The rate limitation in electron transfer ET was not associated with ET into the CCO as cytochrome a was predominantly reduced in the aerobic steady state.. This implies that the rate lim
Trang 1The influence of temperature and osmolyte on the catalytic cycle
Jack A Kornblatt1, Bruce C Hill2and Michael C Marden3
1
Enzyme Research Group, Concordia University, Montreal, Quebec, Canada;2Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada;3INSERM U473, Le Kremlin-Biceˆtre Cedex, France
The influence of temperature on cytochrome c oxidase
(CCO) catalytic activity was studied in the temperature
range 240–308 K Temperatures below 273 K required the
inclusion of the osmolyte ethylene glycol For steady-state
activity between 278 and 308 K the activation energy was
12 kcalÆmol)1; the molecular activity or turnover number
was 12 s)1at 280K in the absence of ethylene glycol CCO
activity was studied between 240and 277 K in the presence
of ethylene glycol The activation energy was 30kcalÆmol)1;
the molecular activity was 1 s)1at 280K Ethylene glycol
inhibits CCO by lowering the activity of water The rate
limitation in electron transfer (ET) was not associated with
ET into the CCO as cytochrome a was predominantly
reduced in the aerobic steady state The activity of CCO in
flash-induced oxidation experiments was studied in the low
temperature range in the presence of ethylene glycol Flash
photolysis of the reduced CO complex in the presence of
oxygen resulted in three discernable processes At 273 K the rate constants were 1500 s)1, 150 s)1and 30s)1and these dropped to 220s)1, 27 s)1and 3 s)1at 240K The acti-vation energies were 5 kcalÆmol)1, 7 kcalÆmol)1, and
8 kcalÆmol)1, respectively The fastest rate we ascribe to the oxidation of cytochrome a3, the intermediate rate to cyto-chrome a oxidation and the slowest rate to the re-reduction
of cytochrome a followed by its oxidation There are two comparisons that are important: (a) with vs without ethy-lene glycol and (b) steady state vs flash-induced oxidation When one makes these two comparisons it is clear that the CCO only senses the presence of osmolyte during the reductive portion of the catalytic cycle In the present work that would mean after a flash-induced oxidation and the start of the next reduction/oxidation cycle
Keywords: cytochrome c oxidase; osmolytes; rate limitations
Cytochrome c oxidase (CCO) is the terminal electron
transfer (ET) enzyme of the mitochondrial electron
trans-port chain and a site of energy transduction During the
catalytic cycle, the enzyme accepts electrons one at a time
from cytochrome c; it stocks electrons in four metal centers
(CuA, cytochrome a, cytochrome a3 and CuB) and finally
transfers four electrons to oxygen The reduced oxygen
combines with four protons to form two molecules of water
At the same time, protons are pumped from one side of the
protein to the other [1] As the CCO is normally inserted in
the mitochondrial membrane, this pumping, added to the
consumption of protons in the mitochondrion, results in the
formation and maintenance of a transmembrane gradient of
protons It, in conjunction with the membrane potential,
powers the synthesis of ATP as well as other energy
requiring functions [2]
The CCO from bovine heart contains 13 different protein
subunits, two hemes in the form of cytochromes a and a3,
two coppers in the form of CuAwith a third as CuB; it also contains Mg, Zn and some tightly bound phospholipid or detergent The three-dimensional structures of the bovine heart CCO [3,4] and the Paracoccus denitrificans CCO [5] are available from the protein data bank The cytochrome c binding site is on the side of the oxidase that faces the cytosolic compartment of the cell Electrons enter the oxidase one at a time from cytochrome c; they enter via CuA [6–8] which is contained within subunit II of the protein The groundwork for establishing the ET sequence was performed by Chance et al [9,10] and by Gibson and Greenwood [11] The sequence of electron transfers into, out of, and within CCO is now more or less defined but there is not universal agreement on the oxidation pathway (see [8,12–14]) The initial ET is from cytochrome c into
CuA[7] which rapidly equilibrates with cytochrome a [15] The second electron rereduces CuA thereby forming the initial two-electron reduced oxidase In the absence of oxygen or the presence of CO, there is probably a two-electron reduction of the cytochrome a3/CuBsite and this is followed by rereduction of the CuA/cytochrome a couple Our scheme for working with the oxidative pathway is based on Hill’s analysis [8] but it is not critical for the data reported here The majority view for the oxidation pathway is summarized in a recent paper by Morgan
et al [14]
CCO acts as a proton pump [1] It actively transfers about one proton across the protein for each electron that is transferred to oxygen Under most conditions, there is no
Correspondence to J A Kornblatt, Enzyme Research Group,
Concordia University, 1455 de Maisonneuve, Montreal,
Quebec, CANADA H3G 1M8.
