(Received for publication, September 7, 1995; and in revised form, November 27, 1995)
From the
Cyanide-binding to the heme-copper binuclear center of bo-type ubiquinol oxidase from Escherichia coli was
investigated with Fourier transform-infrared and EPR spectroscopies.
Upon treatment of the air-oxidized CN-inhibited enzyme with excess
sodium dithionite, a C-
N stretching
vibration at 2146 cm
characteristic of the
Fe
-C=N-Cu
bridging structure was quickly replaced with another stretching
mode at 2034.5 cm
derived from the
Fe
-C=N moiety. The presence
of ubiquinone-8 or ubiquinone-1 caused a gradual autoreduction of the
metal center(s) of the air-oxidized CN-inhibited enzyme and a
concomitant appearance of a strong cyanide stretching band at 2169
cm
. This 2169 cm
species could
not be retained with a membrane filter (molecular weight cutoff
= 10,000) and showed unusual cyanide isotope shifts and a
D
O shift. These observations together with metal content
analyses indicate that the 2169 cm
band is due to a
Cu
CN complex released from the enzyme. The same
species could be produced by anaerobic partial reduction of the
CN-inhibited ubiquinol oxidase and, furthermore, of the CN-inhibited
cytochrome c oxidase; but not at all from the fully reduced
CN-inhibited enzymes. These findings suggest that there is a common
intermediate structure at the binuclear center of heme-copper
respiratory enzymes in the partially reduced state from which the
Cu
center can be easily released upon cyanide-binding.
Cytochrome bo-type ubiquinol oxidase in the aerobic
respiratory chain of Escherichia coli catalyzes the
two-electron oxidation of ubiquinol-8 (QH
) (
)and the four-electron reduction of dioxygen to
water(1, 2) . These redox reactions mechanistically
couple with the formation of an electrochemical proton gradient across
the cytoplasmic membrane not only by scalar protolytic reactions at the
inner and outer surfaces of the membrane but also by a proton pumping
mechanism(3, 4, 5) . Based on the structural
homologies of subunits I, II, and III, cytochrome c oxidases
and some bacterial quinol oxidases including the E. coli cytochrome bo are classified in the heme-copper
respiratory oxidase superfamily(6, 7) . However, there
is a notable difference in electron-donating substrates between the two
enzymes, cytochrome c (a hydrophilic one-electron carrier) and
quinols (hydrophobic two-electron and two-proton
carriers)(8, 9) . As a consequence, the Cu
center is absent in subunit II of quinol
oxidase(5, 10, 11) .
Although the dioxygen reduction mechanism at the heme-copper binuclear center is thought to be identical in both enzymes(12, 13, 14) , these differences raise the questions whether the electron transfer reactions from the substrates to dioxygen and the proton pumping mechanism coupled to these redox reactions are alike or distinct. It is proposed that the last two steps of the four-electron transfer reactions to dioxygen are linked to proton pumping by cytochrome c oxidase(15, 16, 17) . Thus it becomes increasingly important to analyze the mixed-valence states of oxidase as a model for the intermediate species of the dioxygen reduction chemistry, since the understanding of the redox-linked structural change(s) at the metal center appears to be a key point to reveal the redox-linked proton pumping.
In a previous study, Tsubaki (18) analyzed cyanide binding to the Fe-Cu
binuclear center of cytochrome c oxidase. In the resting
(air-oxidized) state, a bound cyanide showed an infrared C-N
stretching band at 2152 cm
, assignable to a bridging
structure,
Fe
-C=N-Cu
.
This assignment was confirmed recently by structural characterizations
and infrared measurements on a series of model complexes containing the
[Fe
-C=N-Cu
]
bridge unit(19) . Upon partial reduction of the CN-inhibited
cytochrome c oxidase an infrared band appeared at 2131
cm
assignable to the
Fe
-C=N structure. Further
reduction resulted in an appearance of two new infrared bands at 2058
and 2045 cm
, concomitantly, assignable to the
Fe
-C=N species. These
observations suggest three kinds of conformational change to occur at
the Fe
-Cu
binuclear site as the
reduction of the metal centers proceeds(18) .
