From the Department of Biological Sciences, University of Essex, Wivenhoe Park, CO4 3SQ Colchester, Essex, United Kingdom
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ABSTRACT |
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The reactions of nitric oxide (NO) with fully
oxidized cytochrome c oxidase (O) and the intermediates P
and F have been investigated by optical spectroscopy, using both static
and kinetic methods. The reaction of NO with O leads to a rapid (~100
s1) electron ejection from the binuclear center to
cytochrome a and CuA. The reaction with the
intermediates P and F leads to the depletion of these species in slower
reactions, yielding the fully oxidized enzyme. The fastest optical
change, however, takes place within the dead time of the stopped-flow
apparatus (~1 ms), and corresponds to the formation of the F
intermediate (580 nm) upon reaction of NO with a species that we
postulate is at the peroxide oxidation level. This species can be
formulated as either Fe5+ = O CuB2+
or Fe4+ = O CuB3+, and it is
spectrally distinct from the P intermediate (607 nm). All of these
reactions have been rationalized through a mechanism in which NO reacts
with CuB2+, generating the nitrosonium species
CuB1+ NO+, which upon hydration
yields nitrous acid and CuB1+. This is followed
by redox equilibration of CuB with
Fea/CuA or Fea3 (in which
Fea and Fea3 are the iron centers of
cytochromes a and a3,
respectively). In agreement with this hypothesis, our results indicate
that nitrite is rapidly formed within the binuclear center following
the addition of NO to the three species tested (O, P, and F). This work
suggests that nitrosylation at CuB2+ instead of
at Fea32+ is a key event in the fast inhibition
of cytochrome c oxidase by NO.
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INTRODUCTION |
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Cytochrome c oxidase (ferrocytochrome c oxidoreductase, EC 1.9.3.1), the terminal enzyme in the mitochondrial respiratory chain, catalyzes the reduction of molecular oxygen to water (1). This process is coupled to proton translocation across the inner membrane. The enzyme contains four redox-active centers. Electron entry from cytochrome c, the natural substrate, occurs via a diatomic copper center, CuA. After rapid equilibrium with Fea,1the electron is transferred to CuB and Fea3, which together constitute the binuclear center, where oxygen is reduced.
Although oxygen binds with low affinity (103
M1) (2) to reduced Fea3, rapid
electron transfer from the two reduced metals comprising the binuclear
center to molecular oxygen ensures that oxygen remains bound as a
peroxy species. In this way, oxygen is kinetically trapped and further
reduction can take place (3, 4). The successive steps leading to the
formation of water have been studied by different spectroscopic
techniques (see Ref. 5 for review). The spectral signatures of two of
these intermediates, which exhibit bands at 607 and 580 nm in the
difference spectrum with respect to the oxidized enzyme, were first
reported by Wikström (6), by reversing the electron transfer
reaction, and further characterized by Wikström and Morgan (7).
These authors assigned the spectral signatures at 607 and 580 nm to a
ferric peroxy (P) and ferryl oxo (F) species, respectively. These
assignments, however, have been challenged by a number of authors
(8-10). For example, Resonance Raman results obtained by Proshlyakov
et al. (10) suggest that the 607 nm band originates from an
oxoferryl structure. On the other hand, the same authors have been
unable to identify the putative peroxy species in their system in
turnover sustained by H2O2 (11). However,
irrespective of the assignments, there seems to exist a general
agreement that compound F (580 nm) is a ferryl oxo species and that it
is one electron more reduced than compound P (607 nm) (11, 12).
One of the ways to solve the problem of the identity of the P intermediate could perhaps be through the use of a suitable probe. The possibility that nitric oxide (NO), a powerful reversible inhibitor of cytochrome c oxidase (13-15), can be used as a probe for the binuclear center has been suggested by recent results using enzyme in slow turnover (16-18). In these experiments, an electron was ejected from the binuclear center, partially reducing cytochrome a. In addition, depletion of the intermediates P and F was also observed on mixing with NO (16, 17). As suggested in these papers, these reactions could be mediated via reaction of NO with CuB, as it has been shown that in addition to binding to reduced Fea3, NO binds to both CuB1+ and CuB2+, albeit with different affinities (19-21). On the other hand, we have suggested previously (15) that binding of NO to CuB1+ could be a key to understanding the mechanism of inhibition of CcO by NO, although this hypothesis was based solely on steady-state kinetic considerations.