Fax: + 33 4 67 52 36 81 (until May 2003), + 1 514 848 2881
(after May 2003), Tel.: + 1 514 848 3404,
E-mail: krnbltt@vax2.concordia.ca
Abbreviations: CCO, cytochrome c oxidase; TMPD, tetramethyl
phenylene diamine; ET, electron transfer.
(Received 2 October 2002, accepted 20 November 2002)
Trang 2slippage ET and proton transfer are tightly coupled [16].
This gives rise to the phenomenon of respiratory control In
terms of the electron transfer reactions, proton transfer
occurs during the reduction of the binuclear center and
during the reduction of oxygen from the peroxy to the
oxyferryl intermediates [17]
A water cycle during turnover has been established
[18,19] The application of hydrostatic-pressure influences
CCO only during turnover [20]; the current view is that
hydrostatic pressure exerts its effects on proteins by
influencing hydration Reducing the activity of water also
influences CCO but only during turnover [18] A series of
small hydrophilic molecules is capable of inhibiting CCO
activity and the inhibition scales with the size of the small
molecule effector [21] The most potent inhibitor is that
whose size is closest to that of water Based on a stochastic
model, the inhibitor studies indicate that around one water
molecule enters and leaves the oxidase for every proton that
is transported Other techniques indicate that between one
and two water molecules enter and leave the CCO for every
proton transported [22] The water cycle is coupled to ET
and proton transfer
The work presented here was based on the view that
pure ET reactions should be relatively insensitive to
temperature changes whereas combined ET, proton and
water transfer should be more sensitive As temperatures
are reduced, the motions of waters internal to protein
structures, are reduced To study enzyme catalysis at low
temperature, it is necessary to include antifreeze or
osmolyte in the enzyme mixture This lowers water
activity and imposes a new rate limitation on catalytic
turnover We use the term rate limitation to indicate
that it is the net effect of an unknown number of slow
steps and that it is not the effect of temperature on a
single rate constant Northrop [23], Brown and Cooper
[24], and Ray [25] have shown that enzymes cannot
usually be analyzed in terms of a single, slowest, rate
determining step All enzymes, though, are rate limited
Temperature effects on the rate limitation have been
exploited over the years through the use of the Arrhenius
equation Temperature sensitivity of the reaction rate
when that rate is not limited by substrate availability
yields an Arrhenius activation energy which is one among
many characteristics of the enzyme For CCO which has
four well defined metal centers that act as electron
acceptors and donors, ET rates into and out of these
centers are influenced by temperature and can be
analyzed using the Arrhenius relation [26]
In this work we show, as have many other studies, that
steady state ET from cytochrome c to oxygen decreases as
temperature decreases Furthermore, steady state
sensiti-vity at low temperature with osmolyte present is greater
than that exhibited for the internal ET reactions during
flash induced oxidations This implies that the rate
limitation in the steady state, low temperature ET reaction
is developed only during the reductive phase of the
catalytic cycle This idea is in keeping with the results of
other studies [12,27–32] In terms of the comparisons
made in this work, it is only with the start of a second
cycle) ET from cytochrome c to the oxidized
oxid-ase) that low water activity causes this rate limitation
to shift from one set of steps to another
Materials and methods
The purification of CCO has been described previously [33] The protein is prepared using cholate, and then suspended
in 1% (v/v) Tween-80 Before use, the protein is mixed with
an equivalent weight of Tween-80, dialyzed to equilibrium
vs 40mM phosphate, pH 6.9 and then frozen The preparation is stable for a period of months
Cytochrome c (prepared without trichloroacetic acid) was purchased from Sigma Tween-80, the detergent used throughout the study, was from Fluka and was their highest grade Ethylene glycol, enzyme grade, was from Fisher All other chemicals were from Fluka and were the highest grade available
Steady state assays of CCO were carried out in 40mM phosphate pH 6.9 The oxidation of cytochrome c was monitored at 550nm The complete assay system contained approximately 40 lMreduced cytochrome c and a variable amount of CCO (with its equivalent weight of Tween-80) depending on the temperature Below 273 K, the assay contained 44% (w/v) 40mM phosphate and 56% (w/v) ethylene glycol The paH of this solution is 7.75 at 273 K and 8.1 at 243 K; the term paH indicates that the activity of hydrogen ions in the mixed solvent solution is known; when
no osmolyte is present, paH and pH are the same [34] The concentration of reduced cytochrome c was the same as in the high temperature samples Temperature inside the assay cuvette was monitored continuously with a T-type thermo-couple (Barrant Co., Barrington, IL, USA)