Subsequently
Tsubaki et al. (11) carried out a combined study using
EPR and FT-IR spectroscopies to clarify the structural differences of
the binuclear center between bo-type ubiquinol oxidase and
cytochrome c oxidase. EPR spectra of bo-type
ubiquinol oxidase in the air-oxidized state showed EPR signals from an
integer spin system confirming the existence of the spin-spin
exchange-coupled binuclear
site(20, 21, 22, 23) . EPR spectra
of the cyanide, azide, and formate complexes in the air-oxidized state
indicated that a gross conformation at the binuclear site seems well
conserved among the heme-copper oxidase superfamily(11) . FT-IR
spectroscopy confirmed these observations: the cyanide that binds to
the air-oxidized enzyme exhibits an infrared band at 2146
cm characteristic to the
Fe
-C=N-Cu
structure(11) .
In the present study we extended the FT-IR and EPR spectroscopic studies to clarify the structure at the heme-copper binuclear center using cyanide as a monitoring probe.
Figure 1:
Cyanide (C
N)
bindings to the wild-type ubiquinol oxidase in the air-oxidized state.
C
N was added to a final concentration of 5
mM to the PEG 4000-treated enzyme (0.53 mM)
containing 1.1 mol of Q
/mol of the enzyme (a and b) and to the untreated enzyme (0.45 mM) containing
2.2 mol of Q
/mol of the enzyme (c and d).
FT-IR spectra in the C-N stretching region were measured just
after (a and c) and 48 h after (b and d) the addition of potassium cyanide. The enzyme at the stage
of (d) was filtered through a membrane filter (MWCO =
10,000), and the filtrate was directly introduced into an infrared
cell. Then, the FT-IR spectrum of the filtrate was measured with a
reference cell containing H
O (e). Conditions:
infrared cell path length, 51 µm; temperature, 4 °C; spectral
accumulation, 200 cycles (40 min); spectral resolution, 4.0
cm
.
Figure 2:
Cyanide (C
)
binding to the fully reduced ubiquinol oxidase (a) and the
effect of carbon monoxide on the CN binding (b and c). a, cyanide (
C
N) was
anaerobically added to a final concentration of 5 mM to the
fully reduced enzyme (0.39 mM) with excess sodium dithionite. b, the enzyme was first fully reduced with excess sodium
dithionite in the presence of carbon monoxide and then
C
N was anaerobically added to the enzyme at a
final concentration of 5 mM. c, the ordinate of b, is reduced by one-fourth to clarify the CO binding to the
enzyme which shows the 1959.7 cm
band. Other
conditions are the same as described in the legend to Fig. 1.
EPR spectra of the
air-oxidized CN-inhibited ubiquinol oxidase and its dithionite-treated
(and quenched at 77 K just after the addition) forms were examined at
15 K. Addition of cyanide to the air-oxidized enzyme reduced the
intensity of a g = 6 high spin signal without affecting the g
= 3 low spin signal (g = 2.98, 2.26, and 1.45) (Fig. 3, a and b) as described
previously(11, 30) . Addition of excess sodium
dithionite to the air-oxidized CN-inhibited enzyme caused a rapid
disappearance of the g = 3 low-spin signal and an appearance of
a new low-spin signal with g = 3.24 (Fig. 3c). Prolonged incubation with sodium dithionite
eventually eliminated the g
= 3.24 low spin signal.
This EPR signal is assignable to the
Fe
-C=N species (22, 23) on the basis of similarity to the
corresponding species (g
= 3.58) of the partially
reduced CN-inhibited cytochrome c oxidase(32) .
Occasionally we observed a weak stretching band at 2123 cm
(for
C
N) which shifted to 2078
cm
upon
C
N substitution (Table 1). This band also appeared in the partially reduced
CN-inhibited states (one-fourth-reduced and one-half-reduced states;
see later) and, therefore, may be assignable to the
Fe
-C=N species.