A mechanism describing the interaction of NO with CuB, leading to the partial reduction of cytochrome a, could be the reverse of that postulated for the reduction of nitrite to NO by non-heme nitrite reductases, which contain only Cu as a metal (see Ref. 22 for review). In this mechanism, depicted in Scheme 1 (solid arrows), Cu1+ of nitrite reductase (A) binds nitrite, forming a complex (B), which after abstraction of one oxygen atom gives an electrophilic nitrosonium (C). This species is analogous to the nitrosonium (Fe2+ NO+), detected by Fourier transform infrared spectroscopy in the heme-containing cytochrome cd1 nitrite reductase of Pseudomonas stutzeri, obtained by incubating the oxidized enzyme with NO (23). The donation of one electron from the metal gives Cu2+ NO (D), which then dissociates as NO and oxidized Cu (E).
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We suggest that in CcO, NO interacts with the oxidized
enzyme at the copper present in the binuclear center,
CuB2+, forming a complex analogous to that
shown at point D in Scheme 1. This eventually yields nitrite
and CuB1+ (Scheme 1, dotted arrows,
E through A). As depicted in Scheme 2, the reduced CuB could
be reoxidized by equilibration with the other redox centers,
i.e. Fea, CuA, and Fea3.
In those cases in which oxygen intermediates are present in the
binuclear center, an obvious possible outcome of the internal electron
transfer from CuB to Fea3 would be the
transition of the oxygen intermediate to the next intermediate in the
catalytic cycle, i.e. P F or F
Ob
(Ob represents the oxidized binuclear center of
CcO, Fea33+
CuB2+). Alternatively,
CuB1+ could bind NO, forming a relatively
stable complex, Cu1+ NO. The formation of this complex
could ultimately be responsible for the inhibition of the enzyme.
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In this study, we have investigated in detail these reactions with a view to test the hypotheses formulated in Schemes 1 and 2. We provide evidence for the mechanisms depicted in these schemes, which are able to explain many of the features encountered in the reaction of NO with CcO.
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MATERIALS AND METHODS |
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Cytochrome c oxidase was prepared by the method of
Soulimane and Buse (24), which yields highly active enzyme (maximal
turnover number, 600 s1). The buffer used throughout was
0.1 M HEPES, 0.5% Tween 80, pH 7.4. Because of the
sluggish reactivity of the binuclear center of this enzyme as
preparation with CO, H2O2 (used to form P and F, respectively), and NO, the enzyme was "pulsed" by reduction and
reoxidation. This was necessary both to prepare the intermediates P and
F and to investigate the reactivity of the oxidized enzyme with NO.
Pulsed Oxidized Enzyme O-- CcO (~45 µM) was fully reduced by incubation with 1 mM sodium dithionite for 2 h at 4 °C. Then, the enzyme was reoxidized by passage through a Sephadex G-25 column equilibrated with the same buffer but containing no dithionite. To ensure that the enzyme was fully reoxidized, the column was loaded, immediately before the addition of the enzyme, with a band of 20 mM potassium ferricyanide. Formation of peroxide upon reoxidation was avoided by addition of 50 µl of 40 mM catalase to the reduced enzyme prior loading onto the column. Following this procedure, the maximum of the Soret band corresponding to the pulsed enzyme was at 423 nm, identical to the maximum observed in the oxidized enzyme as prepared. Formation of oxygen intermediates of the enzyme (e.g. peroxy), due to either incomplete reoxidation or the formation of peroxide, would induce a red shift of the Soret band. The absence of a significant shift in this band after pulsing is indicative of the absence of such oxygen intermediates. The pulsed oxidized enzyme (O) thus obtained was used to prepare the intermediates P and F.
Compounds P and F--
Compound F was formed by incubating O
(see above) in 1 mM H2O2, and the
concentration of F was determined spectroscopically using
580-630 = 5,500 M
1
cm
1 (7) relative to O. Compound P was obtained by two
different methods. In the first method, P was formed when CO gas was
bubbled for a short time (~10 s) through a solution containing
typically ~4 µM O (see above) as described previously
(25). After about 10 min, formation of P reached a steady state, with
conversion of typically ~50% of the enzyme to this form. In the
second method, the oxidized enzyme (pulsing was not necessary) was
incubated with CO overnight at 4 °C to obtain the mixed valence-CO
complex. After briefly degassing the solution, the sample was exposed
to an intense flash of white light in the presence of oxygen. The amount of compound P formed using this method was typically ~80% of
the total enzyme, as determined using the extinction coefficient
607-630 = 11,000 M
1
cm
1 (7) relative to O.
