Nanosecond photoreduction of cytochrome P450cam by channel-specific Ru-diimine electron tunneling wires

Article abstract of DOI:10.1021/ja0294111

We report the synthesis and characterization of Ru-diimine complexes designed to bind to cytochrome P450cam (CYP101). The sensitizer core has the structure [Ru(L₂)L']²⁺, where L' is a perfluorinated biphenyl bridge (F₈bp) connecting 4,4′-dimethylbipyridine to an enzyme substrate (adamantane, F₈bp-Ad), a heme ligand (imidazole, F₈bp-lm), or F (F₉bp). The electron-transfer (ET) driving force (-δG°) is varied by replacing the ancillary 2,2′ -bipyridine ligands with 4,4′,5,5′-tetramethylbipyridine (tmRu). The four complexes all bind P450cam tightly: Ru-F₈bp-Ad (1, K _d = 0.077 μM); Ru-F₈bp-lm (2, K_d = 3.7 μM); tmRu-F₉bp (3, K_d = 2.1 μM); and tmRu-F ₈bp-lm (4, K_d = 0.48 μM). Binding is predominantly driven by hydrophobic interactions between the Ru-diimine wires and the substrate access channel. With Ru-F₈bp wires, redox reactions can be triggered on the nanosecond time scale. Ru-wire 2, which ligates the heme iron, shows a small amount of transient heme photoreduction (ca. 30%), whereas the transient photoreduction yield for 4 is 76%. Forward ET with 4 occurs in roughly 40 ns (k_f = 2.8 × 10⁷ s^-1), and back ET (Fe^II → Ru^III, k_b ≈ 1.7 × 10⁸ s^-1) is near the coupling-limited rate (k _max). Direct photoreduction was not observed for 1 or 3. The large variation in ET rates among the Ru-diimine:P450 conjugates strongly supports a through-bond model of Ru-heme electronic coupling.

Full text of DOI:10.1021/ja0294111

Published on Web 09/23/2003
Nanosecond Photoreduction of Cytochrome P450cam by
Channel-Specific Ru-diimine Electron Tunneling Wires
Alexander R. Dunn, Ivan J. Dmochowski, Jay R. Winkler,* and Harry B. Gray*
Contribution from the Beckman Institute, California Institute of Technology,
Pasadena, California 91125
Received November 19, 2002; E-mail: hbgray@caltech.edu; winklerj@caltech.edu
Abstract: We report the synthesis and characterization of Ru-diimine complexes designed to bind to
cytochrome P450cam (CYP101). The sensitizer core has the structure [Ru(L2)L′]2+, where L′ is a
perfluorinated biphenyl bridge (F8bp) connecting 4,4′-dimethylbipyridine to an enzyme substrate (adaman-
tane, F8bp-Ad), a heme ligand (imidazole, F8bp-Im), or F (F9bp). The electron-transfer (ET) driving force
(-∆G°) is varied by replacing the ancillary 2,2′-bipyridine ligands with 4,4′,5,5′-tetramethylbipyridine (tmRu).
The four complexes all bind P450cam tightly: Ru-F8bp-Ad (1, Kd ) 0.077 µM); Ru-F8bp-Im (2, Kd ) 3.7
µM); tmRu-F9bp (3, Kd ) 2.1 µM); and tmRu-F8bp-Im (4, Kd ) 0.48 µM). Binding is predominantly driven
by hydrophobic interactions between the Ru-diimine wires and the substrate access channel. With Ru-
F8bp wires, redox reactions can be triggered on the nanosecond time scale. Ru-wire 2, which ligates the
heme iron, shows a small amount of transient heme photoreduction (ca. 30%), whereas the transient
photoreduction yield for 4 is 76%. Forward ET with 4 occurs in roughly 40 ns (kf ) 2.8 × 107 s-1), and back
ET (FeII f RuIII, kb ≈ 1.7 × 108 s-1) is near the coupling-limited rate (kmax). Direct photoreduction was not
observed for 1 or 3. The large variation in ET rates among the Ru-diimine:P450 conjugates strongly supports
a through-bond model of Ru-heme electronic coupling.
Introduction
Experimental Section
Electron transfer (ET) is often the rate-determining step in
biological catalysis. The reactions of the cytochromes P450 are
an excellent case in point.1 In the archetypal P450 from
Pseudomonas putida (P450cam), the natural redox partner,
putidaredoxin (Putd), reduces the enzyme far too slowly (kred
≈ 50 s-1) to allow catalytic intermediates to accumulate under
biological conditions (Scheme 1).2
We are studying a variety of Ru-diimine sensitizers designed
to replace the slow biological reduction with a rapid optical
redox trigger.5,6 Each of the most promising sensitizers employs
a perfluorobiphenyl group (F8bp) that couples the Ru-diimine
to a terminal functionality (Chart 1).
In these Ru-diimine:P450 conjugates, the Ru donor and the
ferriheme acceptor are held in position mainly by noncovalent
interactions. Thus, the synthetic flexibility of the sensitizer,
together with the structural framework provided by the enzyme,
makes this an ideal system for exploring basic ET parameters
in a biologically relevant milieu.
General. P450cam and the mutant Y29F were expressed in E. coli
and purified using standard procedures.5,7 Site-directed mutagenesis was
performed using Stratagene QuikChange mutagenesis kits. P450cam
was stored in small aliquots and thawed immediately before use.
