(Received for publication, January 9, 1997, and in revised form, June 2, 1997)
From the Department of Biochemical Sciences A. Rossi-Fanelli and CNR Center of Molecular Biology, University of Rome La Sapienza, I-00185 Rome, Italy
We present novel experimental evidence that,
starting with the oxidized enzyme, the internal electron transfer in
cytochrome c oxidase is kinetically controlled. The
anaerobic reduction of the oxidized enzyme by ruthenium hexamine has
been followed in the absence and presence of CO or NO, used as trapping
ligands for reduced cytochrome a3. In the
presence of NO, the rate of formation of the cytochrome
a32+-NO adduct is independent of
the concentration of ruthenium hexamine and of NO, indicating that in
the oxidized enzyme cytochrome a and
a3 are not in very rapid redox equilibrium; on
the other hand, CO proved to be a poor "trapping" ligand. We
conclude that the intrinsic rate constant for a a3 electron transfer in the oxidized enzyme is
25 s
1. These data are discussed with reference to a model
(Verkhovsky, M. I., Morgan, J. E., and Wikström, M. (1995)
Biochemistry 34, 7483-7491) in which H+
diffusion and/or binding at the binuclear site is the rate-limiting step in the reduction of cytochrome a3 in the
oxidized enzyme.
The three-dimensional structure of cytochrome c
oxidase, the terminal enzyme of the respiratory chain, is now available
for the proteins isolated from Paracoccus denitrificans (1)
and beef heart (2, 3). The core of the active site of the beef heart
enzyme, containing three metal centers bound to subunit I and common to
all terminal oxidases (see Fig. 1) was
predicted correctly on the basis of mutagenesis and spectroscopy (4). The O2 binding site, contributed by the heme of cytochrome
a3 and CuB, is at short distance
from cytochrome a, which is generally believed to be the
electron donor to that site. The two hemes lie across helix X of
subunit I, which provides two His (376 and 378) as protein ligands for
the two metals; the short distance (13 Å) between them supports the
view that the a a3
eT1 is very fast.
Initiating the reaction by photolysis of the CO adduct of the fully
reduced or mixed valence enzyme yields rate constants for internal eT
ranging from 104 to 3 × 105
s1 (5-7). On the other hand, stopped-flow experiments
carried out starting from the fully oxidized (resting or pulsed) enzyme
indicated that the rate of formation of reduced cytochrome
a3 is by comparison very slow (0.1 to >30
s
1 depending on conditions (8-11)); some of these
experiments were carried out also in the presence of CO (10). These
observations led to the hypothesis that, starting with the oxidized
enzyme, internal eT is slow because the pathway to and/or the
coordination of the binuclear center are different from those of the
transient species obtained by photolysis of the CO derivative of the
reduced binuclear site (12). More recently Verkhovsky et al.
(13) have confirmed the observation that the rate of accumulation of reduced cytochrome a3 is slow; however they
proposed that (i) in the oxidized enzyme internal eT is very fast; (ii)
the redox equilibrium favors cytochrome a2+; and
(iii) H+ diffusion and/or binding to the reduced binuclear
site is the rate-limiting step. We have addressed again this crucial
question and carried out new kinetic experiments using a "fast"
enzyme preparation (14) and nitric oxide (NO) to trap reduced
cytochrome a3.
Cytochrome c oxidase was purified from beef heart
according to the method of Soulimane and Buse (14) and stored at
70 °C in 10 mM Tris + 500 mM sodium
chloride + 0.1% (w/v) Triton X-100, pH 7.6. Before use, oxidase was
thoroughly (about 2 days) dialyzed at 4 °C against 100 mM potassium phosphate, pH 7, + 0.1% (w/v) lauryl
maltoside, the same buffer used for the kinetic experiments. This
procedure yields a fraction of the enzyme (
30%) in the slow form,
as shown by the classical cyanide binding experiment (15); nonetheless
almost complete recovery of fast is achieved by "pulsing" (16).