Static Experiments--
In the static experiments, an aliquot of
a solution of NO (50 µl of 2 mM NO) was added to 1.5 ml
of a solution containing compound P (607 nm), F (580 nm), or O (fully
oxidized enzyme). A difference spectrum relative to O was recorded
immediately in a Cary 5E UV-Vis-NIR spectrophotometer. The amount of
cytochrome a reduced was calculated as follows:
605 (reduced minus oxidized) = 17,500 M
1cm
1.
Stopped Flow-- Compounds P, F, and O were rapidly mixed with saturated (~2 mM) or diluted solutions of NO, prepared by diluting the stock saturated solution with different volumes of anaerobic buffer, in a stopped flow apparatus (model SX-18MV Applied Photophysics, Leatherhead, UK). The spectra were collected using a photodiode array detector with a time resolution of 3.3 ms. A global fitting analysis program (Global Analysis, Applied Photophysics) using singular value decomposition of the data was used to model the reactions, the spectra of the kinetic components, and the corresponding rate constants. The spectra of the components constructed in this way were consistent with difference spectra obtained by subtracting spectra taken at different time points during the course of the reaction. The amount of CuA reduced after mixing O with NO was obtained by comparing the amplitude of the change at 830 nm with that obtained after the addition of dithionite (18).
Nitric Oxide-- Nitric oxide (NO) was obtained with a Kipps apparatus by mixing 1 M sulfuric acid with sodium nitrite, essentially as described by Torres and Wilson (26). The NO in the stock solution (usually ~2 mM) and in the dilutions was measured with an NO electrode (Iso-NO Mark II, World Precision Instruments). The level of nitrite in the NO stock solution was also monitored (usually less than 0.1 mM) in the same electrode, by measuring the NO formed after acidification with H2SO4 in the presence of KI.
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RESULTS |
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Static Optical Spectroscopy
Reaction of NO with Species O-- Addition of NO to O generated a difference spectrum (Fig. 1A) that, in the Soret region, exhibited a peak at 445 nm and a trough at 430 nm. At longer wavelengths, a positive band at 605 nm was also observed. Both the positions of the bands and the relative intensities are indicative of reduction of cytochrome a (~40% of the total). These changes were accompanied by a bleached region from 620 to 660 nm.
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Reactions of NO with Species F and P--
Addition of NO to P or F
resulted in the rapid depletion of these intermediates. The spectrum
resulting from the addition of NO to F (Fig. 1B) has a peak
at 415 nm and a trough at 430 nm in the Soret region, and the region
from ~590 to 660 is bleached. Because the scheme presented above (see
Scheme 1, dotted arrows) predicts the formation of nitrite
in the binuclear center of CcO, we tested this prediction by
adding nitrite to O and comparing the final spectrum to that obtained
after addition of NO to F. Fig. 1C shows that exposure of O
to a high concentration of nitrite (10 mM) generated the
same spectral changes as obtained when NO (20 µM) was
added to F. The affinity of the enzyme for nitrite is low and required
a relatively high nitrite concentration to induce spectral changes of
measurable amplitude. The fact that the same effect was obtained with a
much lower NO concentration suggests that when NO is added to F,
nitrite is formed within the binuclear center and interacts with
the oxidized binuclear center (Ob) formed as a result of
the conversion F Ob.
Stopped Flow Spectrophotometry
Reaction of Compound F with NO The reaction of a sample containing compound F (~85%) with NO led, as expected (see Fig. 1B), to the rapid decay of this compound (Fig. 2, curves a and b). The time course was fitted to a biexponential decay, and the rates of the two phases were essentially proportional to the NO concentration (see Fig. 2 legend). Difference spectra relative to O are shown in Fig. 2, inset. The final spectrum was found to be identical to that obtained in the static spectral experiments (Fig. 1B) and thus identical to that observed after the addition of nitrite to the oxidized enzyme (Fig. 1C). Although the time course could be fitted to a biexponential decay, the spectra corresponding to the two kinetic components were indistinguishable (not shown). This indicates that the components corresponding to the decay of compound F and the appearance of the features that we identify with the interaction of the binuclear center with nitrite cannot be separated in this reaction.