Samples were prepared in 50 mM potassium phosphate buffer (pH 7.4)
containing 100 mM KCl. P450 concentration was quantified using the
heme Soret absorption at 416 nm (ꢀ416 ) 115 mM-1 cm-1). All
experiments were performed on samples with a ratio Abs418/Abs280
g
1.55 when camphor free. Spectroscopic experiments used custom quartz
cuvettes fitted with Kontes Teflon stopcocks. Oxygen was removed
from the sample by completing at least 30 cycles of partial vacuum
followed by an influx of argon.
Absorption spectra were taken on an HP-8452A spectrophotometer.
Steady-state luminescence spectra were taken on an ISS K2 fluorometer.
2+
Emission quantum yields were calculated relative to a Ru(bpy)3
standard, whose luminescence quantum yield was taken to be 0.042 in
water.8-10
Reduction of P450cam. P450cam (5.1 µM) was reduced with
sodium dithionite under an atmosphere of carbon monoxide in the
presence of 1.2 equiv of tmRu-F8bp-Im, producing the characteristic
Soret peak at 446 nm. Carbon monoxide was then removed by gently
bubbling argon through the sample for 5 min, resulting in both a change
in shape and a decrease in intensity of the Soret peak (446 nm). This
(1) Ortiz de Montellano, P. R. Cytochrome P450: Structure, Mechanism and
Biochemistry, 2 ed.; Plenum Press: New York, 1995; p 652.
(2) Fisher, M. T.; Sligar, S. G. J. Am. Chem. Soc. 1985, 107, 5018-5019.
(3) (a) Davydov, R.; Makris, T. M.; Kofman, V.; Werst, D. E.; Sligar, S. G.;
Hoffman, B. M. J. Am. Chem. Soc. 2001, 123, 1403-1415. (b) Schlichting,
I.; Berendzen, J.; Chu, K.; Stock, A. M.; Maves, S. A.; Benson, D. E.;
Sweet, R. M.; Ringe, D.; Petsko, G. A.; Sligar, S. G. Science 2000, 287,
1615-1622.
(7) Dmochowski, I. J. Probing cytochrome P450 with sensitizer-linked
substrates; California Institute of Technology: Pasadena, CA, 2000, p 305.
(8) Roundhill, D. M. Photochemistry and Photophysics of Metal Complexes;
Plenum Press: New York, 1994.
(9) van Houten, J.; Watts, R. J. J. Am. Chem. Soc. 1976, 98, 4853-4858.
(10) Cook, M. J.; Lewis, A. P.; McAuliffe, G. S. G.; Skarda, V.; Thomson, A.
J.; Glasper, J. L.; Robbins, D. J. J. Chem. Soc., Perkin Trans. 2 1984,
1293-1301.
(4) Groves, J. T.; McClusky, G. A.; White, R. E.; Coon, M. J. Biochem.
Biophys. Res. Commun. 1978, 81, 154-160.
(5) Dmochowski, I. J.; Crane, B. R.; Wilker, J. J.; Winkler, J. R.; Gray, H. B.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12987-12990.
(6) Wilker, J. J.; Dmochowski, I. J.; Dawson, J. H.; Winkler, J. R.; Gray, H.
B. Angew. Chem., Int. Ed. 1999, 38, 90-92.
9
12450
J. AM. CHEM. SOC. 2003, 125, 12450-12456
10.1021/ja0294111 CCC: $25.00 © 2003 American Chemical Society

Photoreduction of Cytochrome P450cam by ET Wires
A R T I C L E S
Scheme 1 Cytochrome P450cam Catalytic Cyclea
ment possesses a response time of 20 ns (fwhm), and the data are
digitized at 200 megasamples s-1
. For nanosecond lumines-
cence decay measurements, the sample was excited at 10 Hz with 70-
ps, 355-nm pulses from a regeneratively amplified mode-locked Nd:
YAG laser. Luminescence from the cuvette was filtered with a 650-
nm long-pass filter, collected directly by a fiber optic (Fiberguide
Industries) and detected with a Hamamatsu C5680 streak camera. The
data were recorded using Hamamatsu High Performance Digital
Temporal Analyzer 3.1.0 software and fit using Microcal Origin
5.0.
Binding Constants. Luminescence decay profiles were fit to a
biexponential function:
I(t) ) c1e-k t + c2e-k t
(1)
1
2
using nonlinear least squares with iterative reconvolution to account
for finite instrument response. The ratio of enzyme-bound to free
ruthenium complex is c1/c2, where k1 and k2 are the luminescence decay
rate constants for the enzyme-bound and free ruthenium complexes.
This procedure has several advantages over steady-state UV-vis
titrations. The absorption due to the ruthenium complexes complicates
the determination of a dissociation constant from the direct titration of
P450cam with a Ru-wire. Previous results have demonstrated that
camphor and luminescent probe molecules may bind simultaneously
to the enzyme, again complicating the derivation of dissociation
constants from competition binding assays.14
a
Upon binding, the substrate displaces water, converting the heme from
6-coordinate, low-spin 1 to 5-coordinate, high-spin 2. Subsequent reduction
by putidaredoxin produces the ferrous heme, which binds dioxygen (3).