Figure 3:
EPR spectra of ubiquinol oxidase in the
air-oxidized (a) and the air-oxidized CN-inhibited (b) states, and effect of dithionite-treatment on the
air-oxidized CN-inhibited form (c) at 15 K. Cyanide (C
N) was added to the air-oxidized enzyme
(0.39 mM) (a) at a final concentration of 5
mM, and they were incubated on ice overnight to ensure
complete binding of cyanide (b). Then, slight excess sodium
dithionite was added anaerobically to the CN-inhibited enzyme in an EPR
tube through a rubber septum, mixed quickly, and the sample was frozen
in liquid nitrogen (77 K) (c).
The FT-IR
spectra of these autoreduced CN-inhibited enzymes were characterized
with the appearance of a strong cyanide stretching band at 2169
cm (in an H
O buffer) (Fig. 1, c and d). Careful measurements on the time-dependent
spectral change showed the following. 1) There is a preceding phase
forming a 2198 cm
band species (Fig. 1c). 2) The formation of the 2169 cm
band species follows. This phase also shows weak infrared bands
at 2076 and 2038 cm
(Fig. 1d). 3)
The development of the 2169 cm
band intensity
reaches a plateau as the 2146 cm
band disappears,
but the 2034.5 cm
band characteristic to the
Fe
-C=N adduct does not grow so
strong. 4) A further incubation causes a gradual decrease in the
intensity of the 2169 cm
band itself. One of unique
features of this 2169 cm
species is its peculiar
cyanide-isotopic shift pattern that is very different from the usual
metal-bound cyanide species (Table 1). The other is its strong
D
O shift (Table 1). In a D
O medium, the
2169 cm
band shifted to 2161 cm
.
Further, this 2169 cm
species passed through a
membrane filter (MWCO = 10,000) (Fig. 1e)
indicating that this is a small molecular complex released from the
enzyme.
Figure 4:
Cyanide (C
)
bindings to the bound Q
-free ubiquinol oxidase (0.21
mM) in the air-oxidized state. a, the FT-IR spectrum
in the C-N stretching region after incubation of the enzyme with
5 mM
C
N for 150 min on ice. b, the FT-IR spectrum in the C-N stretching region after
incubation of the enzyme with 5 mM
C
N and 1 mM Q
for
46 h on ice. Other conditions are the same as described in the legend
to Fig. 1.
Figure 5:
FT-IR spectra of the CN-inhibited
cytochrome c oxidase at various redox levels in the region
from 2000 to 2200 cm. The partially reduced enzyme
(1.0 mM) in 50 mM Tris-DCl (pD = 8.0) was
incubated with 5 mM K
C
N. a,
CN-inhibited resting state (0/4); b, one-electron
equivalent-reduced CN-inhibited state (1/4); c,
two-electron equivalents-reduced CN-inhibited state (2/4); d, three-electron equivalents-reduced CN-inhibited state (3/4). Other conditions are the same as described in the
legend to Fig. 1.
During these spectral changes we observed
an appearance of a 2162 cm band in a D
O
buffer (Fig. 5b and c). In a similar
experiment carried out in an H
O buffer, the 2162
cm
band in the partially reduced states (one-fourth
and one-half) shifted to 2169 cm
without affecting
the 2152 and 2131 cm
bands (spectra not shown).
These observations strongly suggest that a cyanide species very similar
to that found in the CN-inhibited bo-type ubiquinol oxidase
was produced in the partially reduced state(s) of the CN-inhibited
cytochrome c oxidase. The intensity of the 2169
cm
band (in the D
O buffer, or the 2162
cm
band in the H
O buffer) changed in a
dose- and time-dependent manner. A higher cyanide concentration (20
mM) and a longer incubation time (70 h) in the partially
reduced state (one-fourth-reduced) caused a stronger intensity of the
2169 cm
band (spectra not shown). The 2165
cm
band (in a D
O buffer) observed by
Yoshikawa and Caughey (28, 34) is likely due to the
same species observed in the present study.
