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INTRODUCTION |
Cytochrome bd is a terminal oxidase present in the
respiratory chains of many bacteria (reviewed in Ref. 1, see an
evolutionary tree in Ref. 2). The enzyme catalyzes reduction of
molecular oxygen to water by ubiquinol or menaquinol as natural
electron donors (3-5) but shows no sequence homology to the
heme-copper quinol oxidases. Also, in contrast to these oxidases,
cytochrome bd does not contain copper and, although
generating 
(6-9), does not pump protons (10).
Cytochrome bd-type oxidases purified from Azotobacter
vinelandii or Escherichia coli consist of two subunits
and carry three iron-porphyrin groups: low spin heme
b558, high spin heme
b595, and a chlorin-type high spin
iron-porphyrin group (heme d). All three redox centers are
proposed to be located near the periplasmic side of the membrane (2).
Heme b558 is directly involved in ubiquinol
oxidation (11, 12). Heme d binds O2 and is
involved in the trapping and reduction of oxygen (3). The specific role of heme b595 is still a matter of debate. One
obvious possible role is the transferrence of electrons from
heme b558 to heme d (13-15).
However, because heme b595 is high spin, it is
tempting to consider its involvement in dioxygen reduction; it was
proposed that hemes b595 and d might
form a binuclear dioxygen reduction center analogous to the heme-copper
oxygen-reducing site in the cytochrome aa3- or
bo3-type oxidases (16-19). In such a case, one might expect heme b595 to react with other
exogenous ligands such as CO or NO as is typical of most high spin
hemoproteins. Surprisingly, despite a long history of such studies,
this essential and apparently simple question has not been answered by
the apparently conflicting data. The intricate line shape of the
absorption changes induced by CO binding with the fully reduced
cytochrome bd in the Soret band as observed in conventional
room temperature studies has long been considered to indicate CO
binding with both hemes d and b595
(e.g. see Refs. 20-22 and references therein). However, the
spectral features attributed to CO binding with
b595 could be caused by a small bandshift of
unligated heme b595 induced by CO interaction
with the nearby heme d (23). Moreover, magnetic circular
dichroism spectroscopy has shown that only a small fraction of
heme b595, if any, in the E. coli
cytochrome bd binds CO or NO at room temperature. On the
other hand, low temperature photodissociation studies carried out by
Poole and co-workers (24-26) on cytochrome bd from
different bacteria have revealed consistently a simple pattern of
photoinduced spectral changes assigned to the photodissociation of the
cytochrome b595-CO complex. Recent femtosecond
photobleaching studies have identified the Soret band of ferrous heme
b595 at about 440 nm and support this
interpretation (23). CO binding with about 15% of heme
b595 at cryogenic temperatures was observed with
the aid of Fourier transform infrared spectroscopy in the membrane-bound cytochrome bd from E. coli, but
there was no binding in the isolated enzyme (18).
Conceivably, the enzyme may behave differently at cryogenic and room
temperatures. On the other hand, the results obtained by different
techniques and at different temperatures were also often done with
different preparations and under different conditions, which
complicates direct comparison of the data in the literature.
We considered it worthwhile to address this problem specifically and
compare the spectral changes associated with CO binding and
photodissociation at room and low temperatures in the same preparation
of purified cytochrome bd from A. vinelandii. The results confirm that CO photodissociation from heme
b595 is the sole major process revealed
spectrophotometrically at temperatures around
100 °C. However, the
fraction of heme b595 that shows CO
photodissociation at low temperature corresponds to only about 5% of
the enzyme. These observations resolve the apparent contradiction between the room and low temperature experiments and corroborate the
model of cytochrome bd interaction with exogenous ligands assuming negative cooperativity between hemes
b595 and d (22, 27).
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EXPERIMENTAL PROCEDURES |
Cytochrome bd was isolated from A. vinelandii strain MK8 overproducing cytochrome bd (28)
essentially as reported (21). The cytochrome bd
concentration was determined from the dithionite-reduced minus air-oxidized difference absorption spectra using a

-(628-605) value of 12 mM
1
cm
1 (21). E. coli cytochrome bd was
isolated from the membranes of strain GO105/pTK1 (29) as described
(30), but the final hydroxyapatite chromatography step was omitted.
