(Received for publication, September 6, 1994; and in revised form, November 18, 1994)
From the
A ruthenium-labeled cytochrome c derivative was
prepared to meet two design criteria: the ruthenium group must transfer
an electron rapidly to the heme group, but not alter the interaction
with cytochrome c oxidase. Site-directed mutagenesis was used
to replace His on the backside of yeast C102T
iso-1-cytochrome c with a cysteine residue, and the single
sulfhydryl group was labeled with (4-bromomethyl-4`methylbipyridine)
(bisbipyridine)ruthenium(II) to form Ru-39-cytochrome c (cyt c). There is an efficient pathway for electron transfer from
the ruthenium group to the heme group of Ru-39-cyt c comprising 13 covalent bonds and one hydrogen bond. Electron
transfer from the excited state Ru(II*) to ferric heme c occurred with
a rate constant of (6.0 ± 2.0)
10
s
, followed by electron transfer from ferrous
heme c to Ru(III) with a rate constant of (1.0 ± 0.2)
10
s
. Laser excitation of a complex
between Ru-39-cyt c and beef cytochrome c oxidase in
low ionic strength buffer (5 mM phosphate, pH 7) resulted in
electron transfer from photoreduced heme c to Cu
with a
rate constant of (6 ± 2)
10
s
, followed by electron transfer from Cu
to heme a with a rate constant of (1.8 ± 0.3)
10
s
. Increasing the ionic strength to
100 mM leads to bimolecular kinetics as the complex is
dissociated. The second-order rate constant is (2.5 ± 0.4)
10
M
s
at 230 mM ionic strength, nearly the same as that of
wild-type iso-1-cytochrome c.
Cytochrome c oxidase is a redox-linked proton pump that
transfers electrons from cytochrome c to molecular
oxygen(1, 2, 3) . Three of the redox active
metal centers, heme a, heme a, and Cu
, are
located in subunit I, and their ligands have been identified by
extensive mutagenesis experiments on Rhodobacter sphaeroides cytochrome c oxidase (4, 5) . Cu
is located in subunit II and is liganded by Cys
,
Cys
, His
, and
His
(6, 7, 8, 9) . The
Cu
center has been suggested to consist of a mixed-valence,
binuclear Cu(1.5), Cu(1.5) cluster rather than a single copper
atom(10, 11, 12) . The complex between
cytochrome c and cytochrome c oxidase is stabilized
by electrostatic interactions between the highly conserved lysines
surrounding the heme crevice of cytochrome c and four or more
carboxylates on subunit
II(13, 14, 15, 16, 17) .
One of these carboxylates, Glu-198, is located between the Cu
ligands Cys
and Cys
. The close
spatial relation between Cu
and the binding site led to the
suggestion that Cu
might accept electrons directly from
cytochrome c(17) .
The characterization of the
electron transfer reaction from cytochrome c to the initial
acceptor in cytochrome c oxidase has been a difficult problem.
Stopped-flow spectroscopy has been used to study the bimolecular
kinetics(18, 19) , but does not appear to have
sufficient time resolution to measure the initial electron transfer
step within the complex. Flow-flash photolysis studies of the complex
between ferrocytochrome c and reduced-CO-inhibited cytochrome c oxidase indicated that heme c transferred an electron to
Cu with a rate constant greater than 7
10
s
(20) . In contrast, a rate constant
of only 630 s
was observed for electron transfer
from cytochrome c to heme a in cytochrome c oxidase
using a photoreduced flavin to initiate the reaction(21) . Pan et al.(22) used our new ruthenium photoexcitation
technique(23, 24, 25, 26, 27, 28, 29, 30) to study electron transfer within complexes between
cytochrome c oxidase and derivatives labeled at specific
lysines on the backside of horse cytochrome c with
Ru(bipyridine)
. The Ru(II) group is photoexcited to a
metal-to-ligand charge-transfer state, Ru(II*), which is a strong
reducing agent and rapidly transfers an electron to heme c. The
photoreduced heme c was found to initially transfer an electron to
Cu
with a rate constant greater than approximately 1
10
s
, followed by electron
transfer from Cu
to heme a with a rate constant of 2
10
s
. It was not possible to
measure the rate constant for electron transfer from heme c to Cu
for these derivatives, probably because the rate for electron
transfer from Ru(II*) to heme c is not fast enough compared to the
subsequent electron transfer to Cu
.