Oxidase concentration is expressed as functional units (cytochrome
aa3). Glucose (30 mM) and glucose
oxidase (0.3 mg/ml) were used to achieve complete deoxygenation, in the
presence of catalase. Stock solutions of NO (Air Liquide, Paris,
France) or CO were prepared by equilibrating degassed buffer with the
pure gases ([NO] in solution = 2 mM and [CO] in
solution = 1 mM at 20°C). Lauryl maltoside was from
Biomol (Hamburg, Germany). Ascorbate and glucose oxidase were from
Sigma (St. Louis, MO). Ruthenium hexamine was from Aldrich (Milwaukee,
WI).
Stopped-flow experiments were carried out either with a Durrum-Gibson instrument equipped with a diode array (TN6500; Tracor Northern, Madison, WI) or with a single wavelength apparatus (DX.17MV; Applied Photophysics, Leatherhead, U. K.). The diode array stopped-flow can acquire up to 80 spectra of 1,024 elements; the acquisition time for each spectrum is 10 ms. The dead time of the single wavelength stopped-flow is 1 ms.
Data analysis was carried out with the software MATLAB (MathWorks, South Natick, MA) on an Intel 486 computer. Spectral smoothing was performed by using the singular value decomposition algorithm according to Henry and Hofrichter (17). Spectral deconvolution was obtained starting from reference spectra by using the left division option, provided by MATLAB. Kinetic simulations were carried out using a differential equations solver algorithm implemented by Dr. E. Henry (National Institutes of Health, Bethesda, MD).
The anaerobic reduction of fast oxidized cytochrome c
oxidase has been investigated employing ruthenium hexamine as electron donor because (i) the reduction of cytochrome a and
CuA is sufficiently fast and thermodynamically favorable
(E°
200 mV (18)); and (ii) the spectral changes of the two
cytochromes can be monitored over the whole range without optical
interference by the reductant. Electron entry in cytochrome
c oxidase occurs via the binuclear copper center called
CuA, which is in very rapid redox equilibrium with
cytochrome a (k = 1.8 × 104 s
1), the electron donor to the binuclear
cytochrome a3-CuB center. To
stabilize the reduced state of cytochrome a3, we
used CO and NO. NO is the most efficient "trapping" ligand for
electrons on the cytochrome a3-CuB
center because its combination is very fast and strictly bimolecular
(kon = 1 × 108
M
1 s
1 (19)) and its affinity
very high (Ka = 109
M
1 given a dissociation rate constant
koff = 0.1 s
1 (20)). In this paper
we shall focus our discussion on the internal eT between cytochrome
a and the cytochrome
a3-CuB center.
When degassed oxidized oxidase was anaerobically mixed with a
solution of ruthenium hexamine, ascorbate, and NO, the time-resolved absorption spectra (Fig. 2A)
can be analyzed using the spectral components shown in Fig.
2B, i.e. the oxidized, the half-reduced (cytochrome a2+
CuA+-cytochrome
a33+ CuB2+),
and the fully reduced nitrosylated species. The calculated time courses
of these spectral components are shown in Fig. 2C. Their
optical contribution was back-reconstructed using the calculated time
courses and subtracted from the observed spectral data; the resulting
residuals (Fig. 2D) indicate that the spectral components used are sufficient to describe the experimental data to better than
95%. Within the first 30 ms after mixing, the half-reduced enzyme is
populated transiently, whereas cytochrome a3
remains oxidized and unligated. Later on, the half-reduced species
decays to the fully reduced nitrosylated species; this indicates
that the reduction of cytochrome a3 and NO
binding are synchronous. This is fully consistent with the fact that
the pseudo-first order rate constant for NO binding under these
conditions is indeed very high (k
6,000 s
1). We may therefore conclude that NO acts as an
efficient trapping ligand for reduced cytochrome
a3 and that the observed rate of formation of
the cytochrome a32+-NO adduct
starting with the oxidized enzyme is slow.