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Reactions of Compound P with NO
Reactions within the Dead Time (1.4 ms)--
When a sample
containing a high proportion of compound P (~80%) was mixed with NO
in the stopped flow spectrophotometer, some spectral changes took place
in the dead time of the apparatus (~1.4 ms), whereas the band at 607 nm remained unchanged in this time frame (Fig.
3, arrows). The final spectrum
after ~1 s is consistent with that shown in Fig. 1D
(static experiments), showing reduction of cytochrome a and
depletion of P. The difference spectrum corresponding to the changes
which occurred during the dead time is given in Fig.
4A, solid line (and also in
Fig. 3, inset, solid line). This spectrum (Fig. 4A,
solid line) shows two peaks in the Soret region at about 425 and
445 nm and a trough around 410 nm. At longer wavelengths, a positive
band at ~580 nm, a shoulder at 600 nm, and a negative region centered
at ~615 nm can also be observed. These features are reminiscent of
the formation of compound F, possibly accompanied by some
reduction of cytochrome a. However, both the changes in the
Soret and the regions are slightly different from those expected
for simultaneous formation of F and reduction of cytochrome
a. For example, although a band at 445 nm is clearly present
in the Soret region, there is no concomitant increase in the
band
at ~605 nm. Only a shoulder at 600 nm is observed. We can discount
spectral contributions from a transition of the type P
F, because
the increase at 580 nm is not paralleled by a decrease at 607 nm of an
amplitude approximately twice as large (see the extinction coefficients
given under "Materials and Methods") (Fig. 4A, solid
line). In addition, even in case that reduction of cytochrome
a (increase at 605 nm) compensates for the decrease at 607 nm, the change at 445 nm in the Soret region (which is 3 times larger
in amplitude than at 605 nm; see Fig. 1A) would have to be
about twice as large than observed in Fig. 4A.
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Reactions Following the Dead Time-- Following the dead time, species P (607 nm) and the newly formed species absorbing at 580 nm decayed simultaneously (Fig. 5), with a rate that was found to depend on the NO concentration (Fig. 5, inset). These changes were complete in ~800 ms at 1 mM NO, and together with those changes observed in the Soret region, they are compatible with the decay of compounds P and F. The amplitude of the decrease at 580 nm from 1.4 to 800 ms was found to be identical, within our experimental error, to the amplitude of the increase observed within the dead time (Fig. 3, inset). This indicates that the compound F that decays from 1.4 to 800 ms corresponds to that formed within the dead time. The amplitude of the decrease at 607 nm, however, was smaller than the corresponding amplitude of the P present before mixing (Fig. 3). In fact, one of the troughs in Fig. 5 is centered at 612 nm, not at 607 nm. This apparent shift can be explained by a simultaneous decrease at 607 nm and an increase at 605 nm, which, together with the presence of a positive band at 442 nm in the difference spectrum, indicates that some reduction of cytochrome a takes place simultaneously with the decay of the P and F intermediates.2 Accordingly, the reduction of cytochrome a and the decay of P and F resulted in a single kinetic transition in the global analysis (see "Materials and Methods"). In addition, internal differences between spectra collected at different time points show that the component corresponding to cytochrome a reduction is present at all time intervals (not shown). This indicates that both processes are interconnected, such that the rate of reduction of cytochrome a is limited by the rate of decay of the intermediates P or F. As in the static experiments (Fig. 1D), the final amount of cytochrome a reduced was about 40% of the P initially present.
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Reactions of O with NO
As for compound P, when NO was mixed with O some changes occurred
within the instrumental dead time (<1.4 ms) (Fig.
6A). The difference spectrum
recorded (t = 1.4 ms minus t = 0 ms) is
compatible with the appearance of approximately equal percentage
(~15%) of reduced cytochrome a and compound F. Although
the reduction of cytochrome a observed may be expected (in
~1.4 ms, a process with k = 100 s1
completes ~15% of its total amplitude), the formation of F was not
anticipated. We attribute the formation of F to heterogeneity in the
sample, suggesting that preparations of O (after reduction and
reoxidation; see "Materials and Methods") contain a small proportion of the O-like
species3 (described above).