Reduction of 3 produces the ferrous, peroxide-bound heme (4), which rapidly
protonates (5).3 In the prevalent model, the peroxide then undergoes
heterolysis to produce water and a ferryl [FeIV)O]•+ species (6), which
oxidizes the substrate.4
Fitting errors for c1, c2, k1, and k2 were determined by systematically
varying one parameter while optimizing the other three to minimize
the sum of the absolute values of the difference between the model
and the data. Limits on a particular variable were set when the presence
of obvious residuals indicated that the model did not fit the data. In
practice, the fitting error on c1 and c2 was found to be about 10% of
the total amplitude: err(c1) ) 0.1(c1 + c2). Propagation of this error
through the determination of Kd, assuming the worst-case perfect
correlation of c1 and c2, shows that the fitting error is 20% when c1 )
c2, but becomes substantial when one phase predominates. For instance,
when c1 and c2 account for 20 and 80% of the amplitude, the resulting
Kd becomes uncertain to within a factor of 2.3.
Chart 1. Ru-diimine Wiresa
ET Rate Constants. The raw transient absorption kinetics contain
contributions from both heme/Ru redox processes and the bleach
associated with the Ru-diimine excited state (*Ru2+). The observed
kinetics at 420 and 445 nm were corrected for the contribution of *Ru2+
prior to fitting. The *Ru2+ decay was recorded at 427 nm (the ferrous/
ferric heme isosbestic). This trace was then scaled to account for the
differences in *Ru2+/Ru2+ extinction coefficients at 420, 427, and 445
nm (*Ru2+/Ru2+∆ꢀ445/∆ꢀ427 ) 1.06, ∆ꢀ420/∆ꢀ427 ) 0.83) and subtracted
from the kinetics at 420 and 445 nm.
Transient absorption kinetics were interpreted according to the model
shown in Scheme 2. The change in optical density (∆OD) at time t is
given by eq 2:
a
1 Ru-F8bp-Ad; 2 Ru-F8bp-Im; 3 tmRu-F9bp; 4 tmRu-F8bp-Im.
species was assigned as the imidazole-ligated ferrous heme, in
agreement with the previously determined spectrum of N-phenylimi-
dazole-ligated ferrous P450cam.11 Subsequent addition of carbon
monoxide to the cuvette resulted in the restoration of the Soret band
of CO-ligated P450cam.
kf[*Ru]0∆ꢀ
kb + ksep - kL
ksep
ksep
kL
(∆OD)(t) )
1 -
exp(-kLt) -
(
)
[
ksep
ksep
1 -
exp[-(kb + ksep)t] +
-
(2)
(
)
]
kb + ksep
kL
kb + ksep
As a control, the same procedure was performed with 50 µM
camphor replacing tmRu-F8bp-Im. Five minutes of argon purging were
sufficient to shift the Soret peak from 446 to 408 nm, indicative of the
complete conversion of CO-bound to five-coordinate ferrous heme.
Transient Spectroscopy. Transient absorption and emission data
were collected using instruments described previously.12,13 The instru-
where [*Ru2+]0(M-1) is the concentration of excited ruthenium complex
at time zero and ∆ꢀ is the change in molar extinction coefficients:
(12) Dmochowski, I. J.; Winkler, J. R.; Gray, H. B. J. Inorg. Biochem. 2000,
81, 221-228.
(13) Low, D. W.; Winkler, J. R.; Gray, H. B. J. Am. Chem. Soc. 1996, 118,
117-120.
(11) Dawson, J. H.; Andersson, L. A.; Sono, M. J. Biol. Chem. 1983, 258,
13637-13645.
(14) Dunn, A. R.; Hays, A.-M. A.; Goodin, D. B.; Stout, C. D.; Chiu, R.;
Winkler, J. R.; Gray, H. B. J. Am. Chem. Soc. 2002, 124, 10254-10255.
9
J. AM. CHEM. SOC. VOL. 125, NO. 41, 2003 12451

A R T I C L E S
Dunn et al.
II
III
III
II
Scheme 2. ET Processes in Photoexcited Ru-diimine:P450cam
∆ꢀ(λ) ) ꢀFe - ꢀFe + ꢀRu - ꢀRu
(3)
Conjugatesa
Sufficient laser power was used to ensure that all photosensitizer
molecules were excited; [*Ru2+]0 ) [Ru]tot. The values ∆ꢀ445 ) 90
mM-1 cm-1 and ∆ꢀ420 ) -72 mM-1 cm-1 were derived from the
steady-state spectra of reduced and oxidized P450cam bound to tmRu-
F8bp-Im plus the known RuII/RuIII ∆ꢀ values.15,16 The rate constant
kL(s-1) is the observed decay rate of *Ru2+ in the presence of P450:
kL ) k0 + kf + kE
(4)
a *Ru2+ reduces the heme (kf) or decays to the ground state through a
combination of intrinsic decay (k0) and energy transfer to the heme (kE),
which decays non-radiatively to the ground state. The charge-separated
state (Ru3+‚‚‚Fe2+) undergoes back electron transfer (kb) or decays to form
a long-lived ferrous heme (ksep).21
where the other rate constants are for the intrinsic decay (k0), forward
electron transfer (kf), and Fo¨rster energy transfer to the heme (kE).
Because the rates of forward and back ET are comparable to the
response time of our instrument, the instrumental response function
was deconvolved from the observed kinetics.17 The recorded ∆OD was
converted into an intensity:
Scheme 3. Synthesis of Ru-diimine Wiresa
I ) (I0)(10-∆OD
)
(5)
The response function was then deconvolved from the observed
intensity I by iterative reconvolution. The algorithm used was written
in MatLab 5.3 and relies on the built-in simplex minimization
algorithm.
