From the
Current understanding of the oxygen reduction and proton
translocation processes in cytochrome c oxidase is largely
derived from the data obtained by a nonphysiological method for
initiating the catalytic reaction: photolyzing carbon monoxide (CO)
from the CO-inhibited enzyme in the presence of oxygen (O
Cytochrome c oxidase, the terminal enzyme in the
mitochondrial electron transport chain, reduces O
The most recent applications of the CO-flash method were the
resonance Raman studies of intermediates in the reaction of the enzyme
with O
Both optical absorption and
resonance Raman studies show that possible artifacts are induced by the
CO-flash method, since the spectra of the enzyme at the end of the
reaction depend on the method of initiation: the CO-flash method
results in the ``resting'' enzyme and the direct mixing
method results in the ``pulsed'' form
(5, 26) . Recent experiments suggest that Cu
The time limit of a conventional stopped flow mixer (several ms)
results from the need for relatively large solution volumes (
The mixing point in our rapid mixer is a simple
pinhole nozzle as illustrated in Fig. 1( inset). Two
solutions are introduced into the mixer co-axially but do not mix until
they reach the pinhole, where the flow becomes turbulent due to the
sudden increase of the flow speed and the geometry of the aperture.
Since the mixing time depends on the particular nozzle, we picked a
nozzle with a 20-µs mixing time for the time resolved measurements.
In Fig. 1(plot) the mixing time of the nozzle was determined by
comparing the intensities of Raman lines from HPO42- and
H
Resonance Raman measurements were
obtained by focusing the output from a Krypton ion laser on the sample
jet. The Raman scattering was dispersed and measured by a single
spectrometer equipped with a liquid N
The Fe-O
An
unexpected result of the CO-flash experiments was that cytochrome a was oxidized simultaneously with the decay of the primary
Fe-O
The electron
transfer rate for the direct mixing method was estimated by following
the oxidation state of cytochrome a (19, 22) in the high frequency resonance Raman spectra
(Fig. 3). In the top trace (10 µs) the two lines in the
electron density marker line (
).
However, considerable evidence suggests that the use of CO introduces
artifacts into the reaction mechanism. We have therefore developed a
rapid solution mixer with a mixing time of 20 µs to study the
catalytic reaction by directly mixing the enzyme with O
without using CO. Unexpectedly, the resonance Raman scattering
detected for the first 120 µs after the mixing show that the CO
influences neither the structure of the primary oxy-intermediate, its
rate of decay, nor the rate of oxidation of cytochrome a. This
implies that CO has an effect on the later stages of the catalytic
process, which may involve the proton translocation steps, and calls
for the re-examination of the catalytic process by using the direct
mixing method. In addition, these results demonstrate the feasibility
of using the rapid mixing device for the study of biological reactions
in the microsecond time domain.
to water
in a binuclear center formed by cytochrome a
and a
nearby copper atom, Cu
. Two additional redox centers,
Cu
and cytochrome a, supply electrons to the
binuclear center. The enzyme harnesses the chemical energy released by
the O
reduction to translocate four protons across the
inner mitochondrial membrane and thus contributes to mitochondrial ATP
synthesis
(1, 2) . The investigation of the catalytic
process of the enzyme by directly mixing the enzyme with O
( direct mixing method) has been impossible, because with
a conventional stopped flow apparatus, the mixing dead time (several
milliseconds) is comparable with the enzyme's turnover time
(3) . Dynamic information such as the structure and kinetics of
the intermediates and the proton uptake and release kinetics, on which
the present understanding of the catalytic mechanism is largely based
(1, 2, 4, 5) has therefore been obtained by the CO-flash
method: initiating the reaction by photolyzing carbon monoxide
from the CO-inhibited enzyme in the presence of O
(6) . This method was first developed by Gibson and
Greenwood for transient absorption studies of intermediates generated
at room temperature
(6) and by Chance et al. for both
absorption and EPR studies of intermediates trapped at cryogenic
temperatures
(7) . The CO-flash method has been utilized for
several decades under the general assumption that the use of CO does
not affect the catalytic process
(4, 6, 7, 8, 9, 10, 11, 12, 13) .
(1, 5, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) .
Identification of iron-oxygen stretching Raman lines from many of the
key intermediates have enabled the elucidation of their active site
structures and the determination of their formation and decay kinetics.