In this study we
have prepared a new Ru-cyt c derivative specifically designed
to measure the initial electron transfer reaction from cytochrome c to cytochrome c oxidase. Two design criteria had to be
satisfied to achieve this goal. First, the Ru-cyt c derivative
must interact with cytochrome c oxidase in the same fashion as
wild-type cytochrome c. Second, the rate of electron transfer
from Ru(II*) to the heme group must be fast compared to the rate of
electron transfer from the heme group to the initial acceptor in
cytochrome c oxidase. This requires an efficient pathway for
electron transfer from Ru(II*) to the heme group, and optimum redox
properties for Ru(II*). Beratan et al.(31) have
identified an efficient pathway for electron transfer from residue 39
on the backside of cytochrome c to the heme group involving 13
covalent bonds and one hydrogen bond (Fig. 1). A single cysteine
residue was introduced at position 39 of yeast C102T iso-1-cytochrome c by site-directed mutagenesis, and labeled with a
sulfhydryl-selective trisbipyridineruthenium reagent (26) to
form Ru-39-cyt c (Fig. 1). This new derivative
satisfied both design criteria listed above, and was used to measure
the rate constant for intracomplex electron transfer from cytochrome c to Cu. The reaction was also studied as a
function of ionic strength in order to provide information about the
mechanism under physiological conditions.
Figure 1:
Pathway for electron transfer in Ru-39-cyt c. The x-ray crystal structure of oxidized yeast
iso-1-cytochrome c was obtained from the Brookhaven protein
data base (File 2Ycyt c). His was substituted
with a Cys and the ruthenium complex was attached to the sulfur atom of
Cys
. The proposed pathway for electron transfer extends
from Ru-39 through the peptide backbone of Ser
and
Gly
where there is a hydrogen bond from the amide nitrogen
of Gly
to the heme propionate
group.
Figure 2:
Purification of Ru-39-cyt c. The
crude reaction mixture of Ru-39-cyt c was eluted on a 1
10 cm Waters SP 8HR cation exchange column with a linear
gradient from 10 to 500 mM sodium phosphate at pH 7 at a flow
rate of 1 ml per min. The major peak eluting at 46 min (325 mM sodium phosphate) contained pure Ru-39-cyt c.
Scheme 1: Scheme 1
Figure 3:
Photoinduced electron transfer within
Ru-39-cyt c (12 µM in 100 mM sodium
phosphate, pH 7). A, the Fe(II) transient was obtained from
the difference between the 550-nm transient and the 556.5-nm transient
as described in the text. B, 434-nm transient represents the
photoexcitation and recovery of Ru(II). The smooth lines are the
theoretical curves for Fe(II) (A) and Ru(II) (B) in Fig. S1, with k = 6.0
10
s
, k
=
1.0
10
s
, and k
= 5.4
10
s
, using the equations given in Durham et
al.(24) .
Figure 4: Photoinduced electron transfer from Ru-39-cyt c to cytochrome c oxidase. The solution contained 12 µM Ru-39-cyt c and 18 µM cytochrome c oxidase in 5 mM sodium phosphate, pH 7, 10 mM aniline, 1 mM 3CP. A, 550-nm transient; B, 830-nm transient; C, 604-nm transient. The solid lines are the best fits to described in the text.