After the dead time, about 40% of cytochrome a became
reduced (k ~ 100 s
1), as indicated by
bands at 442 and 605 nm (intensity ratio, 3:1) (Fig. 6B), in
agreement with the results obtained in the static spectral experiments
(Fig. 1A). Reduction of cytochrome a was accompanied by reduction of 15-20% CuA (not shown)
(18).
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DISCUSSION |
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The results presented above may be rationalized by reference to a simplified reaction mechanism presented in Scheme 3. The basic feature of this scheme is that the different reactions, observed upon mixing NO with derivatives of CcO, can all be explained by a single electron donation from NO to the binuclear center. Each step in Scheme 3 comprises a sequence of events analogous to those indicated by dotted arrows in Scheme 1, i.e. NO binds to CuB2+ and results in the formation of HNO2 and reduction of CuB. The latter would permit electron transfer from CuB either to cytochrome a/CuA or to oxygen intermediates bound to Fea3. A detailed reaction mechanism is given in Scheme 4, in which the two parts, A and B, differ in the chemical assignment of P. We have assumed in part A that the species P (607 nm) and F (580 nm) are the ferric peroxy and oxoferryl derivatives of Fea3, respectively, as suggested previously by Wikström and Morgan (7).
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Because we propose that NO provides the binuclear center with one reducing equivalent, it follows that the precursor of the F formed during the dead time (spectrally O-like and labeled X in Scheme 4) is at the formal oxidation level of peroxide. Therefore, if P (PM) is the ferric peroxy species, then X could contain either CuB3+, with the iron as a ferryl oxo species (Fe4+ = O), or alternatively as CuB2+, with Fe5+ = O (see Scheme 4). The presence of either of these species, suggested previously by other authors (9-12, 29, 30), would explain the fast formation of compound F solely by reduction of CuB3+ to CuB2+ or Fe5+ to Fe4+. Our data suggest that PM coexists with species X. possibly in an equilibrium, because they are at the same oxidation level.
An analogous and equally fast reaction may be expected to occur between
NO and PM, generating a peroxy species with CuB
reduced. Such a species, postulated previously, has been termed
PR in the literature
(31).4 The conversion from
PM to PR would not result in observable optical changes, because these species have similar spectra (31). This is in
agreement with our results, because the band at 607 nm corresponding to
P remains unchanged on addition of NO during the dead time (Fig. 3).
Thus, after mixing a sample containing P (PM and X) with
NO, we obtained two different products: one was PR, from the transition PM PR, and the other was F,
from the transition X
F. The mechanism in Scheme 4 accounts,
therefore, for the spectral changes that occur within the dead time
after mixing P with NO, namely, the apparent nonreactivity of
PM (Fig. 5) and the formation of F from a species
spectrally similar to O.
Furthermore, our results show that the species F (absorbing at 580 nm)
and PR (assumed to absorb at 607 nm) (31) decay
simultaneously after mixing with NO (Fig. 5). This suggests that they
are in rapid equilibrium, one of them reacting with NO and recruiting the other into the reactive form. From our experiments (Fig.
1B), it is clear that it is compound F that reacts with NO
yielding O, and we thus incorporated this into Scheme 4. Thus,
PR would react not with NO per se, but through
an equilibrium established between PR and F. For the
reaction between F and NO, a reaction analogous to that described for
PM and species X was suggested, after which, an internal
electron transfer from CuB could facilitate the transition
F Ob. Support for the role of CuB in this
reaction comes from the fact that although F is known to react with CO (33) and other ferryl groups (e.g. in myoglobin) react with NO (34), these processes are much slower than those presented here.
After the formation of species O, a further electron donation from NO
would then lead to the observed reduction of cytochrome a.
In agreement with this model, our results show that this process is
fast when mixing O with NO (k = 100 s
1)
but is rate-limited by the decay of P and F (~8 s
1)
when NO reacts with these intermediates. This is expected if the
substrate for this reduction (Ob) is the product of the
reaction of P and F with NO, as Scheme 4 shows.
The Identity of Compound X--
It seems generally agreed that F
(580 nm) has an oxo ferryl structure, containing
CuB2+, but the assignment of P (607 nm) is more
problematic. Basically, the discussion in the literature revolves
around whether the oxygen O---O bond can be broken following addition
of only two electrons to the enzyme. This could be achieved by means of
an additional electron, recruited from elsewhere in the protein, to
produce an oxo ferryl derivative. Four main sources for this electron have been suggested, i.e. the porphyrin ring, an amino acid,
the copper atom CuB, or the iron atom itself. The donation
of an electron from the porphyrin ring or an amino acid, leading to the
formation of a radical, as observed in other enzymes (i.e.
peroxidases and catalases), seems to be unlikely (9, 29, 31), although a radical has been observed to occur in the enzyme in turnover (35).