The rate of electron transfer from cytochrome a to cytochrome
a
was determined by following the oxidation states
of cytochrome a (19, 22) . These studies, along
with site-directed mutagenesis studies of bacterial oxidases
(25) , are helping to clarify the microscopic mechanism for
oxygen reduction and proton translocation in the enzyme. It is
important in such studies of the catalytic process to approach
physiological conditions as closely as possible. However, initiation of
the catalytic process by the CO-flash method is clearly
nonphysiological, and, in fact, the question of the introduction of
artifacts in the enzymatic reaction due to the photolytic initiation
has been raised
(5, 26) .
is the first binding site for O
(10, 11, 27, 28) , but it is known
that the photolyzed CO coordinates to Cu
where it is not
released until a few microseconds
(28) , a time scale similar to
that for O
binding to cytochrome a
(
= 8 µs)
(11) . Moreover, time-resolved
resonance Raman measurements have shown that cytochrome
a
is unrelaxed until several microseconds after
the photolysis of CO
(29) . The CO-flash method could have a
faster O
on-rate than that of the direct mixing method if
the O
is already in the vicinity of active site prior to
the CO dissociation. On the other hand, if the active site cannot
accommodate two ligands, the presence of CO at Cu
could
delay O
association. Last, the structure of the reduced
enzyme in the region of the binuclear center could be quite different
if generated from a CO-bound form versus a ligand-free form
(26) . These arguments suggest that anomalous results might be
introduced by the use of CO especially in the early stages of the
reaction. We have therefore developed a rapid solution mixer with a
mixing/dead time of 20 µs and followed the first 120 µs of the
reaction of cytochrome c oxidase with O
by
resonance Raman scattering.
10
µl) required for optical absorption. When the reaction is being
probed by a laser beam to obtain resonance Raman or fluorescence
spectra, large volumes are not necessary. Volume elements on the order
of 10 µm
10 µm
10 µm (
1 pl) are
sufficient to obtain high quality spectra. Moreover, by using a small
volume mixer with the ``continuous flow'' method, the flow
speed can be made very high, which accelerates the generation of
turbulence in the sample flow and thus promotes efficient mixing. Using
these concepts, a method for mixing two solutions on the microsecond
time scale to study chemical dynamics by fluorescence was first
developed by Regenfuss et al. (30, 31) and
subsequently used by Paeng et al. (32, 33) with resonance Raman scattering. We have adapted their
general ideas and constructed a rapid mixing device with several
modifications.
PO4- in the acid/base reaction between
Na
HPO
and HCl. From this we determined that
mixing was 90% complete in less than 20 µs. The nozzle aperture is
25 µm in diameter and 600 µm long. The holes in the stainless
steel nozzles were made by laser drilling. The parent solutions were
filtered to remove dust and transferred to gas-tight syringes with
equal volumes (25 ml) where they were driven by a syringe pump at a
rate of 0.64 ml/min per syringe. The time after mixing was estimated
from the flow speed of the jet (43 µm/µs) which is calculated
by assuming a homogeneous 25-µm diameter jet over the short
distance (5 mm) between the entrance of the aperture of the nozzle and
the longest time point reported here (120 µs). Time-resolved Raman
scattering measurements are obtained by moving the focus point of the
laser away from the nozzle exit along the sample jet. Full details of
the mixing device will be described elsewhere.
Figure 1:
The relative intensity of a Raman line
from HPO42- is plotted as a function of time after mixing with
HCl. Mixing is 90% complete in 20 µs. A schematic illustration of
the rapid mixing device used for these measurements is shown in the
inset and described in the text.
Cytochrome c oxidase was purified from beef heart by the method described by
Yoshikawa et al. (34). The enzyme (95 µ
M),
dissolved in 50 m
M sodium phosphate buffer (pH 7.4), was
reduced by adding cytochrome c and sodium ascorbate to final
concentrations of 5 µ
M and 20 m
M, respectively.