The large difference of the
bound C-N stretching vibration between bo-type ubiquinol
oxidase and cytochrome c oxidase in the reduced state is
likely due to a specific character of the cyanide-binding to the
binuclear center(36) . Cyanide binding to other typical ferrous
hemoproteins is extremely weak, except for horseradish peroxidase in
which the electrostatic interaction (or hydrogen bond) between a
protonated distal His residue and a ferrous heme-bound cyanide plays a
substantial role in the stabilization(37) . Thus, it is
possible that the ferrous heme-bound cyanide at the binuclear center is
stabilized by a protonated His residue in the vicinity of the heme in
the fully reduced state. The protonation of the His residue may be
directly coupled to the uptake of a proton upon binding of cyanide to
the reduced oxidase(38) . Among three invariant His residues
(His-284, His-333, and His-334) on the distal side of the high spin
heme(6, 7) , His-284 is likely to have such a role
since it is probably not an obligatory ligand to Cu unlike
His-333 and His-334(12, 24, 30) .
Alternatively, His-333 may perform such a part in cytochrome c oxidase, since it seems to be disordered or to have multiple
conformations in an x-ray crystal structure for the azide-inhibited
air-oxidized cytochrome c oxidase from Paracoccus
denitrificans(39) , but not for the air-oxidized
cytochrome c oxidase from bovine heart
mitochondria(40) . The x-ray crystal structures revealed also
that His284 can form hydrogen bond with Tyr-288 and Trp-280 and His-333
with Thr-352 or the carbonyl oxygen of
Phe-348(39, 40) . Thus the greater difference in the
Fe
-C=N stretching vibration is likely
due to the difference in the interaction between the protonated distal
His residue and the Fe
-C=N moiety.
The binuclear center mutant oxidases showed neither CN-bridging
infrared band in the air-oxidized state nor
Fe-C=N infrared band in the fully
reduced state, although several Fe
-C=N
species seemed to be formed. It is clear that presence of the Cu
center is essential for the binding of cyanide to the ferrous
heme since these mutant oxidases did somehow bind CO (although with a
very broad infrared band around 1970 cm
(His333Ala (24) and Y288L)
or with very weak affinity
(H284A)). However, we could not evaluate the specific role of the
imidazole group of His-284 and His-333 and the phenol group of Tyr-288
in the present study.
This 2169
cm species showed unusual cyanide isotope shifts and
a D
O shift (Table 1) and was able to pass through a
membrane filter (MWCO = 10,000). Metal content analyses of the
enzyme before and after the anaerobic cyanide treatment in the presence
of excess Q
revealed that a substantial amount (30%) of
copper ions was released in the filtrate after the treatment (Table 2). These observations suggest that the 2169
cm
band arose from a low molecular weight
copper-cyano species (i.e. a Cu
CN complex)
released from bo-type ubiquinol oxidase. EPR analysis revealed
no indication of a Cu
ion in the enzyme preparation
that showed the 2169 cm
band, suggesting that this
Cu
CN complex was in reduced state (i.e. Cu
state).
There are several reports
describing the release of copper ions from copper proteins in the
presence of cyanide. For cytochrome c oxidase both the
Cu and Cu
centers of the air-oxidized enzyme
could be removed by dialysis against a CN-containing
solution(41, 42) . It must be noted, however, that the
conditions are very different from the one in the present study. The
dialysis was done at alkaline pH (i.e. pH 10), and a much
higher concentration (50 mM
1.0 M) of cyanide
was required(41, 42) . Among the previous reports, our
particular interest is the cyanide binding study for Cu/Zn-superoxide
dismutase from bovine erythrocyte(43) . Raman and FT-IR
spectroscopic studies revealed that the native
Cu
-superoxide dismutase binds one cyanide showing a
band at 2137 cm
. With increased concentration of
cyanide the 2137 cm
band became weaker, and strong
vibrational modes (2123, 2093, and 2075 cm
)
developed concomitantly. These bands were due to di-, tri-, and
tetracyano Cu
complexes, respectively, arising from
copper removed from the protein. Simultaneously a new band appeared at
2169 cm
having an abnormally large
CN
shift of -60 cm
(43) . This 2169
cm
species was also observed in the filtrate through
a membrane filter(43) . All of these observations strongly
suggest that the 2169 cm
species found for the
superoxide dismutase-CN system is identical with the 2169
cm
species in the present study. Han et al. (43) concluded that the 2169 cm
species was
neither a protein species nor a microcell of CuCN solid because of its
abnormal cyanide isotopic shifts.