Low temperature experiments were performed using a dual-wavelength
scanning spectrophotometer with low temperature facilities and
photolysis arrangements as described (31-33). A typical experiment was
performed as follows. Purified cytochrome bd in a buffer
containing 100 mM potassium phosphate, 0.5 mM
EDTA, 0.02% n-dodecyl-
-D-maltoside, and 30%
(v/v) ethylene glycol, pH 7.2, was reduced with a small amount of solid
sodium dithionite for 15 min or by 10 mM ascorbate/50 µM
N,N,N',N'-tetramethyl-1,4-phenylenediamine
for 25 min in a 2-mm path-length cuvette (total volume, 1 ml) and then
bubbled with CO for 2 min at room temperature. The cuvette was
transferred in total darkness to an ethanol/dry-ice bath at
78 °C
and frozen rapidly. After equilibration in the sample compartment of
the spectrophotometer, a baseline was recorded and stored in the
memory. The sample was subsequently photolysed for 3 min with the
focused beam from a 150-watt tungsten lamp, and the difference
absorption spectra (versus the stored baseline) were
recorded. A reduced CO-untreated sample was prepared and frozen in a
parallel manner to provide a baseline for the low temperature
measurements of the CO binding-induced absorption changes and compared
with the photodissociation-induced spectral changes.
Laser flash-induced CO photodissociation/recombination time-resolved
measurements at room temperature were done as described (34).
Deconvolution of the kinetic curves of CO recombination with cytochrome
bd at room temperature was done with the software package
GIM (subroutine "discrete") developed by A. L. Drachev in the
A. N. Belozersky Institute of Physico-Chemical Biology (Moscow).
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RESULTS |
Room Temperature Static Spectra--
Fig. 1 compares absorption
changes induced by CO in the dithionite-reduced cytochrome
bd from A. vinelandii (spectrum
a) and E. coli (spectrum b)
at room temperature. This side-by-side comparison of the two enzymes is
important because some of the essential CO binding studies were
performed with cytochrome bd purified from E. coli, whereas others were done with the A. vinelandii enzyme. Therefore, for generalization of the conclusions it is desirable to ensure that in the same study, the two enzymes show the
same CO-induced spectral changes.
The two difference spectra are very similar and agree well with the
published data (21, 22, 35). In the visible region, a typical red shift
of the absorption band of ferrous heme d at ~630 nm
(presumably, the Qy transition) is observed, giving
rise to a symmetric first derivative-shaped curve with a maximum at 642 nm and a minimum at 622 nm. This shift is accompanied by an increased
absorption at 538 nm that could report concurrent modulation of the
Qx band of heme d. The intricate line shape of the Soret band changes induced by CO binding is not yet fully
understood, but as discussed in Refs. 22 and 23, it may result from an
overlay of two effects. First there is a large blue shift of the
ferrous heme d
-band that gives rise to a maximum at
~400 nm (formation of CO-ligated heme d) and a broad
minimum centered around 432 nm (decrease in free heme d)
with an inflection point around 420 nm between them. This major effect
overlaps with a sharp first derivative-shaped feature with a
maximum at 436 nm and a minimum at 444 nm dominated by perturbation of
the ferrous heme b595 band at ~440 nm induced
indirectly by CO binding to heme d (23). As discussed in
Ref. 22, room temperature absorption spectra provide no clear evidence
for direct CO binding by heme b595.
Low Temperature CO Photodissociation--
A difference absorption
spectrum (CO-reduced minus reduced) recorded at
100 °C
is shown in Fig. 2a. Changes in the 500-700-nm range are
virtually identical to those at room temperature (Fig. 1a) and reflect mainly CO
binding with heme d. In the Soret band, the changes recorded
at low temperature are slightly different in line shape and showed some
variability between the samples. Very similar results were obtained
with the samples reduced by ascorbate + N,N,N',N'-tetramethyl-1,4-phenylenediamine
instead of dithionite (data not shown).

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Fig. 1.
Room temperature difference absorption
spectra induced by CO binding with cytochrome bd from
A. vinelandii (a) and E. coli (b). a, 3 µM cytochrome bd purified from A. vinelandii in 100 mM potassium phosphate, pH 7.2, with
0.5 mM EDTA, 0.02%
n-dodecyl- -D-maltoside, and 10% (v/v)
glycerol. b, the difference spectrum was recorded with 3.5 µM cytochrome bd purified from E. coli and has been normalized by a A-(642-622) value
to spectrum a; 0.025% sodium
N-lauroylsarcosinate was used in the buffer instead of
0.02% n-dodecyl- -D-maltoside.
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Photoinduced Absorption Changes--
Illumination of the frozen
CO-ligated cytochrome bd with white light for 3 min brings
about absorption changes indicative of a red shift of the Soret band
(Fig. 2, spectrum
b). The line shape (a maximum around 438 nm and a minimum at
about 420 nm) is in good agreement with earlier data (25, 26) and is
typical of CO photodissociation from a high spin heme b
(36). No photoinduced changes of heme d can be discerned in
the far red region under these conditions (cf. also Refs.