Flash photolysis at 532 nm was carried out with a Quantel laser with a 10-ns pulse The cuvette holder was cooled with a double Peltier junction, the lower of which was cooled with a refrigerated bath It was relatively easy to get as low as 240K The buffer system contained 44% (w/v) 40mM phosphate pH 6.9 and 56% (w/v) ethylene glycol, 10mMascorbate and 50 lMtetramethyl phenylene diamine (TMPD) The oxidase, final concentration 5 lM, was added to the buffer system kept at about 270K The total volume was 1 mL The solution was gassed with CO and allowed to sit on ice until such time as it was completely reduced The cuvette was placed in the refrigerated holder When the cuvette attained thermal equilibrium, 87 lL of oxygenated solution of 44% (w/v) 40mMphosphate/56% (w/v) ethylene glycol was added with a chilled syringe; the cuvette contents were mixed with the same syringe and the
CO flashed off The temperature of the cuvette wall was monitored before and after flashing The samples were monitored at a wavelength of 442 nm and the data stocked
in a LeCroy 9400 digital oscilloscope; they were treated as described below
Because these are single runoff experiments, it was not possible to average multiple flash-induced events A total of
32 000 data points were collected for each flash and these were converted to fewer than 200, with the points at greater times being the average of neighboring points centered at the times indicated For example, if the data are collected
at 1 ls per point, the total scan covers 32 ms The data point
at 10ms is the average of 64 points from 9.969 ms to 10.032 ms Shorter times use fewer points to avoid a large spread compared to the time after the flash [35] An equation containing the sum of three exponential terms was
fit to the data using
Trang 3Figure 1 shows the response of CCO to temperature
changes The assay used is spectrophotometric and
meas-ures the disappearance of reduced cytochrome c absorbance
at 550nm The assay consists of cytochrome c, CCO with
its equivalent weight of Tween-80, phosphate buffer and
oxygen The data were collected from 280to 306 K Above
this value the slope of the Arrhenius plot started to change
As we were not interested in the elevated temperatures, we
did not take data far above the straight portion of the
Arrhenius plot We note that many other workers have
found a break in the curve at about the same temperature
[36,37] In standard assays at 273 K, the oxidase turnover
number is close to 10s)1which is similar to that found in
the older work Kinetic and thermodynamic parameters are
summarized in Table 1 The Arrhenius activation energy
calculated from the data of Fig 1 is 12 kcalÆmol)1, also
about the same as found in earlier work
Under conditions similar to those of Fig 1, there is little
indication that the rate limitation is a slow ET step between
cytochrome a and the binuclear center In steady state
spectra in the presence of cytochrome c and TMPD/
ascorbate, the predominant form of the oxidase appears
to be a mixture of the pulsed form of the oxidized oxidase
and cytochrome a2a3
Figure 2 shows the spectral approach to the anaerobic
state as electrons are transferred from TMPD/ascorbate to
oxidase to oxygen Qualitatively, the significant aspect of the
spectra is that there is only a minor peak that grows in at
443 nm between 2 min and 6 min If cytochrome a were
completely reduced, the 443 nm peak would be
consider-ably higher as would the 605 nm peak (vida infra)
The addition of ethylene glycol to the buffers at 56%
(w/v) allows one to work at temperatures as low as 230K
Provided the phosphate concentration is kept < 0.1M,
there is no precipitation of phosphate even at the lowest temperatures At temperatures < 290K the response of the oxidase to changing temperature appears to follow a linear Arrhenius relation (Fig 3) Above 290K there is a slow (minute time scale) precipitation of the CCO which complicates the kinetic analysis and prevents us from being able to make a direct comparison between CCO at high temperature (> 290K) with and without ethylene glycol The bending of the curve in Fig 3 at the higher temperatures
Fig 1 CCO was assayed spectrophotometrically in 40 m M phosphate
pH 6.9 The initial reduced cytochrome c concentration was 40 l M ; its
oxidation was followed at 550nm The temperature in the cuvette was
monitored during the assay with a T-type thermocouple; the
tem-perature was constant within ± 0.1 K over the course of the
meas-urement The rate constant on the ordinate is a turnover number,
expressed on a per second time scale.
Table 1 Kinetic and thermodynamic constants for the steady state activity and flash induced oxidation activity of cytochrome c oxidase.
No ethylene glycol
56% ethylene glycol Steady state turnover
number at 280K
12 s)1 1 s)1
Steady state E
(T > 273 K) 12 kcalÆmol)1 (T < 273 K) 30kcalÆmol)1 Single kinetic constants
for the three identified kinetic processes
T ¼ 300 K a
T ¼ 273 K
25 000 s)1 1500 s)1
10 000 s)1 150s)1
800 s)1 30s)1 Flash induced oxidation
Efor the three distinguished processes
T > 273 Ka T < 273 K
3 kcalÆmol)1 5 kcalÆmol)1
7 kcalÆmol)1 7 kcalÆmol)1
13 kcalÆmol)1 8 kcalÆmol)1
a
Data taken from Oliveberg et al 1989 [26].