where c represents the oxidized form
of Ru-39-cyt c present before the laser pulse, and
(Cu
a
)
represents the resting form of cytochrome c oxidase. The
transients at the three wavelengths were fit to the complete kinetic
equations for , assuming k
k
:
The fast phase of the reoxidation of Ru-39-cyt c had k = (6 ± 2)
10
s
, and the amount of heme c
reoxidized was 0.09 ± 0.01 µM. The 830-nm
transient had k
= (6 ± 2)
10
s
, k
=
(1.6 ± 0.3)
10
s
, and the
amount of Cu
reduced and reoxidized was 0.11 ± 0.02
µM. The 604 nm transient had k
= (7 ± 3)
10
s
, k
= (1.8
± 0.3)
10
s
and the
amount of heme a reduced was 0.10 ± 0.01 µM. The
transients at the three wavelengths are thus consistent with , with k
= (6 ± 2)
10
s
and k
= (1.8 ± 0.3)
10
s
. The transients indicate that each of the
reactions in proceed in the forward direction with
equilibrium constants of at least 5. The small slow phase in the 550-nm
transient could be due to a minor form of the complex that is not
properly aligned for rapid electron transfer from heme c to cytochrome c oxidase.
Figure 5:
Bimolecular electron transfer from
Ru-39-cyt c to cytochrome c oxidase. The solutions
contained 5 µM Ru-39-cyt c, 0 to 7 µM cytochrome c oxidase, 2 mM sodium phosphate, pH
7, 10 mM aniline, 1 mM 3CP, and 100 mM NaCl.
The pseudo first-order rate constant k, in
s
, obtained from the 550- and 604-nm transients is
plotted as a function of the concentration of cytochrome c oxidase in µM. The solid line is the best
fit to a second-order rate constant of 1.75
10
M
s
.
Figure 6:
Dependence of the second-order rate
constant of the reaction between Ru-39-cyt c and cytochrome c oxidase on ionic strength. The solutions contained
1-12 µM Ru-39-cyt c, 0-20 µM cytochrome c oxidase, 2 mM sodium phosphate, pH
7, 10 mM anline 1 mM 3CP, and 70-240 mM NaCl. The second-order rate constants, in units of M s
, were measured as
described in Fig. 5and are plotted as a function of the square
root of the ionic strength, in units of M.
The kinetic results over the entire ionic strength range are consistent with the mechanism shown in :
At low ionic strength, all of the Ru-39-cyt c will be complexed with cytochrome c oxidase, and the reaction will proceed according to the top line
of . At intermediate ionic strength (55-105
mM), there will be an equilibrium between complexed and
uncomplexed Ru-39-cyt c
represented by the
dissociation constant K
. The complexed Ru-39-cyt c
will react according to the top line of , while the uncomplexed, photoreduced Ru-39-cyt c
will first have to bind to cytochrome c oxidase and then transfer an electron. At ionic strengths
above 100 mM, all of the Ru-39-cyt c
will be uncomplexed before the laser flash, and will have to bind
to cytochrome c oxidase before electron transfer can occur.
The dissociation constant K
of the complex between
Ru-39-cyt c
and cytochrome c oxidase was determined from the fraction f of the fast
phase of the 604-nm transients using :
where E is the total concentration of
cytochrome c oxidase and C
is the total
concentration of Ru-39-cyt c. is based on a 1:1
stoichiometry for complex formation. The value of K
increased from less than 1 µM at low ionic strength
to 8 µM at 75 mM ionic strength, and to greater
than 100 µM at ionic strengths above 105 mM (Table 1). It was only possible to determine actual values
for K
at ionic strengths between 55 and 105 mM where both the slow phase and the fast phase could be observed
simultaneously.
The flavin flash photolysis method of Hazzard et
al.(21) was used to compare the second-order rate
constant of Ru-39-cyt c to that of wild-type iso-1-cytochrome c and horse cytochrome c at high ionic strength (100
mM sodium phosphate, pH 7, 5 mM EDTA). The
second-order rate constants are the following: Ru-39-cyt c,
(2.5 ± 0.4) 10
M
s
; wild-type yeast iso-1-cytochrome c, (1.7 ± 0.4)
10
M
s
; horse
cytochrome c, (8.3 ± 2)
10
M
s
. The rate
constant of Ru-39-cyt c is thus slightly larger than that of
wild-type iso-1-cytochrome c. The same rate constant was
obtained for Ru-39-cyt c using either lumiflavin excitation or
ruthenium excitation.