24-26) because of geminate recombination of CO with the heme
d, which is fast enough even at
100 °C (3). Much lower
temperatures (4-5 K) are required to observe the photolysis and
subsequent recombination of CO with heme d (37). This
property makes the low temperature photodissociation studies fairly
selective for cytochrome b595, whereas at room
temperature the changes from heme d in the Soret region
overlap those of heme b595.

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Fig. 2.
Absorption changes induced by CO binding and
photodissociation from the reduced cytochrome bd at
100 °C. a, static difference
absorption spectrum (1 mM CO minus reduced);
b, photodissociation spectrum (expanded 4-fold). The sample
cell contained 0.8 µM cytochrome bd from
A. vinelandii reduced with a few grains of solid dithionite.
Other conditions were as described in the Fig. 1 legend for
spectrum a except that 30% (v/v) ethylene glycol
was used instead of 10% (v/v) glycerol.
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At a 25-fold higher concentration of the enzyme, the matching
absorption changes in the visible region can be resolved clearly (Fig.
3); the difference spectrum of the
photoinduced changes shows peaks at 595 and 556 nm and a trough at 571 nm, diagnostic of CO dissociation from a high spin rather than low spin
heme b. Together, the data show that CO photodissociation
from high spin heme b595 is the sole effect
revealed by static spectra in cytochrome bd at
100 °C.

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Fig. 3.
CO photodissociation from heme
b595 as revealed by changes in the visible
region of the absorption spectrum. Conditions were as described in
the Fig. 2b legend, but the cytochrome bd
concentration was 20 µM. The temperature was
100 °C.
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Quantitation of the Absorption Changes--
The photoinduced
absorption changes at low temperatures cannot be quantitated directly
with the use of known extinction coefficient values for the
chromophores because of the well known effect of intensification of the
absorption bands at cryogenic temperatures (typically ~10-fold) (38,
39). This phenomenon is mainly caused by an increase in the effective
optical pathway because of the multiple reflection of light within the
frozen sample, with a minor contribution from band narrowing. The
extent of band intensification depends critically on the buffer/solvent
system used, the exact procedure for freezing the sample, and other
details that render comparison of the different series of experiment
samples problematic (38, 39).
However, when the experiment is repeated under the same conditions,
fairly reproducible results can be obtained. Therefore, to calibrate
the absorption changes induced by photodissociation of CO at cryogenic
temperatures, we compared their magnitude with that of the well
resolved changes induced in the same samples by CO binding with heme
d and recorded at the same low temperatures. In each
experiment, a set of three low temperature spectra was recorded. First,
a spectrum of a control sample reduced with dithionite was taken.
Second, a spectrum of a similar sample that was treated with CO at
room temperature in the dark before freezing was recorded (reduced + CO
state). Third, after taking the low temperature spectrum of the reduced + CO state, the frozen sample was illuminated, and a spectrum of the
photodissociated state was taken (reduced + COlight). Each
of the low temperature spectra was recorded versus a frozen
sample of buffer alone to compensate for light scattering. Subsequently, low temperature difference spectra were constructed for
CO photodissociation at low temperature (reduced + COlight minus reduced + CO) compared with low
temperature recordings of a sample prepared by CO binding at room
temperature (reduced + CO minus reduced). In this way, the
magnitude of the heme b595 response induced by
low temperature photodissociation of CO was evaluated by comparison
with the well separated absorption changes of heme d in the
same frozen sample induced by CO binding.
Eight such experiments were performed, and the results are summarized
in Table I. The normalized observed
photoinduced absorption changes of heme b595 are
almost two orders of magnitude smaller than a typical extinction
coefficient of
215 mM
1 cm
1
for a difference spectrum induced by CO binding with reduced high spin
heme b proteins (36) and correspond to photodissociation of
~4.5% of heme b595 at
70 °C and
even less (~3%) at
100 °C. The actual values may be slightly
higher because of somewhat lower intensification of the Soret band
changes relative to the
-range. In our samples, the
/
intensification ratio was about 1.4. The corresponding values for the
percentage of b595-CO photodissociation are
given in Table I in brackets and give an upper limit for the effect
(~4 and 6% of the heme at
100 °C and
70 °C,
respectively).
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Table I
Quantitation of the absorption changes induced by CO photodissociation
from heme b595 at cryogenic temperatures
Conditions were as described in the Fig. 2 legend. Cytochrome
bd concentration, 0.8 µM. R,
denotes the fully reduced form of cytochrome bd.