Fig 2 The CCO aerobic steady state at 276 K The oxidase concen-tration was 5 l M , the cytochrome c was 3 l M The buffer was 40m M phosphate pH 6.9 Spectrum 1, before the addition of TMPD and ascorbate; spectra 2–6, after the addition of 3 m M ascorbate and
300 l M TMPD; spectra 7–10, progression to the totally reduced oxidase.
Trang 4reflects the fact that multiple processes are occurring Below
290K there is only one discernable process and it has an
activation energy of 30kcalÆmol)1
Qualitatively, the bottleneck that slows the catalytic
activity is between cytochrome a and cytochrome a3
(Fig 4) as was shown in earlier work [18] The data were
collected at 250K in the presence of low concentrations of
cytochrome c plus TMPD and ascorbate As the
concen-tration of cytochrome c is increased, the fraction of
cytochrome a that is reduced increases The fraction of
cytochrome a that is reduced is, in part, represented by the
peak that grows in at 443 nm and that which disappears at
416 nm The spectra of Figs 2 and 4 are compared in Fig 5
where the emphasis is on the changes occurring at 443 nm
and 605 nm
The progression to and out of the steady state is shown in
Fig 5 Both monitoring wavelengths show that the steady
state is reached within 5 min of TMPD/ascorbate addition
to either high or low temperature samples The steady state
is then maintained over the course of 6 min (276 K) or
600 min (250 K) until the preparations become anaerobic
The time course of the absorbance changes at 276 K (d)
and 250K (s) are shown in (A) and (B) based on the data
of the absorption spectra of Figs 2 and 4 In both panels, the
data have been normalized to a concentration of 1 lM
oxidase and have been corrected at 605 nm for the
absorbance contributed by oxidized cytochrome c
(< 5%) Figure 5A shows the time course of the
absorb-ance change at 443 nm The difference between the steady
state values of the 276 K sample and the totally reduced
sample at 276 K are clearly much larger than the
compar-able difference seen for the 250K sample This difference
reflects the extent to which cytochrome a is reduced; it is
more reduced in the 250K sample than in the 276 K
sample An approximation [38] of the extent to which
cytochrome a is reduced can be obtained from the data of (B) which shows the time course of changes at 605 nm This approximation is based on the fact that the 605 nm band is almost exclusively the result of cytochrome a absorption Under the conditions used here the extinction coefficient for the oxidized oxidase is 26 mM )1Æcm)1; this is the pulsed form The extinction coefficient for the totally reduced oxidase at 60 5 nm is 40 mM )1Æcm)1 A605 for the 276 K sample starts at 0.034 in the steady state and yields a value
of 0.042 for the totally reduced oxidase This corresponds to
50% reduction of cytochrome a in the steady state at
276 K At 250K, in the presence of ethylene glycol, A605is
0.038 in the steady state and is 0.042 when the sample goes totally reduced This corresponds to 75% of the cytochrome a reduced in the steady state The presence of ethylene glycol inhibits the oxidase by reducing electron flow between cytochrome a and cytochrome a3
In order to study the nature of the block, we carried out flash induced oxidation experiments in the presence of 56% (w/v) ethylene glycol at temperatures below 273 K Reduced CO oxidase was mixed with oxygen and the CO flashed off with a 10-ns pulse (532 nm) Fig 6 shows the evolution of absorption changes at 442 nm as a function of time Two typical data sets are shown One was collected at
268 K (s) and the second at 238 K An equation containing the sum of three exponential rates was fit to the data thereby yielding three rate constants at each temperature The fitted lines are included in the figure The typical R2was 0.998 The three sets of rate constants from temperatures between 278 and 240K were plotted as shown in Fig 7 The Arrhenius energies (Table 1) are 5 kcalÆmol)1(fastest
Fig 4 The CCO aerobic steady state at 250 K The oxidase concen-tration was 6.3 l M , the cytochrome c was 3 l M The buffer was 44% (w/v) 40m M phosphate pH 6.9, 56% (w/v) ethylene glycol Spectrum
1, before the addition of TMPD and ascorbate; spectra 2–19, after the addition of 3 m M ascorbate and 300 l M TMPD; spectra 20–30, pro-gression to the totally reduced oxidase.
Fig 3 CCO was assayed spectrophotometrically in a mixed solvent
system consisting of 44% (w/v) 40 m M phosphate, pH 6.9 and 56%
(w/v) ethylene glycol The initial reduced cytochrome c concentration
was 40 l M ; its oxidation was followed at 550nm The temperature in
the cuvette was monitored during the assay with a T-type
thermo-couple and was constant within ± 0.1 K over the course of the
measurement The rate constant on the ordinate is a turnover number,
based on a per minute time scale.