The 8 cm D
O downshift observed for the 2169 cm
species is not likely due to a direct hydrogen bonding of an
H
O (or D
O) molecule in medium to the CN moiety
of the Cu
CN complex, as hydrogen bonding
becomes weakened by an H-D exchange(44) . For the horseradish
peroxidase (Fe
)-CN system, an 8 cm
D
O upshift (from 2029 to 2037 cm
)
of the C-N stretching frequency has been attributed to the
formation of a hydrogen bond between the heme-coordinated cyanide anion
and a protonated (or deuterated) distal His residue(37) . It is
more likely, therefore, that an H
O (or D
O)
molecule itself also participates in forming the
Cu
CN complex and the resulting
intramolecular interactions between cyanide(s) and a water ligand(s)
may be essential for the appearance of the 2169 cm
band and its unusual vibrational mode pattern.
We have tried
to prepare the CuCN complex by mixing copper(I)
chloride and/or copper(I) cyanide with varying amounts of cyanide in
aqueous solution, but without success. It was reported that a reaction
involving copper(I), cyanide, and water under conditions of high
temperature and pressure gave a by-product species with
(Cu
(CN)
(H
O))
structure,
comprising a two-dimensional polymer(45) . We propose that this
kind of species may be responsible for the 2169 cm
band.
Mechanism of the Formation of the
CuCN Complex-The partially
reduced conditions seem essential for the formation and release of the
Cu
CN complex. Indeed the 2169
cm
species could be quickly produced by anaerobic
partial reduction of the CN-inhibited enzyme but not at all from the
fully reduced form. The absence of the 2034.5 cm
band that is characteristic to the
Fe
-C=N adduct may be noticed
when the 2169 cm
species developed fully by the
autoreduction of the CN-inhibited enzyme. This observation is
consistent with the notion that the presence of the Cu
center is essential for the binding of cyanide to the reduced
enzyme as discussed in the previous section.
The CN-inhibited bound
Q-free oxidase resulted in neither the development of the
2169 cm
band nor the release of the Cu
center. A simultaneous addition of excess Q
with
cyanide to the bound Q
-free oxidase was not so effective.
These observations suggest that the precise structure around the
binuclear site or the quinone binding site(s) may be essential for the
formation of the Cu
CN complex. The role of the
loosely bound ubiquinones (Q
or Q
) is not
clear, but is likely only for providing electron equivalents to the
metal centers of ubiquinol oxidase by an unknown mechanism. This is
based on the observation of the 2169 cm
species in
the one-fourth-reduced or one-half-reduced conditions of the
PEG-treated CN-inhibited ubiquinol oxidase, which contains only one
molecule of Q
at the high affinity binding site.
It is
of great importance that the same 2169 cm species
could be produced by anaerobic partial reduction of the CN-inhibited
cytochrome c oxidase in which no ubiquinone molecule is bound.
This observation suggests that there is a common intermediate structure
at the binuclear center of the heme-copper respiratory oxidases in the
partially reduced CN-inhibited state which is very susceptible to the
cyanide binding(s) and release of the Cu
center. The
greater formation of the 2169 cm
species for bo-type ubiquinol oxidase than cytochrome c oxidase
may be related to the instability of the
Fe
-C=N species (although its
presence was confirmed with the g
= 3.24 EPR signal)
in the partially reduced CN-inhibited enzyme. This difference is also
likely due to a slight difference(s) of the cyanide coordination
structure (including distal His residues) at the binuclear center. The
unique property of the heme-copper oxidase revealed in the present
study may provide a clue for understanding a mechanism of the dioxygen
reduction chemistry and the redox-linked proton pumping.
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