As shown in Fig. 2, the time course of formation of the cytochrome a32+-NO adduct is not monophasic; it can be fitted to two exponential processes, the amplitude of the rapid phase being approximately 80% of the total. This biphasic time course has been observed with all preparations and tentatively explained assuming that a fraction of the enzyme is in the "resting" state even in a fast preparation. Consistent with this hypothesis, the amplitude of the slow phase decreases significantly upon pulsing the enzyme (16) by reduction and subsequent exposure to oxygen (data not shown). Moreover, cyanide binding to the oxidized enzyme is also biphasic (15).
Fig. 3 shows the results of an experiment
carried out with a single wavelength double mixing stopped-flow. The
time courses at 438 nm indicate that reduction of cytochrome
a3 in the presence of NO is somewhat faster
(k = 22 s
1) than in the presence of CO at the
same concentration (k
= 13 s
1). At 431 nm the
first observable event is a fast absorbance decrease corresponding to
reduction of cytochrome a; this is followed by the formation
of the reduced cytochrome a3-ligand adduct,
proceeding at k
= 19 s
1 with NO and
k
= 4.5 s
1 with CO. Given the relatively
small combination rate constant for CO (k = 8 × 104 M
1 s
1 (21)),
the formation of the CO-bound derivative (at 431 nm) lags behind the
reduction of cytochrome a3 (at 438 nm), whereas NO binding is synchronous to cytochrome a3
reduction, in agreement with the experiment of Fig. 2.
Effect of Ruthenium Hexamine Concentration
The anaerobic
reduction of oxidized oxidase was followed at different concentrations
of ruthenium hexamine (from 125 µM to 2 mM)
in the presence of NO. The measured rate constants for the reduction of
cytochrome a and the formation of the cytochrome a32+-NO adduct are shown in Fig.
4. The rate of reduction of cytochrome a increases linearly with the concentration of ruthenium
hexamine (k = 1.2 × 105
M1 s
1), whereas the formation
of cytochrome a32+-NO is essentially
independent. This finding is consistent with the hypothesis that the
reduction of cytochrome a3 is controlled kinetically.
Effect of NO and CO Concentration
In Fig.
5 the rate constant for the formation of
the NO and CO adducts of cytochrome
a32+ is reported as a function of
ligand concentration (from 7 to 500 µM). The cytochrome
a32+-NO adduct is formed at a rate
that is essentially independent of NO concentration and slightly higher
than the average rate of reduction of cytochrome
a3 in the absence of ligands (k = 16 s
1, arrowhead). On the contrary, the rate of
formation of the cytochrome a32+-CO
adduct is slower than the rate of cytochrome a3
reduction and increases with CO concentration. The different behavior
can be rationalized on the basis of the different combination rate constants for the binding of the two ligands to reduced cytochrome a3.
These findings demonstrate that using fast oxidase preparations, CO is inadequate to the role of trapping ligand, whereas NO is definitely suitable.
The structure of the active site of cytochrome c
oxidase, including cytochrome a and the (oxygen-binding)
binuclear center cytochrome a3-CuB,
is shown in Fig. 1. Helix X (one of the transmembrane helices of
subunit I) provides ligands to both cytochrome a
(His378) and cytochrome a3
(His376). Laser photolysis and flow-flash experiments
starting from the CO complexes of bovine oxidase (5-7, 19, 22-25)
have shown that the a a3 eT is
very fast (kF > 105
s
1 and kR > 104
s
1). These very rapid rates (µs) are consistent with
the short distance between the two metals (13 Å) which are connected
via a possible pathway involving 16 covalent bonds (26). On the other
hand, it has been observed repeatedly (8-10, 13, 27) that the apparent rate of formation of reduced cytochrome a3, both
in the presence and absence of CO, is considerably slower (0.1 to > 30 s
1 depending on experimental conditions). Despite
some complexities caused, for instance, by the fraction of resting and
pulsed or fast enzyme, the apparent rate constant for complete
reduction of the cytochrome a3-CuB
site was found to correlate under selected conditions with the turnover
number; thus Malatesta et al. (10) concluded that internal
eT to the oxidized binuclear center is the rate-limiting step in
turnover.