Trang 5process), 7 kcalÆmol)1and 8 kcalÆmol)1(slowest process).
These numbers are much smaller than those obtained in the
steady state assay; the activation energies obtained in the
flash induced oxidation experiments cannot account for
the rate limitation in the steady state
Discussion
The catalytic activity of CCO varies from preparation to
preparation and from laboratory to laboratory It is a
function of ionic strength, pH, detergent and detergent
concentration, temperature, cosolvents, hydrostatic
pres-sure, osmotic pressure and probably other factors
None-theless, catalytic activity is still one of the few characteristics
that reflect the actual role of the oxidase in the tissues
What is that role? The oxidase catalyzes the transfer of
electrons from cytochrome c to oxygen It couples the
energy liberated in the process to the generation of an
electrochemical gradient of protons The two appear to be
tightly coupled under most circumstances Even under conditions where the oxidase is not contained within a membrane, protons are still pumped from one side of the protein to the other There is also a water cycle that is coupled to electron and proton transfer Water must enter and leave the protein for the oxidase to turnover [18,21] The function of this last cycle is unknown but it may be part of the proton transfer machinery
The overall rate constant for any process is determined by all the rate constants that contribute to the reaction [23–25]
In the case of ET from cytochrome c to oxygen catalyzed by CCO, rate constants as a function of temperature have been
Fig 6 Flash photolysis of Cu1A a 2 Cu1B a 2 CO + O 2 Five l M reduced
CO CCO was in 44% (w/v) 40m M phosphate pH 6.9, 56% (w/v) ethylene glycol The temperature was 268 K (s) or 238 K (d) Oxygen was added with a prechilled syringe and the CO was flashed off with a 10-ns pulse at 532 nm; 32 000 data points were collected and treated as detailed in Materials and methods An equation containing the sum of three exponentials was fit to the data The R 2 for the fit was better than 0.998; the fit is shown as the solid line through each set of data points.
Fig 5 The time course of the absorbance changes at (A) 276 K (d) and
(B) 250 K (s) These data are taken from the absorption spectra of
Figs 2 and 4 In both panels, the data have been normalized to a
concentration of 1 l M oxidase and have been corrected for the
absorbance contributed by cytochrome c (< 5%) (A) Time course of
the absorbance change at 443 nm (B) Time course of absorbance
changes at 60 5 nm The data in (B) can be used to approximate the
extent of cytochrome a reduction during the steady state It is 50%
reduced at 276 K and 75% reduced at 250K.
Fig 7 Evaluation of Arrhenius activation energies of the three processes discernible in the low temperature flash photolysis experiments The activation energies are: k1, 5 kcalÆmol)1(d); k2, 7 kcalÆmol)1(s); k3: 8.0kcalÆmol)1(.) based on the assumption that the individual data sets could be fit to straight lines.
Trang 6measured several times An early study by Smith and
Newton [37] showed that simple Arrhenius behavior was
not followed over an extended temperature range Two
processes were evident with a break at about 30°C At
temperatures above the break, the activation energy was
8 kcalÆmol)1; at temperatures below the break the
acti-vation energy was 12.5 kcalÆmol)1 The Smith/Newton
data was not substantially different from that collected
earlier by Minnaert (quoted in [36]) Other workers have
subsequently reported similar activation energies in the
same temperature range [39,40] Under similar conditions,
we find 12 kcalÆmol)1 at temperatures < 30°C The
turnover numbers determined in the above mentioned
papers and ours are about the same Interestingly, the
acti-vation energies for the bimolecular rate constants between
cytochrome c and CCO are also about 16 kcalÆmol)1for the
temperature range < 20°C [41]
The inclusion of high concentrations of the
cryoprotec-tant, ethylene glycol, inhibits the catalytic activity of CCO
[18] In order to work at temperatures as low as 235 K, it
was necessary to include 56% (w/v) ethylene glycol in the
solution; this results in 90% inhibition of the overall
activity of the cytochrome c oxidase, an energetic difference
of 1.2 kcalÆmol)1 Part of the inhibition probably stems
from a slight weakening of the interaction between
cytochrome c and the oxidase [42]; however, halving the
cytochrome c concentration in the steady state assays had
no effect on the measured activity Ethylene glycol and low
temperature also increase the viscosity of the medium; if
there are large conformational changes that occur in the
oxidase during the catalytic cycle, these would be influenced
by the increased viscosity Certainly, there are
conforma-tional changes that occur here Lowering temperature
increases both the dielectric coefficient of the medium and
the dielectric coefficient inside the protein: both can be
expected to influence catalytic activity The majority of the
inhibition arises from blocking an internal ET step located
between cytochrome a and cytochrome a3 [18]; this is
clearly shown in Figs 4 and 5