The location of the ruthenium group on the backside of Ru-39-cyt c was designed to allow normal interaction with cytochrome c oxidase. The second-order rate constant for the reaction of Ru-39-cyt c with cytochrome c oxidase at high ionic strength is nearly the same as that of wild-type iso-1-cytochrome c. This is one of the most important criteria for the functional integrity of the derivative. In addition, the redox potential and visible spectra, including the 695-nm band, are the same as for wild-type cytochrome c, indicating that the conformation of the heme crevice is not altered.
The kinetics of the photoinduced reaction between
Ru-39-cyt c and cytochrome c oxidase remain unchanged
as the ionic strength is increased from 5 to 40 mM, and the
equilibrium dissociation constant K of the c
:(Cu
a
) complex remains less than 1
µM (Table 1). The reaction observed in the present
experiments can therefore be correlated with the high affinity phase of
the steady-state reaction, which has a K
of 9
10
M for native yeast
iso-1-cytochrome c in 25 mM ionic strength buffer at
pH 7.8(15, 61) . The K
value
determined from the steady-state kinetics is essentially the same as
the equilibrium dissociation constant K
determined
from direct binding studies of the oxidized proteins(61) .
Furthermore, the turnover number for the high affinity phase has been
shown to be rate-limited by the dissociation of the product complex at
low ionic strength(62, 63) .
In some respects the
most revealing results of the ruthenium photoreduction experiments are
observed at intermediate ionic strength (55-105 mM),
where both intracomplex and bimolecular kinetics are present
simultaneously (Table 1). The fact that the rate constant for the
fast phase of electron transfer from c to
Cu
does not change as the ionic strength increases from 5
to 105 mM indicates that the dissociation rate constant k
is much smaller that k
(). If k
were larger than k
, then rapid equilibrium conditions would apply,
and separate slow and fast phases would not be observed. Assuming that
the bimolecular reaction proceeds according to , the
second-order rate constant determined from the slow phase will be given
by :
is based on the steady-state assumption, which
should be valid in the present case because the concentration of
cytochrome c oxidase is much larger than the concentration of
photoreduced Ru-39-cyt c. Since k
is much smaller than k
at ionic strengths up to 105 mM, the second-order rate
constant will be equal to k
under these
conditions. The value of k
is thus 2.2
10
M
s
at 75
mM ionic strength, and 1.7
10
M
s
at 105 mM ionic strength. If it is assumed that the equilibrium dissociation
constant K
measured for the c
:(Cu
a
) complex is the same for the c
:(Cu
a
) complex, then K
= k
/k
.
This is a reasonable assumption, since the Minnaert mechanism IV that
accounts for the first-order time course of the steady-state reaction
is based on equal dissociation constants for oxidized and reduced
cytochrome c(64, 65) . With this assumption,
the values of k
are 1.4
10
s
at 75 mM ionic strength, and 8.5
10
s
at 105 mM ionic
strength, which are significantly less than k
.
At the lower limit of physiological ionic strength, 105 mM,
the reaction can be described by complex formation with rate constant k = 1.7
10
M
s
, intracomplex
electron transfer with rate constant k
= 6
10
s
, and complex dissociation
with rate constant k
= 8.5
10
s
. Since k
is
small compared to k
, essentially all of the
ferrocytochrome c that binds to the high affinity site will
react before dissociation. As the ionic strength is increased above 105
mM, k
will increase and k
will decrease. At some ionic strength it is
likely that k
will become comparable to k
, and k
will be a function
of all three rate constants as given in . Since k
is unchanged over the ionic strength range from
5 to 105 mM, it will probably remain unchanged as the ionic
strength is increased further. The decrease in k
with increasing ionic strength is thus probably due to an
increase in k
and/or a decrease in k
rather than to changes in k
. Ionic strength dependence studies have been
used extensively to characterize electrostatic interactions between
redox proteins, and it is generally agreed that the slope of a plot of
log k versus the square root of the ionic strength is
proportional to the strength of the electrostatic
interaction(14, 66, 67) . The slope of the
plot for k
shown in Fig. 6is essentially
the same as that of a plot of the steady-state parameter V
/K
for the reaction
between horse cytochrome c and beef cytochrome c oxidase(14) . This indicates that the electrostatic
interactions are similar for yeast Ru-39-cyt c and native
horse cytochrome c.