To account for the slow reduction of cytochrome a3 starting with the oxidized enzyme, two different mechanisms have been proposed. The following simplified scheme may help discussion,
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The alternative mechanisms proposed are as follows.
Kinetic ControlIn the oxidized enzyme, the rate of eT from cytochrome a to cytochrome a3 is in the ms time range. If the reduction of cytochrome a and the binding of the trapping ligand are sufficiently fast, the reduction of cytochrome a3 will rate limit the binding of X, making reduction and ligation synchronous. Under these conditions, the rate of cytochrome a3 reduction should be independent of the concentration of both the reductant and the trapping ligand. We have developed (9, 10) an experimental protocol to probe eT to cytochrome a3 by mixing the oxidized (fast or pulsed) enzyme with a reductant containing X = NO or CO, known to bind quickly and tightly to reduced cytochrome a3. In this paper we have shown that the observed rate constant for the formation of cytochrome a3+2-NO is independent of the concentration of ruthenium hexamine (Fig. 4) and of NO (Fig. 5), implying a rate-limiting monomolecular process, which we assign to k2.
Thermodynamic ControlVerkhovsky et al. (13) have
suggested that eT is very fast (µs) even in the oxidized enzyme, but
the apparent rate constant for reduction of cytochrome
a3 appears slow because thermodynamics favors
reduced cytochrome a. If this holds, only a fraction
(10%) of reduced cytochrome a3 will be
populated on a short time scale (at the rate of cytochrome a
reduction); nevertheless this fraction should be available for
combination with a trapping ligand. Since equilibrium measurements have
shown that low pH stabilizes cytochrome a32+ (28, 29), Verkhovsky et
al. (13) postulated that (i) H+ is the trapping ligand
X, driving the reaction in Scheme 1 to the right; and (ii) the rate of
diffusion and/or binding of H+ to the reduced site is slow,
accounting for the relatively slow (ms) rate of reduction of cytochrome
a3 vis-à-vis a very rapid (µs) eT.
Verkhovsky et al. (13) observed that the pH dependence of
the process (already documented by Malatesta et al. (10)) was not inconsistent with their hypothesis, although the apparent rate
constant increases at acidic pH by a factor of only 3/pH unit.
The pH dependence of the redox potential implies that low pH stabilizes reduced cytochrome a3, without kinetic implications. Verkhovsky et al. (13) also observed that the time course of formation of reduced cytochrome a3 and of H+ dissociation by phenol red (used as a pH indicator in unbuffered medium) is synchronous; this observation, however, is consistent with both mechanisms, since synchrony would be expected also if eT per se was rate-limiting, with reduction of cytochrome a3 coupled to rapid H+ uptake by a redox-linked ionizable group.
In summary, there is substantial agreement about the bare experimental
observation, i.e. that starting from oxidized cytochrome c oxidase, the rate of formation of reduced cytochrome
a3 is in the ms time range even with a large
excess of reductant; nevertheless, two alternative mechanisms have been
proposed. The experiments reported in this paper are consistent with a
kinetic control of internal eT, but they appear difficult to reconcile
with the hypothesis that cytochrome a and
a3 are in fast redox equilibrium and that uptake
of protons is the rate-limiting step in the reduction of cytochrome
a3 (and CuB). The data in Fig. 5
show that the rate constant for the formation of cytochrome
a32+-NO is independent of [NO]
over a large range; this provides unequivocal evidence that NO binding
is rate-limited by a monomolecular process, which we assign to a slow
eT to cytochrome a3, excluding that the two
cytochromes are in very rapid equilibrium in the oxidized enzyme. If a
fraction (10%) of cytochrome
a32+ was populated within µs after
reduction of cytochrome a, then the apparent rate constant
for reduction of cytochrome a3 should (i)
increase as a hyperbolic function of [NO] to a plateau
represented by the pseudo-first order rate constant for the reduction
of cytochrome a, and (ii) increase linearly with the
reductant concentration at a sufficiently high concentration of NO. As
shown above (Figs. 4 and 5), this is not what we observed. In Fig. 5 we
also show the CO concentration dependence of the rate constant for the
formation of the cytochrome a32+-CO
complex. Given the relatively slow second order rate constant (k = 8 × 104
M
1 s
1, (21)), CO binding lags
behind cytochrome a3 reduction, and the
formation of the cytochrome a32+-CO
adduct is CO concentration-dependent. Simulations of the
kinetic model reported in Scheme 1 predict this behavior and yield an estimate of the equilibrium constant for the a
a3 eT (see legend to Fig. 5). Thus both sets of
data are quantitatively consistent with a kinetic control mechanism. To
maintain Verkhovsky's hypothesis (13), one should postulate that NO
cannot bind to reduced cytochrome a3
unless a H+ is already bound at that
site; in this case proton binding and/or diffusion would limit NO
binding. This possibility seems difficult to reconcile with information
available on oxidase and other reduced hemeproteins, keeping in mind
that binding of NO to reduced cytochrome c oxidase is very
rapid indeed, follows bimolecular kinetics, and has a very high
affinity (Ka = 109
M
1).