The influence of temperature on the ethylene
glycol-inhibited protein was studied in both the steady state
condition and the flash induced oxidation condition
Steady state condition During steady state turnover, the
activation energy for ET from cytochrome c to oxygen is
30kcalÆmol)1 (Fig 3), 2.5 times the value found in the
temperature range between 273 and 300 K in the absence
of ethylene glycol The block is not at the delivery of
electrons into CuA as the steady state spectrum of the
cytochrome a shows it to be about 75% reduced (Figs 4
and 5) Low temperature that results in freezing is capable
of inducing the same inhibition Nicholls and Kimelberg
[43] showed that a solution of oxidase and TMPD/
ascorbate would yield a mixed valence oxidase
(cyto-chrome a reduced, cyto(cyto-chrome a3 oxidized) at 77 K It
took between 40and 240s for the samples to freeze;
during the freezing process, the sample undergoes osmotic
stress The vapor pressure of the ice surrounding the
oxidase is lower than the vapor pressure of water in the
protein The Nicholls/Kimelberg experiment is therefore
similar to the ones carried out here using ethlyene glycol
as the osmotic stress agent [44]
Flash induced oxidation condition During the oxidative phase of CCO, three processes can be easily seen after flash induction of oxidation The three steps have energy barriers which are substantial (5–8 kcalÆmol)1) These energy barriers are relatively independent of the presence
or absence of ethylene glycol and the temperature at which the measurements are made (Table 1) The reac-tions occurring during the oxidative phase cannot account for the increased energy barrier occurring during steady state turnover
The influence of temperature and ethylene glycol on the three rate constants was also studied The rate constants decrease by > 90% when ethylene glycol is added [8,28] an energetic difference of about 1.5 kcalÆmol)1These decrease
by another 80% as the temperature is lowered from 273 to
248 K [45] or 240K (this work)
Our rate constants for the oxidation phase are still faster than the overall rate constant during the steady state As the latter is the complex function of all the internal and intermolecular rate constants, the difference between the two sets of numbers is expected
In summary, at room temperature when no osmolyte is present, the activation energies of the individual steps in ET are comparable to the activation energy for the overall reaction The limitation on the steady state, catalytic rate, is
a reasonably fast reaction associated with ET The inclusion
of 56% (w/v) ethylene glycol changes that The individual
ET steps are slowed by the imposition of low temperature The activation energies are about the same as they are in the absence of ethylene glycol but the activation energy for steady state turnover is far higher than it is for any of the individual oxidative steps This can only mean that a new rate limitation has been introduced into the catalytic cycle and that this new rate limitation occurs only after the completion of the flash induced oxidation of the reduced oxidase
The situation is somewhat, but not quite, analogous to that seen by Karpefors et al [46] when they found that the onset of a large deuterium isotope effect was not seen immediately after mixing with D2O but rather occurred only after a lag time It is more in keeping with the idea that something rate limiting occurs at the onset of a second catalytic cycle of reduction of CCO by cytochrome c Mitchell and Rich [47] proposed that two protons were taken up on reduction of CCO and that these were taken up concomitantly with reduction of the binuclear cytochrome
a3-CuBsite Our results could also be explained by proton uptake during reduction of the cytochrome a-CuA site as proposed by Capitano et al [48]
Ethylene glycol is a cryoprotectant It acts by influen-cing the colligative properties of water The inclusion of ethylene glycol in our solutions lowers not only the freezing point of our solutions, but also the activity of water If water entry and exit are necessary for catalytic
ET to occur, then we have shown here that this cycle is initiated only during the reductive phase of the catalytic cycle Reduced oxidase would therefore start as a hydrated molecule There would be no impediment to
ET within the oxidase nor from the oxidase to oxygen The rate limitation in the overall steady state process would be the entry of water accompanying reduction and
ET within the protein
Trang 7We wish to thank M J Kornblatt for helpful discussions This work
was generously supported by Natural Sciences and Engineering
Research Council (Canada) and the Institut National de la Sante´ et
la Recherche Medicale (France).
References
1 Wikstrom, M.K (1977) Proton pump coupled to cytochrome c
oxidase in mitochondria Nature 266, 271–273.
2 Mitchell, P (1979) Keilin’s respiratory chain concept and its
chemiosmotic consequences Science 206, 1148–1159.
3 Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T.,
Yam-aguchi, H., Shinzawa-Itoh, K., Nakashima, R., Yaono, R &
Yoshikawa, S (1996) The whole structure of the 13-subunit
oxidized cytochrome c oxidase at 2.8A˚ Science 272, 1136–1144.