Fitting errors for kf, kb, and ksep were determined by systematically
adjusting one variable while leaving the other two free to adopt whatever
values minimized the sum of the absolute values of the difference
between the model and the data. Limits on a particular variable were
set when the presence of a clear residual indicated that the model did
not fit the data. Error in the rates is best expressed as a multiplicative
factor. The errors are estimated to be: kf(445 nm) 2.1; kf(420 nm) 1.8;
kb(445 nm) 2.3; kb(420 nm) 2.0; ksep(445 nm) 1.1; and ksep(420 nm)
1.5. These errors are in accord with those expected for a multiexpontial
fit to moderate quality data.17
Fo1rster Energy Transfer. The rate constant kE was calculated from
standard theoretical expressions:18
6
R0
k ) k
(6)
(7)
0( r )
E
R60 ) 8.8 × 10-5(κ2n-4φ0J)
∞F (λ)E (λ)λ4 dλ
∫
0
A
0
J )
(8)
a Deprotonation of 4,4′-dimethyl-2,2′-bipyridine with lithium diisopropyl
amide (LDA) followed by nucleophilic attack on decafluorobiphenyl results
in the ET bridge 7.
∞F (λ) dλ
∫
0
0
where J is the overlap between the luminescence spectrum of the donor
and absorption spectrum of the acceptor (weighted by λ4), φ0 is the
luminescence quantum yield in the absence of energy transfer, n is the
index of refraction, and κ is an orientation factor dependent on the
alignment of the donor and acceptor dipoles (κ2 ) 2/3 for random
alignment).
desired conjugated compounds (Scheme 3). Absorption and
emission maxima at 456 and 620 nm (1 and 2) and 444 and
654 nm (3 and 4) are consistent with the previously reported
spectra of [Ru(bpy)2(Me2bpy)]2+ and [Ru(tmbpy)2(bpy)]2+ 19
.
Binding. All of the Ru-diimine wires (1-4) bind to P450.
Binding of Ru-F8bp-Ad induces a shift in the Soret absorption
maximum from 416 to 414 nm, consistent with partial displace-
ment of water from the heme iron. Similarly, coordination of
both Ru-F8bp-Im and tmRu-F8bp-Im shifts the Soret peak to
420 nm (Figure 1), consistent with the value of 421 nm reported
for the ferric P450cam:N-phenylimidazole complex.20 The
measured extinction coefficient at 446 nm in the spectrum of
Calculation of Buried Surface Area. The solvent-exposed surface
areas of Ru-F8bp-Ad, P450cam, and the P450cam:Ru-F8bp-Ad
conjugate (pdb code 1k2o) were calculated with the Solvation module
of InsightII using a 1.4 Å probe. Buried surface area was computed by
subtracting the surface area of the conjugate from that of Ru-F8bp-
Ad and P450cam alone. The difference in buried surface areas for the
∆ and Λ stereoisomers of Ru-F8bp-Ad is negligible.
Results
the tmRu-F8bp-Im:FeII-P450cam conjugate is 117 mM-1 cm-1
,
in agreement with the value of 116 mM-1 cm-1 reported for
the N-phenylimidazole complex.11 All of the absorption spectra
are consistent with predominantly low-spin hemes in the Ru-
wire:P450cam conjugates.
Synthesis. Sequential nucleophilic substitution of decafluo-
robiphenyl proved to be an especially efficient route to the
(15) Kotkar, D.; Joshi, V.; Ghosh, P. K. J. Chem. Soc., Chem. Commun. 1987,
1, 4-6.
(16) Miller, R. R.; Brandt, W. W.; Puke, M. J. Am. Chem. Soc. 1955, 77, 3178-
3180.
(19) Opperman, K. A.; Mecklenburg, S. L.; Meyer, T. J. Inorg. Chem. 1994,
(17) Demas, J. N. Excited State Lifetime Measurements; Academic Press: New
York, 1983.
33, 5295-5301.
(20) Dawson, J. H.; Andersson, L. A.; Sono, M. J. Biol. Chem. 1982, 257, 3606-
(18) Wu, P.; Brand, L. Anal. Biochem. 1994, 218, 1-13.
3617.
9
12452 J. AM. CHEM. SOC. VOL. 125, NO. 41, 2003

Photoreduction of Cytochrome P450cam by ET Wires
A R T I C L E S
Figure 1. UV-vis absorption spectra of ferric P450cam (black, open
circles), 5.2 µM tmRu-F8bp-Im (red, open squares), and ferrous P450cam
ligated by tmRu-F8bp-Im (green).
Figure 3. Transient absorption spectrum measured 20 µs after 470 nm
excitation of equimolar tmRu-F8bp-Im and P450cam (9.6 µM). Observed
changes in optical density are chiefly due to the conversion of ferric to
ferrous heme, with comparatively minor contributions from RuII to RuIII
oxidation.
Figure 2. Luminescence decay. (A) 10 µM 1:1 tmRu-F8bp-Im:P450cam
luminescence decay (tmRu-F8bp-Im, black; tmRu-F8bp-Im + 1 equiv
P450cam, red, open circles). (B) Nanosecond time scale luminescence decay
of 1:1 tmRu-F8bp-Im:P450cam (4.5 µM) (instrument response ca. 70 ps;
see Experimental Section). The initial rapid (k > 1 × 109 s-1) decay is
intrinsic to P450cam and likely represents a trace impurity. The slower
decay on this time scale corresponds to the rapid decay in Figure 2A (kL )
3.7 × 108 s-1). Green, P450cam; black, P450cam + tmRu-F8bp-Im; red,
monoexponential fit.
