twice the measured value of
the present preparation and also for the final preparation of Kolonay et al.(6) . Both methods used the overproducing strain
of A. vinelandii MK8. Contaminating protein of molecular
weight different from the two main bands on the electrophoretic gel was
estimated at no more than 12%. We suggest that a significant amount of
(totally heme-free) apoprotein is also present in these membranes and
copurifies with the active complex. Although this would lower the
apparent value of heme per mg of complex, the relative stoichiometry of
the different hemes per complex would be unaffected.
-region of reduced heme d. A value of 12.0 ± 1.0
mM
cm
for the wavelength
pair 628 nm minus 605 nm was calculated for the reduced minus
air-oxidized complex. It should be noted that this wavelength pair is
unaffected by the amount of oxygenated species present. The
corresponding value reported for the E. coli enzyme is 7.4
mM
cm
(5) .
Stoichiometry of the Redox Centers
The apparent
simplicity of the cytochrome bd complex belies many
experimental anomalies. The first concerns the number of redox centers.
It is generally assumed that the complex incorporates two classes of
redox-active heme, b and d. Previous experiments have clearly
identified two species of b-type heme, heme b558 and heme
b595(5, 7) . However, 2 mol of heme d/complex have been
suggested on the basis of CO titration experiments(8) , and
support for this stoichiometry is provided by the presence of two
binding affinities of nitrite to heme d(10) . The present data
from metal and heme analysis, as well as ligand binding titration
results are summarized in . The ratio of total iron to
b-type heme is 1.7. This result indicates the stoichiometry of two
b-type hemes plus one remaining heme, namely heme d. The small excess
of iron, in addition to an expected value of 1.5 Fe/b-heme, may be due
to some nonspecific iron as indicated by the EPR spectrum at g =
4.3. This stoichiometry is further supported by the results of CO and
NO titrations, which do not depend on quantitation of b-type heme. Both
CO and NO bind with high affinity to reduced heme d at a stoichiometry
of 1 mol of ligand/mol of complex. With this binding, the optical
absorbance of reduced heme d is fully removed. Finally, the spin
concentration of the high affinity heme-nitrosyl signal gives a value
of one per complex. We therefore conclude that cytochrome bd contains only one heme d per complex.
Ligand Binding to Hemes b595 and d
Although CO and
NO bind to reduced heme d with high affinity, some low affinity binding
to reduced heme b595 also takes place. It is possible to use these
different affinities to identify spectral changes in the Soret region
due to the two hemes. Changes in the Soret region with high affinity
binding of CO and NO indicate that the weak band at 443 nm must
represent changes in heme d absorption alone. The assignment of the
Soret band of reduced heme d to 443 nm would be in agreement with the
findings of Poole et al.(18) , who noted a small change
at 445 nm following low temperature photolysis of bound CO in E.
coli. Flash experiments using a helium/neon laser at 632.8 nm (36) have also indicated that a single absorbance band in the
Soret spectrum at 443 nm can be assigned to heme d. High affinity CO
binding also induces spectral changes at 415 nm and around 540 nm (Fig. 4). Similar changes have been noted in low temperature
CO-photodissociation spectra in the presence of oxygen by d'Mello et al.(4) . These changes are not seen with NO binding (Fig. 6). At present we have no obvious explanation for these
effects. The parallel changes around 595 nm and in the Soret region,
following low affinity binding of CO or NO, indicate binding to heme
b595 although the position of the trough in the Soret region is not the
same for the two ligands.
(37).
). None could be seen under the conditions of the
present experiments. Direct ligation of hydroxide to ferric heme d does
not seem to occur despite total loss of oxygen from ferrous heme d. The
low spin signal at g = 2.55 from ferric heme d is still present
at alkaline pH. Rothery and Ingledew (7) have noted that
alkaline pH lowers the total intensity in the high spin region of the
spectrum by approximately 20% and drastically increases the relative
size of the rhombic component. Unfortunately the low spin region of the
EPR spectrum was not presented, but the authors suggested that there is
a pH-dependent equilibrium between the rhombic and axial components of
the aerobic spectrum. This equilibrium would be affected as changes in
pH titrate an ionizable group associated with heme b595. The present
results indicate that this must occur without direct ligation of
hydroxide to heme.
The Nature of the Oxygen Binding Site
Previous
work on CO binding (29) and CO photodissociation (23) has
provided some evidence for the presence of a heme-heme binuclear center
in the cytochrome bd complex. Some interaction between heme
b595 and heme d is also indicated from the fact that binding of
molecular oxygen to anoxically oxidized heme d perturbs the EPR line
shape of heme b595(7) . Similar perturbations are seen in the
present work when oxygen is removed from heme d by exposure to alkaline
pH, but as discussed above this may simply be due to the effects of
changes in an ionizable group close to heme b595. The results with NO
and cyanide binding show that any cooperative interaction between the
redox centers in cytochrome bd must be weak. There is no
strong anti-ferromagnetic coupling when NO is bound to both heme b595
and heme d. The apparently simple EPR characteristics of NO binding are
good evidence for two binding sites that are some distance from each
other and hence not part of a binuclear center. Spectral evidence
clearly identifies heme d and heme b595 as the two sites. The close
distance exchange coupling found in the copper-containing oxidases is
not present in cytochrome bd and is probably the reason why
the oxygenated heme d species is so stable. In cytochrome aa, this species is very short lived at room
temperature(40) . Similarly, cyanide binding to heme d is only
slightly influenced by the presence of substrate. This contrasts to the
cyanide affinity of heme a
in cytochrome c oxidase, which is greater by over a factor of 10
in
the partially reduced enzyme compared with the fully oxidized or fully
reduced states(37) . Overall, the results argue against the
presence of a binuclear center where the two hemes act in a strong
cooperative manner to facilitate oxygen reduction.
Table: Purification of cytochrome bd from A. vinelandii
Table: Analysis of purified cytochrome bd complex from
A. vinelandii
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.