4 Yoshikawa, S., Shinzawa-Itoh, K., Nakashima, R., Yaono, R.,
Yamashita, E., Inoue, N., Yao, M., Fei, M.J., Libeu, C.P.,
Mizushima, T., Yamaguchi, H., Tomizaki, T & Tsukihara, T.
(1998) Redox-coupled crystal structural changes in bovine heart
cytochrome c oxidase Science 280, 1723–1729.
5 Ostermeier, C., Harrenga, A., Ermler, U & Michel, H (1997)
Structure at 2.7 A˚ resolution of the Paracoccus denitrificans
two-subunit cytochrome c oxidase complexed with an antibody FV
fragment Proc Natl Acad Sci USA 94, 10547–10553.
6 Hill, B.C (1988) Electron transfer from cytochrome c to O2 Ann.
NY Acad Sci 550, 98–104.
7 Hill, B.C (1991) The reaction of the electrostatic cytochrome
c-cytochrome oxidase complex with oxygen J Biol Chem 266,
2219–2226.
8 Hill, B.C (1994) Modeling the sequence of electron transfer
reactions in the single turnover of reduced, mammalian
cytochrome c oxidase with oxygen J Biol Chem 269, 2419–
2425.
9 Chance, B., Saronio, C & Leigh, J.S Jr (1975) Functional
inter-mediates in reaction of cytochrome oxidase with oxygen Proc.
Natl Acad Sci USA 72, 1635–1640.
10 Chance, B., Saronio, C., Leigh, J.S Jr, Ingledew, W.J &
King, T.E (1978) Low-temperature kinetics of the reaction
of oxygen and solubilized cytochrome oxidase Biochem J 171,
787–798.
11 Gibson, Q.H & Greenwood, C (1963) Reactions of cytochrome c
oxidase with oxygen and carbon monoxide Biochem J 86, 541–
555.
12 Branden, M., Tomson, F., Gennis, R.B & Brzezinski, P (2002)
The entry point of the k-proton-transfer pathway in cytochrome c
oxidase Biochemistry 41, 10794–10798.
13 Einarsdottir, O (1995) Fast reactions of cytochrome oxidase.
Biochim Biophys Acta 1229, 129–147.
14 Morgan, J.E., Verkhovsky, M.I., Palmer, G & Wikstrom, M.
(2001) Role of the PR intermediate in the reaction of cytochrome c
oxidase with O2 Biochemistry 40, 6882–6892.
15 Antalis, T.M & Palmer, G (1982) Kinetic characterization of the
interaction between cytochrome oxidase and cytochrome c J Biol.
Chem 257, 6194–6206.
16 Chance, B., & Williams, G.R (1956) The respiratory chain and
oxidative phosphorylation Adv Enzymol 17, 65–134.
17 Fabian, M & Palmer, G (2001) Proton involvement in the
tran-sition from the peroxy to the ferryl intermediate of cytochrome c
oxidase Biochemistry 40, 1867–1874.
18 Kornblatt, J.A & Hoa, G.H (1990) A nontraditional role
for water in the cytochrome c oxidase reaction Biochemistry 29,
9370–9376.
19 Kornblatt, J.A., Kornblatt, M.J., Rajotte, I., Hoa, G.H & Kahn,
P.C (1998) Thermodynamic Volume cycles for electron transfer in
the cytochrome c oxidase and for the binding of cytochrome c to cytochrome c oxidase Biophys J 75, 435–444.
20 Kornblatt, J.A., Hui Bon, H.G & Heremans, K (1988) Pressure-induced effects on cytochrome oxidase: the aerobic steady state Biochemistry 27, 5122–5128.
21 Kornblatt, J.A (1998) The water channel of cytochrome c oxidase: inferences from inhibitor studies Biophys J 75, 3127–3134.
22 Kornblatt, J.A & Kornblatt, M.J (1992) Cytochrome c oxidase: the presumptive channel holds at least four waters Biochim Bio-phys Acta 1099, 182–184.
23 Northrop, D.B (1981) Minimal kinetic mechanism and general equation for deuterium isotope effects on enzymic reactions: uncertainty in detecting a rate-limiting step Biochemistry 20, 4056–4061.
24 Brown, G.C & Cooper, C.E (1993) Control analysis applied to single enzymes: can an isolated enzyme have a unique rate-limiting step? Biochem J 294, 87–94.
25 Ray, W.J Jr (1983) Rate-limiting step: a quantitative definition Application to steady-state enzymic reactions Biochemistry 22, 4625–4637.
26 Oliveberg, M., Brzezinski, P & Malmstrom, B.G (1989) The ef-fect of pH and temperature on the reaction of fully reduced and mixed-valence cytochrome c oxidase with dioxygen Biochim Biophys Acta 977, 322–328.