Table 1. Dissociation Constants
Figure 4. Transient absorption at 445 (top) and 420 nm (bottom) for 10
µM 1:1 tmRu-F8bp-Im:P450cam (black, data; blue, fit; red, convolved fit).
The kinetics were corrected for both free and bound *Ru2+ by measuring
the transient absorption of *Ru2+ at a P450cam FeII/FeIII, RuII/RuIII isosbestic
(427 nm). This spectrum was then scaled and subtracted from the kinetics
recorded at 420 and 445 nm (Experimental Section). The data were fit to
the kinetics model in Scheme 2 using iterative reconvolution to account
for instrument response. The fit yielded the following rate constants: kf )
2.9 × 107, kb ) 1.6 × 108, ksep ) 8.6 × 106 s-1 (445 nm); and kf ) 2.6 ×
107, kb ) 1.9 × 108, ksep ) 9.3 × 106 s-1 (420 nm). The same procedure
could not be applied to the transient absorption spectra of Ru-F8bp-Im
because the signal due to *Ru2+ is much larger than the signal due to the
heme.
a
Ru-wire
µM
Ru-F8bp-Ad
Ru-F8bp-Im
tmRu-F8bp-Im
tmRu-F9bp
0.077 ( 0.011
3.7 ( 0.5
0.48 ( 0.18
2.1 ( 1.3
a
Uncertainties are standard deviations derived from independent analysis
of at least three measurements.
All of the Ru-wires show biphasic luminescence decays in
the presence of P450cam. The fast phase results from partial
quenching due to energy transfer to the heme, and in the case
of Ru-F8bp-Im and tmRu-F8bp-Im, electron transfer (see
Scheme 2 and following sections). Although the heme Soret
does not shift in the presence of tmRu-F9bp, the biphasic
luminescence decay of the Ru-wire due to energy transfer
indicates that it too binds P450cam. Typical biphasic lumines-
cence decays for a Ru-wire in the presence of P450 are shown
in Figure 2. The ratio of the amplitudes of the fast (bound) and
slow (free Ru-wire) phases was used to calculate binding
constants (Table 1).
(Figure 3). Neither *Ru-F8bp-Ad (*1) nor *tmRu-F9bp (*3)
reduces P450cam, as judged by the lack of a transient absorption
signal.
Photoexcitation of equimolar tmRu-F8bp-Im and P450cam
shows complex early kinetics (Figure 4, Scheme 2). The sharp
rise and fall at the beginning of the trace recorded at 445 nm
are attributed to fast forward (kf) and back (kb) ET. The rates
of accumulation and decay of FeII are comparable to the rise
time of the instrument. Deconvolution was necessary to
eliminate the instrument response contribution from the observed
kinetics. Optimization of the parameters kf, kb, and ksep at 420
Electron Transfer. Upon 470-nm excitation, both tmRu-
F8bp-Im and Ru-F8bp-Im reduce P450cam. The bleach at 420
nm and increase in optical density at 445 nm confirm the
conversion of (ImN)(CysS){PïrN4FeIII} to (ImN)(CysS){PïrN4FeII}
and 445 nm yielded the following rate constants: kf ) 2.8 ×
21
107; kb ) 1.7 × 108; ksep ) 9.0 × 106 s-1
.
9
J. AM. CHEM. SOC. VOL. 125, NO. 41, 2003 12453

A R T I C L E S
Dunn et al.
Table 2. Derivation of kf and Ru-Fe Distances from Luminescence Decay Measurementsa
Ru-wire
kL × 10-6 (s-1
)
k0 × 10-6 (s-1
)
kE × 10-6 (s-1
)
k × 10-6 (s-1
)
Ru−Fe (Å)
R (Å)b
f
0
tmRu-F8bp-Im
Ru-F8bp-Im
Ru-F8bp-Ad
37
13
5.5
4.6
2.0
2.0
4.4c
6.6b
3.5c
28d
4.4
18.1b
18.0
22.1
24.3
18.1e
22.1b
cf. 21.8f
17.0b
tmRu-F9bp
13
4.6
8.4c
18.8
a
b
c
Variation in R0 stems from variation in the heme Q-bands and the emission spectrum of the complex. Calculated from Fo¨rster theory (eqs 6-8). kE
d
e
f
) kL - k0 - kf. From transient absorption kinetics. In accord with the calculated Ru-Fe distance for tmRu-F8bp-Im. From the crystal structure of
Ru-F8bp-Ad:P450cam (ref 23).
In the absence of competing electron transfer (Ru-F8bp-Ad
and tmRu-F9bp), the Ru-Fe distance can be calculated using
Fo¨rster theory from kE, the ruthenium emission spectrum, and
the heme absorption spectrum (Table 2). The Ru-Fe distance
(22.1 Å) calculated for Ru-F8bp-Ad is in excellent agreement
with the value from the crystal structure. The distance of 17 Å
calculated for tmRu-F9bp agrees well with structural modeling
of the tmRu-F9bp:P450cam conjugate and corresponds to a ∼2
Å gap between the end of the perfluorinated biphenyl bridge
and the heme.