The oxygen-saturated buffers were prepared by shaking the buffer
solution vigorously under 1 atmosphere of either O
or
O
gas. All the experiments were
performed at room temperature.
cooled charge-coupled
device camera. A holographic filter was used to remove the laser
scattering.
stretching frequency of the
O
-bound enzyme generated by the CO-flash method was
identified at 570 cm
and had a lifetime of about 50
µs
(14, 18, 21) . To locate the mode by our
direct mixing method, we compared the resonance Raman spectra from the
heme groups after mixing the fully reduced enzyme with
O
or
O
saturated
buffer. As illustrated in Fig. 2, we detect a strong line at 570
cm
for
O
which shifts to
546 cm
for
O
, thus
identifying this line as the Fe-O
stretching mode in exact
agreement with the frequencies obtained by the CO-flash protocol. We
conclude that any possible residual CO or unrelaxed protein in the
CO-flash experiments has no effect on the structure of the Fe-O
unit of cytochrome a
. We observed that the
intensity of the Fe-O
stretching mode decayed to the
background level within 100 µs (data not shown), also consistent
with the CO-flash measurements
(18, 21) .
intermediate
(19, 22) . This result
suggests that electron transfer from cytochrome a to
oxy-cytochrome a
( k
3
10
s
), where the iron atoms of the two
hemes have a separation of
18 Å, occurs faster than electron
transfer from Cu
to oxy-cytochrome a
( k
4
10
s
)
where the oxygen points toward the Cu
and the iron-copper
distance is only 3-5 Å (35, 36). The electron transfer from
cytochrome a to the binuclear center is known to be quite
sensitive to the enzyme preparation
(37) , and thus it, as well
as the electron transfer dynamics between Cu
and the
cytochrome a
heme, could be modulated by a
conformational state set by the photodissociation process or the
presence of photodissociated CO near the active site.
) region (1356 and 1372
cm
) show that the enzyme is largely in the reduced
ligand-free state with the contribution from 1372 cm
coming from some O
-bound cytochrome
a
. At 30 µs, the intense contribution at 1372
cm
indicates that cytochrome a
is almost completely O
-bound. That the high intensity
of the 1372 cm
line at this time comes primarily
from O
coordination to cytochrome a
and not oxidation of cytochrome a is confirmed by
examining the 1600-1650 cm
region. The slight
decrease in intensity at 1609 and 1624 cm
, marker
lines for reduced cytochrome a, with the concomitant increase
in intensity at 1641 cm
, a marker line for oxidized
cytochrome a, indicates that only a small amount of electron
transfer from cytochrome a to the binuclear site has occurred
at this early time. At the next time point (50 µs) nearly half of
cytochrome a has become oxidized, and at 80 µs some
further oxidation of cytochrome a has taken place. No
additional change is detected at 120 µs. Since cytochrome a and Cu
are in equilibrium
(38) , the fractional
oxidation of cytochrome a is consistent with one electron
transferred to the binuclear site. These results show two important
features. First, O
coordination to cytochrome
a
occurs within the mixing time of our apparatus.
Thus, the rapid coordination of O
does not depend on it
being in or near the heme pocket, as it might be in the CO-flash
experiments. Second, the oxidation of cytochrome a occurs just
as rapidly in the direct mixing experiments as it does in the CO-flash
experiments and occurs simultaneously with the decay of the primary
Fe-O
intermediate.
Figure 3:
Time
resolved resonance Raman spectra in the high frequency region obtained
at several different delay times (as indicated) from the samples after
directly mixing fully reduced cytochrome c oxidase with
O
saturated buffer. Experimental conditions
are the same as those in Fig. 2, except for the use of a nozzle with a
20-µs mixing time, and the laser power was 80 milliwatts. Each
spectrum was accumulated for 20 s.
The experiments reported here
demonstrate the structural identity of the O-bound
cytochrome c oxidase prepared by the direct mixing and the
CO-flash methods. The kinetic properties, including the decay of the
primary intermediate and the rate of oxidation of cytochrome
a, are also the same as those found in the CO-flash
experiments. Thus, the photodissociation of the CO does not affect
these early events, although we cannot exclude the possibility that the
O
on-rate is faster in the direct mixing method.
Considering the reported differences at the end of the reaction between
the direct mixing and the CO-flash methods (5, 26), there must be
structural changes in the enzyme that will only become evident in the
properties of the later intermediates which must be clarified by
further investigation. It is especially important to delineate the
properties of the later intermediates, since it has been shown that
they are involved in the coupling between the redox processes and
proton translocation (39). The experiments also demonstrate the
feasibility of the rapid mixing device to address a problem of current
biophysical interest. We expect that this method for the study of
microsecond reaction dynamics will become important in several other
fields of chemical physics and biophysics.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.