27 Adelroth, P., Gennis, R.B & Brzezinski, P (1998) Role of the pathway through K (I-362) in proton transfer in cytochrome c oxidase from R sphaeroides Biochemistry 37, 2470–2476.
28 Adelroth, P., Karpefors, M., Gilderson, G., Tomson, F.L., Gennis, R.B & Brzezinski, P (2000) Proton transfer from glu-tamate 286 determines the transition rates between oxygen inter-mediates in cytochrome c oxidase Biochim Biophys Acta 1459, 533–539.
29 Ruitenberg, M., Kannt, A., Bamberg, E., Ludwig, B., Michel, H.
& Fendler, K (2000) Single-electron reduction of the oxidized state is coupled to proton uptake via the K pathway in Paracoccus denitrificans cytochrome c oxidase Proc Natl Acad Sci USA 97, 4632–4636.
30 Junemann, S., Meunier, B., Gennis, R.B & Rich, P.R (1997) Effects of mutation of the conserved lysine-362 in cytochrome c oxidase from Rhodobacter sphaeroides Biochemistry 36, 14456– 14464.
31 Konstantinov, A.A., Siletsky, S., Mitchell, D., Kaulen, A & Gennis, R.B (1997) The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer Proc Natl Acad Sci USA 94, 9085– 9090.
32 Namslauer, A., Branden, M & Brzezinski, P (2002) The rate of internal heme-heme electron transfer in cytochrome c oxidase Biochemistry 41, 10369–10374.
33 Yonetani, T Cytochrome oxidase from beef heart Biochemical Preparations 11, 14–20(1966).
34 Douzou, P (1977) Cryobiochemistry Academic Press, New York, USA.
35 Marden, M.C., Kister, J., Bohn, B & Poyart, C (1988) T-state hemoglobin with four ligands bound Biochemistry 27, 1659–1664.
36 Minneart, K (1961) The kinetics of cytochrome c oxidase Bio-chim Biophys Acta 50, 23–34.
37 Smith, L & Newton, N (1968) Structure and Function of Cyto-chromes University of Tokyo Press, Tokyo, Japan.
38 Kornblatt, J.A & Luu, H.A (1986) The interactions of cyto-chrome c and porphyrin cytocyto-chrome c with cytocyto-chrome c oxidase The resting, reduced and pulsed enzymes Eur J Biochem 159, 407–413.
39 Malatesta, F., Sarti, P., Antonini, G., Vallone, B & Brunori, M (1990) Electron transfer to the binuclear center in cytochrome
Trang 8oxidase: catalytic significance and evidence for an additional
intermediate Proc Natl Acad Sci USA 87, 7410–7413.
40 Wilms, J., Dekker, H.L., Boelens, R & van Gelder, B.F (1981)
The effect of pH and ionic strength on the pre-steady-state
reac-tion of cytochrome c and cytochrome aa3 Biochim Biophys Acta
637, 168–176.
41 Veerman, E.C., Wilms, J., Dekker, H.L., Muijsers, A.O., van
Buuren, K.J., van Gelder, B.F., Osheroff, N., Speck, S.H &
Margoliash, E (1983) The presteady state reaction of chemically
modified cytochromes c with cytochrome oxidase J Biol Chem.
258, 5739–5745.
42 Kornblatt, J.A., Kornblatt, M.J., Hoa, G.H & Mauk, A.G.
(1993) Responses of two protein-protein complexes to solvent
stress: does water play a role at the interface? Biophys J 65, 1059–
1065.
43 Nicholls, P & Kimelberg, H.K (1968) Cytochromes a and a3.
Catalytic activity and spectral shifts in situ and in solution.
Biochim Biophys Acta 162, 11–21.
44 Parsegian, V.A., Rand, R.P., Fuller, N.L & Rau, D.C (1986) Osmotic stress for the direct measurement of intermolecular for-ces Methods Enzymol 127, 400–416.
45 Morgan, J.E., Verkhovsky, M.I & Wikstrom, M (1996) Observation and assignment of peroxy and ferryl intermediates
in the reduction of dioxygen to water by cytochrome c oxidase Biochemistry 35, 12235–12240.
46 Karpefors, M., Adelroth, P & Brzezinski, P (2000) The onset of the deuterium isotope effect in cytochrome c oxidase Biochemistry
39, 5045–5050.
47 Mitchell, R & Rich, P.R (1994) Proton uptake by cytochrome c oxidase on reduction and on ligand binding Biochim Biophys Acta 1186, 19–26.
48 Capitanio, N., Capitanio, G., Minuto, M., De Nitto, E., Palese, L.L., Nicholls, P & Papa, S (2000) Coupling of electron transfer with proton transfer at heme a and Cu(A) (redox Bohr effects) in cytochrome c oxidase Studies with the carbon monoxide inhibited enzyme Biochemistry 39, 6373–6379.