Using eq 4, we calculated that kE for tmRu-F8bp-Im is 4.4
× 106 s-1 (Table 2). This rate of energy transfer corresponds
to a Ru-Fe distance of 18.1 Å, a reasonable value given the
geometric constraints of the fluorobiphenyl bridge. A Ru-Fe
distance of 18.1 Å can in turn be used to calculate a kE of 6.6
× 106 s-1 for Ru-F8bp-Im, corresponding to kf ) 4.4 × 106
Figure 5. The Ru-F8bp-Ad wire is partially buried upon binding to
s-1, which is 6 times slower than photoinduced reduction of
P450cam. The buried surface (gray, 56% of the total surface area) was
computed with the program GRASP using a 1.4 Å radius probe.
ferric P450cam by tmRu-F8bp-Im. With φ ) (kf/kL), we find
total ferrous heme yields of 76% for tmRu-F8bp-Im and
roughly 30% for Ru-F8bp-Im.
Discussion
The observed binding constants suggest that the interaction
between the ruthenium complex and the enzyme is primarily
hydrophobic in nature. Ru-F8bp-Ad, which has the largest
hydrophobic surface area, binds best, and tmRu-F8bp-Im binds
better than its nonmethylated analogue Ru-F8bp-Im. Previous
work suggests that the binding energy derived from burying
hydrophobic surfaces is around 15 cal Å-2 for protein-protein
interactions.22 The crystal structure of Ru-F8bp-Ad bound to
P450cam shows extensive contacts between the Ru-wire and
the hydrophobic substrate access channel,23 resulting in 1.2 ×
103 Å2 of buried surface area (Figure 5), corresponding to 8.2
(21) Although most of the photoproduced ferrous heme decays in 50 ns, a small
percentage remains. The Soret maximum at 445 nm is observed on all the
time scales we can measure, suggesting that dissociation does not explain
the long-lived charge-separated state. The mutations Y29F and Y96A do
not affect the observed kinetics, indicating that the transiently generated
Ru(bpy)33+ does not oxidize these nearby residues. Tyrosine oxidation by
Figure 6. (A) Cutaway view of the 1.55 Å resolution crystal structure of
[Ru-C9-Ad]2+ bound to P450cam.20 Photochemically generated [Ru-C9-
Ru-diimine complexes has been previously observed: (a) Sjodin, M.;
Styring, S.; A° kermark, B.; Sun, L. C.; Hammarstro¨m, L. J. Am. Chem.
Ad]+ reduces ferric P450cam with a time constant of about 50 µs (-∆G°
Soc. 2000, 122, 3932-3936. (b) Magnuson, A.; Berglund, H.; Korall, P.;
≈ 1.0 eV).6 (B) tmRu-F8bp-Im modeled into the active site of P450cam.
Hammarstro¨m, L.; A° kermark, B.; Styring, S.; Sun, L. C. J. Am. Chem.
The perfluorobiphenyl bridge improves the electronic coupling between
*Ru2+(L2)L′ and the heme, resulting in direct photoreduction with a time
constant of 36 ns even at lower driving force (-∆G° ≈ 0.45 eV).
Soc. 1997, 119, 10720-10725. Several studies have shown that [Ru-
(bpy)3]3+ decays through multiple pathways in aqueous solution: (a) Brune,
S. N.; Bobbitt, D. R. Talanta 1991, 38, 419-424. (b) Ledney, M.; Dutta,
P. K. J. Am. Chem. Soc. 1995, 117, 7687-7695. (c) Ghosh, P. K.;
Brunschwig, B. S.; Chou, M.; Creutz, C.; Sutin, N. J. Am. Chem. Soc.
1984, 106, 4772-4783. (d) Arce-Sagues, J. A.; Gillard, R. D.; Lancashire,
R. J.; Williams, P. A. J. Chem. Soc., Dalton Trans. 1979, 1, 193-198. (e)
Creutz, C.; Sutin, N. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 2858-2862.
The yield of reduced P450 at 5 µs is about 1.7% for tmRu-F8bp-Im and
0.5% for Ru-F8bp-Im, as judged by transient absorption at 445 nm. Thus,
the processes leading to the long-lived ferrous heme constitute a minor
side reaction compared to the initial forward and back electron transfer.
(22) Eisenberg, D.; McLachlan, A. D. Nature 1986, 319, 199-203.
(23) Dunn, A. R.; Dmochowski, I. J.; Bilwes, A. M.; Gray, H. B.; Crane, B. R.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 12420-12425.
cal Å-2. A similar calculation based on the crystal structure of
Ru-C9-Ad bound to P450cam (Figure 6) yields similar binding
energies (∆ isomer: 9.13 kcal mol-1, 8.4 cal Å-2; Λ isomer:
9.69 kcal mol-1, 9.3 cal Å-2).12 The gain in binding for
hydrophobic burial is lower for our complexes than that
observed at protein interfaces. In part this result must reflect
the energetic cost of “opening” the enzyme.23
9
12454 J. AM. CHEM. SOC. VOL. 125, NO. 41, 2003

Photoreduction of Cytochrome P450cam by ET Wires
A R T I C L E S
Table 3. Reduction Potentials
0.13 eV (Table 3). Semiclassical theory predicts a 4-fold increase
in the rate of forward electron transfer, in qualitative agreement
with the ET rates calculated from transient absorbance and
luminescence decay rates (Table 2).
redox couple
potential (V, NHE)
P450cam (Fe3+/2+
[Ru(bpy)3]3+/2+*
)
∼-0.3a
-0.62b
-0.75c
1.26b
[Ru(tmbpy)3]3+/2+*
[Ru(bpy)3]3+/2+
Structural variations in the Ru-wires allowed us to test the
role of the intervening medium on the rate of electron transfer.