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CO Recombination--
Independent evidence for CO binding with a
small fraction of heme b595 in the same
preparation of the enzyme is provided by the time-resolved studies of
CO recombination with the reduced cytochrome bd after laser
flash-induced photolysis of the ligand complex at room temperature
(Fig. 4). The absorption changes induced by CO photodissociation were fully reversible in the dark, and their
decay revealed several phases in general agreement with the data of
Jünemann et al. (40). The rapid phase is well
separated kinetically and is characterized by
of 18 ± 3 µs
at ~1 mM CO (second order rate constant,
k1 = 5.5 × 108
M
1 s
1). A difference spectrum
of this phase is shown in Fig. 4 by spectrum a,
which is typical of CO binding with heme d (including a
spectral perturbation of ferrous heme b595
induced by CO binding with heme d (23)). The slower part of
recombination included 2-3 phases with
values in the range of
0.2-2.3 ms, but the difference spectra of the phases were rather
similar and indicated CO binding with a b-type heme in
agreement with the findings in Ref. 40. Heterogeneity of the slow part
of the response could reflect CO recombination with both
b595 and low spin heme
b558. In any case, the overall magnitude of the
slow phase of absorption changes allows us to evaluate the upper limit
of the CO-reactive fraction of heme b595.

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Fig. 4.
Room temperature CO recombination after laser
flash photolysis of the cytochrome bd-CO complex.
a, difference spectrum of the first phase ( = 18 ± 3 µs); b, spectrum of the combined slower
phases (0.2-2.3 ms). Cytochrome bd was from A. vinelandii, 2.4 µM; CO concentration, ~1
mM. The buffer was as described in the Fig. 2 legend.
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An overall difference spectrum for the slow phases is given
in Fig. 4 by spectrum b, which reveals a maximum
near 440 nm and a minimum around 420 nm, similar to the photoinduced
absorption changes at low temperatures. Its amplitude
(
420-440 = 4-6 mM
1
cm
1) allows an estimate of the upper limit for
b595 reactivity toward CO and corresponds to
2-3% of this redox center. This value may be even less if part of the
slow phase of CO recombination reflects binding of the ligand with the
low spin heme b558 (40).
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DISCUSSION |
This work resolves the long standing controversy as to whether
high spin heme b595 in cytochrome bd
reacts with exogenous ligands like CO. Although the absorption changes
induced by illumination of the CO complex of cytochrome bd
at low temperatures pointed consistently to CO photodissociation from
heme b595 (3, 24-26, 41), room temperature
magnetic circular dichroism measurements allowing resolution of
individual signals of ferrous heme b595 in the
visible have shown that the heme does not respond significantly to
either CO or NO (22). The present work shows that 1) there is CO
binding with ferrous b595 as evidenced by both
low temperature photodissociation of the bd-CO complex and
time-resolved studies of CO recombination at room temperature, but 2)
the binding involves only a small fraction of the heme, some 2-5%,
that could well go unnoticed in magnetic circular dichroism studies.
Two alternative explanations for our finding can be considered. First,
the results may provide support for the model of exogenous ligand
binding with cytochrome bd suggested in Refs. 22 and 27.
This model (A) implies that the two high spin hemes d and b595 are located very close to each other and
form a diheme binuclear center with a capacity for only one molecule of
exogenous ligand like CO. This molecule distributes between the two
heme irons within the binuclear center in accordance with their
intrinsic affinities for the ligand, heme d having a higher
affinity (cf. Fig. 9 in Ref. 22). Within the framework of
model A, our work indicates that the Kd of
heme d for CO is about 20-30-fold lower that that of
b595, so that about 95% of the ligand bound to
cytochrome bd reposes on heme d (i.e.
the ligand molecule trapped in the diheme binuclear center of the
oxidase spends about 95% of time at heme d and ~5% at
heme b595).
Model B proposes that the cytochrome bd population
may be heterogeneous so that in ~95% of the enzyme the ligand can
bind to heme d only, whereas in the remaining ~5% the
ligand reacts with heme b595 (whether heme
d in this 5% of the enzyme can also bind CO is difficult to deduce).
Model B may be more consistent with the recombination data that show
heme b595-associated changes to be slower than
those of heme d. Indeed, if there were free rapid (say,
nanosecond) equilibration of CO between hemes d and
b595 within the same heme-binding pocket, one
might expect synchronous development of the absorption changes of the
two hemes induced by CO recombination.
However, the above alternatives are not necessarily mutually exclusive
and rather may pose a question as to what the time constant for
equilibration between the states is.
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(Eq. 1)
|
If the characteristic time is short relative to an
observation period in a particular type of experiment, model A applies. If the equilibration is slow, model A will transform to model B. The
process may be slow if limited, for instance, by an exchange of the
endogenous ligands in the coordination sphere of heme d (42). It must be emphasized that the above discussion refers to
cytochromes bd from A. vinelandii and E. coli, showing much the same CO reactivity (e.g. Fig.
1). It is interesting that in cytochrome bd from
Bacillus stearothermophilus, the major part of heme
b595 binds CO at room temperature as can be
inferred from the difference spectra in Fig. 1B of Ref.
43.