The three-dimensional structure of cytochrome c oxidase now available (1-3) may help further discussion and elicit some speculation. It is intriguing that separate channels for diffusion of oxygen and protons to the active site have been postulated. Access of protons to the cavity in between the iron of cytochrome a3 and CuB may involve diffusion through pore A and/or pore B (1); on the other hand, oxygen (and other uncharged ligands) may have access to the binuclear center predominantly through yet another proposed channel coated with hydrophobic side chains (3). Assuming also that NO and CO preferentially diffuse to the cytochrome a3-CuB center via this hydrophobic channel, why should binding of NO to reduced cytochrome a3 be impossible unless a (rate-limiting) proton has already diffused to this site via a separate channel? This seems somewhat peculiar given that NO is thermodynamically and kinetically a very efficient trapping ligand for reduced cytochrome a3, possibly more effective than protons.
In conclusion, the new kinetic data on reduction of cytochrome a3 and NO binding are difficult to reconcile with the hypothesis that in the oxidized enzyme cytochrome a and a3 are in very fast (µs) redox equilibrium and that H+ diffusion and/or binding to the reduced binuclear site is the unique rate-limiting step in the buildup of reduced cytochrome a3. Our hypothesis is that starting from the oxidized enzyme, internal eT to cytochrome a3 is slow (ms) and rate limiting the turnover (10), and only starting from the reduced configuration of the binuclear center (with or without a bound ligand) is internal eT very rapid (µs). This difference may be rationalized if the introduction of electrons into the cytochrome a3-CuB binuclear site was associated with a local structural changes, resulting in a high reorganizational energy term.
Insofar as we have established that in the oxidized enzyme internal eT is not in the µs time range, we should attempt to reconcile this finding with the structure. The reorganizational energy term in the Marcus theory (see Ref. 30) is known to affect eT at fixed D-A distance as discussed by Gray and Malmström (31) and Brzezinski (32); a large reorganizational energy associated with eT to the cytochrome a3-CuB center is expected to slow down eT considerably. As suggested before (12, 26), a slow eT may be accounted for if the coordination of cytochrome a3 was different in the two oxidation states; that was just an example among other possible mechanisms, having in common a reorganization of the electron-accepting site. Given that pH controls the redox potential of cytochrome a3 (28, 29) and that transient H+ uptake has been observed synchronous with eT (13, 29, 32, 33), it is possible that such a structural change may involve protons. Understanding the structural basis of the reorganizational energy term associated with eT to cytochrome a3-CuB remains an open question, and possibly kinetic experiments with mutants of the proton channels and higher resolution crystallographic data of the unligated oxidized and the reduced enzymes may help our understanding of this crucial mechanistic feature, which we believe to be general for all terminal oxidases.
We thank Prof. M. T. Wilson (Colchester, U. K.) for stimulating discussions and Prof. G. Buse (Aachen, Germany) for collaboration in setting up the preparation of the fast enzyme. We also express our thanks to Dr. Eric Henry for stimulating discussions about simulations of kinetic models.