The assignment of the 607 nm form to an oxoferryl Fe4+ = O structure with CuB3+ has been postulated by
other authors (5, 9, 30), and this unusually high oxidation state for
CuB has been recently detected in an inorganic model (36).
Proshlyakov et al. (10) also associate the 607 nm absorbing
species to an iron oxo group on the basis of Resonance Raman
16O-18O mixed-isotope experiments with 607 nm
excitation. These authors, however, attribute the band that appears at
804 cm1 to Fe5+ = O (and
CuB2+).
Physiological Implications of the Reactions Observed between NO and CcO-- Our finding that nitrite is rapidly formed in the binuclear center of CcO suggests that Scheme 1 is likely to be one of the pathways through which NO is metabolized and detoxified. In addition, this mechanism is consistent with the fact that mitochondrial NO metabolism by CcO also occurs anaerobically (43), because the mechanism in Scheme 1 does not require oxygen. These results also have implications regarding the mechanism of inhibition of CcO by NO. It has been suggested that this process can be explained by reversible binding of NO to ferrous cytochrome a3 (42), and this is consistent with the competitive nature of the inhibition with respect to oxygen. However, consideration of the "on" and "off" rates of NO with ferrous a3 (44, 42) is not in agreement with the observed low Ki for the inhibition of cellular respiration by NO (43). This apparent conflict can be explained if, as we have shown, an additional interaction of NO with the binuclear center (i.e. with CuB2+) takes place, thus decreasing the Ki of NO for CcO. Thus, although formation of ferrous a3-nitrosyl complex has been detected in the inhibitory process (15) and has been suggested as the primary event in the inhibition (42), this work suggests that formation of ferrocytochrome a3 may be a consequence rather than a cause of the inhibition. A possible mechanism by which inhibition could take place is suggested by the fast reduction of CuB2+ to CuB1+ by NO. The competition of NO and oxygen would not be at the ferrocytochrome a3 but instead at CuB1+. Furthermore, binding of NO to CuB1+ could block the redox state of this metal, which would then constitute the real inhibitory site. Alternatively, NO may transfer rapidly from CuB1+ to cytochrome a3 on addition of an electron to this site in turnover.
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ACKNOWLEDGEMENT |
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We acknowledge M. Sharpe for helpful discussions.
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FOOTNOTES |
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* This work was supported by the Wellcome Trust, the Royal Society, the Spanish government, and the Biotechnology and Biological Science Research Council, UK.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-1206-872538;
Fax: 44-1206-872592; E-mail: wilsmt{at}essex.ac.uk.
1 The abbreviations used are: Fea and Fea3, iron centers of cytochromes a and a3, respectively; CcO, cytochrome c oxidase; NO, nitric oxide; O, fully oxidized cytochrome c oxidase formed after reduction and reoxidation of the enzyme; Ob, oxidized binuclear center of CcO; P and F, oxygen intermediates of CcO at an oxidation state two and three electrons more reduced, respectively, than the oxidized binuclear center; PM, species formed immediately after CO-photodissociation from mixed valence-CO in the presence of oxygen; PR, species formed immediately after CO-photodissociation from the fully reduced CO complex in the presence of oxygen.
2
Because the spectra of P and reduced cytochrome
a are similar in the band (P has a band at 607 nm and
cytochrome a at 605 nm) the simultaneous decay of P and a
smaller increase of reduced cytochrome a led only to a small
perturbation in this region (see extinction coefficients under
"Materials and Methods").
3 Other authors have previously suggested the presence of heterogeneities even in "fast" oxidase preparations, based on the presence of two phases in the kinetics of ligand binding (32), especially formate.
4 PR is the form obtained after photolysis, in the presence of oxygen, of the Fea3---CO bond in CO-bound fully reduced oxidase. In this case, the second reducing equivalent for oxygen is provided directly from cytochrome a, and not by CuB, which remains reduced. In contrast, the species obtained from CO-bound mixed valence enzyme, in which the second reducing equivalent for oxygen is provided through oxidation of CuB, is called PM (31).
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REFERENCES |
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