Taking into account the differences in Ru-heme distances and
driving forces, a coupling model with a uniform distance decay32
of 1.1 Å-1 and λ ) 0.8 eV27a,30 predicts only 12-fold faster ET
for tmRu-F8bp-Im compared to Ru-C9-Ad, instead of the
1400-fold rate difference that is observed (Figure 6). Similarly,
tmRu-F8bp-Im efficiently reduces P450cam while tmRu-
F9bp does not, despite the similarity in donor-acceptor distances
and driving forces. The saturated bonds in Ru-C9-Ad and the
through-space jump in tmRu-F9bp likely weaken electronic
couplings compared to those associated with the imidazole-
terminated Ru-wires and, hence, greatly slow ET.33 Our results
thus strongly support a through-bond model for coupling the
Ru and heme centers.34
The biological reduction of P450cam by Putd (50 s-1) is
slow for two reasons: the driving force is low and the coupling
to the deeply buried heme is weak. The coupling to the ferriheme
was enhanced in enzyme conjugates containing the first genera-
tion of ruthenium sensitizer-linked substrates, which featured a
direct ET pathway through a saturated alkyl chain. As a
result, ET occurs on a submillisecond time scale (2 × 104 s-1).6
Both theory and experiment indicate that incorporating
aromatic groups into the linker will further enhance the
electronic coupling.37 By employing a more direct, largely
conjugated path, tmRu-F8bp-Im is able to photoreduce P450cam
in nanoseconds (2.8 × 107 s-1), 103 times faster than the Ru-
wire with an alkyl chain linker and 5 × 105 times faster than
putidaredoxin.
[Ru(tmbpy)2(dmbpy)]3+/2+
1.07d
a
b
c
d
Low spin (ref 35). Reference 8. Reference 36. In MeCN vs SSCE
(ref 19).
The imidazole-functionalized complexes weakly ligate the
ferric heme, as tmRu-F8bp-Im binds with only 0.87 kcal mol-1
greater affinity than tmRu-F9bp. The small energetic contribu-
tion of coordination may result from steric effects or poorer
σ-donating ability stemming from the electron-withdrawing
perfluorobiphenyl unit.
These results and previous work14 suggest that designing a
small molecule to bind in a given enzyme active site can be
relatively straightforward. Hydrophobic interactions are nondi-
rectional, predictable, and hence easily engineered: 1000 Å2
of buried surface area should result in a low micromolar
dissociation constant. Of course, this simple strategy does not
include considerations such as target specificity or water
solubility, two important qualities in drug design.
ET Kinetics. According to semiclassical theory, coupling-
limited electron tunneling (kmax) will occur when the driving
force (-∆G°) equals the reorganization energy (λ).24,25 Back
electron transfer in the P450cam:tmRu-F8bp-Im conjugate
(-∆G° ≈ 1.5 eV) should be in the inverted region for λ in the
range 0.7-0.9 eV; the reaction should be 10 (λ ) 0.9 eV) to
5000 (λ ) 0.7 eV) times slower than forward electron transfer.26
The inverted effect has been observed in several biological27
and synthetic ET systems.28 We found, however, that back ET
is 10 times faster than the forward reaction. One possible
explanation is that electron transfer initially produces an
electronically excited product;29,30 another is phonon-modified
inelastic tunneling, which can be activationless in the classical
inverted region.31
Concluding Remarks
Photoreduction of the enzyme by the channel-specific Ru-
imidazole wires occurs on the nanosecond time scale, fully 5
orders of magnitude faster than reduction by the natural redox
partner putidaredoxin. Fast electron injection was only observed
in the imidazole-terminated Ru-wires. However, calculations
based on simple electronic coupling models suggest that
The transient absorption data show that tmRu-F8bp-Im
injects electrons into the ferriheme of P450cam more efficiently
than Ru-F8bp-Im. The methyl groups in tmRu-F8bp-Im
increase the driving force for forward electron transfer by
(32) Langen, R.; Colon, J. L.; Casimiro, D. R.; Karpishin, T. B.; Winkler, J.
R.; Gray, H. B. J. Biol. Inorg. Chem. 1996, 1, 221-225.
(33) Beratan, D. N.; Skourtis, S. S. Curr. Opin. Chem. Biol. 1998, 2, 235-243.
(34) (a) Winkler, J. R. Curr. Opin. Chem. Biol. 2000, 4, 192-198. (b) Onuchic,
J. N.; Beratan, D. N.; Winkler, J. R.; Gray, H. B. Annu. ReV. Biophys.
Biomol. Struct. 1992, 21, 349-377. (c) Beratan, D. N.; Onuchic, J. N.;
Winkler, J. R.; Gray, H. B. Science 1992, 258, 1740-1741.
(35) (a) Gunsalus, I. C.; Meeks, J. R.; Lipscomb, J. D. Ann. N.Y. Acad. Sci.
1973, 212, 107-121. (b) Sligar, S. G.; Gunsalus, I. C. Proc. Natl. Acad.
Sci. U.S.A. 1976, 73, 1078-1082.
(36) Elliott, C. M.; Freitag, R. A.; Blaney, D. D. J. Am. Chem. Soc. 1985, 107,
4647-4655.
(37) (a) Smalley, J. F.; Feldberg, S. W.; Chidsey, C. E. D.; Linford, M. R.;
Newton, M. D.; Liu, Y.-P. J. Phys. Chem. 1995, 99, 13141-13149. (b)
Warman, J. M.; Smit, K. J.; de Haas, M. P.; Jonker, S. A.; Paddon-Row,
M. N.; Oliver, A. M.; Kroon, J.; Oevering, H.; Verhoeven, J. W. J. Phys.
Chem. 1991, 95, 1979-1987. (c) Langen, R.; Chang, I.-J.; Germanas, J.
P.; Richards, J. H.; Winkler, J. R.; Gray, H. B. Science 1995, 268, 1733-
1735. (d) Creager, S.; Yu, C. J.; Bamdad, C.; O’Connor, S.; MacLean, T.;
Lam, E.; Chong, Y.; Olsen, G. T.; Luo, J. Y.; Gozin, M.; Kayyem, J. F. J.
Am. Chem Soc. 1999, 121, 1059-1064. (e) Sikes, H. D.; Smalley, J. F.;
Dudek, S. P.; Cook, A. R.; Newton, M. D.; Chidsey, C. E. D.; Feldberg,
S. W. Science 2001, 291, 1519-1523. (f) Pollard, W. T.; Felts, A. K.;
Friesner, R. A. AdV. Chem. Phys. 1996, 93, 77-134. (g) Davis, W. B.;
Wasielewski, M. R.; Ratner, M. A.; Mujica, V.; Nitzan, A. J. Phys. Chem.
A 1997, 101, 6158-6164. (h) McConnell, H. M. J. Chem. Phys. 1961, 35,
508-515.
(24) Marcus, R. A. J. Chem. Phys. 1956, 24, 966-989.
(25) Marcus, R. A.; Sutin, N. Biochim. Biophys. Acta 1985, 811, 265-322.
(26) We assume that λRu ) 0.57 eV and λheme falls between 0.7 and 1.2 eV
(Winkler, J. R.; Wittung-Stafshede, P.; Leckner, J.; Malmstrom, B. G.; Gray,
H. B. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4246-4249).
(27) (a) Scott, J. R.; Willie, A.; McLean, M.; Stayton, P. S.; Sligar, S. G.;
Durham, B.; Millett, F. J. Am. Chem. Soc. 1993, 115, 6820-6824. (b) Turro,
C.; Zaleski, J. M.; Karabatsos, Y. M.; Nocera, D. G. J. Am. Chem. Soc.
1996, 118, 6060-6067.
(28) (a) Gould, I. R.; Moser, J. E.; Armitage, B.; Farid, S.; Goodman, J. L.;
Herman, M. S. J. Am. Chem. Soc. 1989, 111, 1917-1919. (b) Wasielewski,
M. R.; Niemczyk, M. P.; Svec, W. A.; Pewitt, E. B. J. Am. Chem. Soc.
1985, 107, 1080-1082. (c) Ohno, T.; Yoshimura, A.; Mataga, N. J. Phys.
Chem. 1986, 90, 3295-3297. (d) Chen, P.; Duesing, R.; Tapolsky, G.;
Meyer, T. J. J. Am. Chem. Soc. 1989, 111, 8305-8306. (e) Closs, G. L.;
Miller, J. R. Science 1988, 240, 440-447. (f) Fox, L. S.; Kozik, M.;
Winkler, J. R.; Gray, H. B. Science 1990, 247, 1069-1071.
(29) Beitz, J. V.; Miller, J. R. J. Chem. Phys. 1979, 71, 4579-4595.
(30) (a) Mines, G. A.; Bjerrum, M. J.; Hill, M. G.; Casimiro, D. R.; Chang,
I.-J.; Winkler, J. R.; Gray, H. B. J. Am. Chem. Soc. 1996, 118, 1961-
1965. (b) Makinen, M. W.; Churg, A. K. Structural and analytical aspects
of the electronic spectra of heme proteins. In Iron Porphyrins; Lever, A.
B. P., Gray, H. B., Eds.; Addison-Wesley: Reading, MA, 1983; Vol. 1,
pp 141-236.
(31) Daizadeh, I.; Medvedev, E. S.; Stuchebrukhov, A. A. Proc. Natl. Acad.
Sci. U.S.A. 1997, 94, 3703-3708.
9
J. AM. CHEM. SOC. VOL. 125, NO. 41, 2003 12455

A R T I C L E S
Dunn et al.
improved conjugation could overcome the loss of a direct bond
to the heme.33
Acknowledgment. This work was supported by the Fannie
and John Hertz Foundation (A.R.D.), the National Institutes of
Health predoctoral program (I.J.D.), NSF CHE-0111416, and
NIH DK19038.
Hydroxylation catalyzed by P450cam is only one example
of numerous biological processes, including photosynthesis and
respiration, that involve oxidation and reduction steps. Current
methods for studying enzyme reactions, for instance, stopped-
flow mixing and photocaged substrates, have time resolutions
limited by diffusion. Intramolecular ET in Ru-substrate:enzyme
conjugates dramatically improves the accessible time resolution.
Supporting Information Available: Synthesis and charac-
terization of Ru-diimine complexes (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
JA0294111
9
12456 J. AM. CHEM. SOC. VOL. 125, NO. 41, 2003