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Edited by Jack Halpern, University of Chicago, Chicago, IL and accepted January 28, 2005 (received for review January 5, 2005)
Recent investigations have shed much light on the nuclear and electronic factors that control the rates of long-range electron tunnel-
ing through molecules in aqueous and organic glasses as well as through bonds in donor– bridge–acceptor complexes. Couplings
through covalent and hydrogen bonds are much stronger than those across van der Waals gaps, and these differences in coupling
between bonded and nonbonded atoms account for the dependence of tunneling rates on the structure of the media between re-
dox sites in Ru-modified proteins and protein–protein complexes.
I
ndependent efforts by Gamow (1) tunneling energy gap (⌬), defined as the
in late 1928 and Gurney and Con- vertical energy required to remove an Longer tunneling distances can be exam-
don (2) in early 1929 to explain electron from the donor, or a hole from ined if D and A are immobilized. In a typ-
radioactive decay using the ‘‘new the acceptor, and place it on the bridge. If ical experiment, a small concentration of
quantum mechanics’’ produced an ex- the donor-acceptor separation (d) is a lin- electron or hole donors is embedded in a
pression for the probability that a particle ear function of n (d ⫽ do ⫹ n␦), then glassed solvent amid a higher concentra-
冉 冊
will tunnel through a square potential tion of randomly distributed acceptors.
n⫺1
energy barrier. The transmission coeffi- h Ab h bb The donor is a photoexcited chromophore
cient () decays exponentially with in- HA B ⫽ h bB
⌬ ⌬ or a radiolytically generated radical. The
creasing barrier width (r) and the decay time-dependent survival probability of the
constant varies as the square root of the
product of the barrier height (⌬E) times ⬀ e ⫺冉 ␦冊 ln
1
冉 冊.
⌬⑀
h bb d [3] donor depends on the concentration of
acceptors, the rate constant for electron兾
the mass (m) of the particle (Eq. 1). HAB will decay exponentially with dis- hole transfer when D and A are in van der
⫺共2兾 –h兲 冑2m⌬Er tance (hbb ⬍⬍ ⌬; Eq. 3). Using the result Waals contact (ko), and the distance de-
⬀e . [1] from semiclassical theory that ET rates cay factor . Extracting reliable values for
are proportional to HAB 2
(Eq. 2), it is pos- ko and  from time-resolved spectroscopic
This theory rationalized the microsec- measurements, however, can be rather
ond tunneling of an ␣-particle through a sible to define effective tunneling barriers
(⌬Eeff) in terms of superexchange param- difficult because the two parameters are
3 ⫻ 10⫺4-Å, 6-MeV barrier (2) and pre-
eters, as well as the exponential decay highly correlated (19, 22). In the case of
dicted that in the same time period an
electron could tunnel 19 Å through a constant () describing the variation of photoinitiated ET in glasses, measure-
rates with distance (Eq. 4). ments of luminescence decay kinetics and
1-eV barrier. In the intervening years,
luminescence quantum yields at several
冉 冊冋冉 冊 冉 冊册
electron tunneling has been found to –h2 2
play pivotal roles in solid-state physics 2 ⌬ different quencher concentrations provide
⌬Eeff ⫽ ln enough information to decouple ko and ,
(3), chemistry (4, 5), and biology (5–7). 8m e ␦ h bb
permitting reliable values to be deter-
冉 冊
A Landau-Zener treatment of the re-
actant-product transition probability –h2 mined for each parameter (19).
produces the familiar semiclassical ex- ⫽ 2 Our experimental investigation of
8m e Ru(tpy)2⫹
pression for the rate of nonadiabatic 2 luminescence quenching by
Fe(OH2)3⫹ 6 in aqueous acidic glasses
electron transfer (ET) between a donor ⫽ 共0.952 eV A2兲  2. [4]
(D) and acceptor (A) held at fixed dis- placed rigorous limits on the distance de-
tance (Eq. 2) (5). Saturated hydrocarbon spacers typically cay constant for tunneling through water
exhibit  ⬇1.0 Å⫺1 (⌬Eeff ⫽ 0.95 eV) (23). The luminescence lifetime of
kET ⫽ 冑 43
H2
h RT AB
2
(10–12). The decay constant for phe-
nylene bridges depends on the dihedral
angle between adjacent aromatic rings
*Ru(tpy)2⫹2 in aqueous glasses is long
enough to allow a significant distance
range (⬇25 Å) to be probed. A distance
䡠exp ⫺再 共⌬G⬚ ⫹ 兲 2
4 RT
. 冎 [2]
[0.4–0.8 Å⫺1 (13, 14); ⌬Eeff ⫽ 0.15–0.61
eV]. Polyene and phenylenevinylene
bridges exhibit remarkably efficient ET
decay constant of 1.58(5) Å⫺1 was ob-
tained for H2SO4兾H2O, HSO3F兾H2O, and
D2SO4兾D2O glasses (⌬Eeff ⫽ 2.4 eV) (14).
The electronic coupling matrix element over very long distances: -values as We also have determined  and ⌬Eeff
(HAB) reflects the strength of the interac- low as 0.04 Å⫺1 (⌬Eeff ⫽ 0.002 eV) have values for electron tunneling from
tion between reactants and products at been reported (15). electronically excited [Ir(-pyrazolyl)(1,5-
the nuclear configuration of the transition cyclooctadiene)]2 to 2,6-dichloro-1,4-
state. McConnell (8), building on prior Aqueous and Organic Glasses
work by Halpern and Orgel (9), argued Several experimental investigations have
This paper was submitted directly (Track II) to the PNAS
that the interaction energy (2HAB) be- demonstrated that solvent hole and elec- office.
tween two redox centers separated by a tron states can mediate long-range elec-
Abbreviations: ET, electron transfer; cyt, cytochrome; ccp,
covalent bridge composed of n identical tron tunneling (16–21). In fluid solution, cyt c peroxidase; MTHF, 2-methyltetrahydrofuran; Hb,
repeat units depends on the coupling when the positions of D and A are not hemoglobin.
strength between the redox sites and the constrained by a covalent bridge, diffusion †Towhom correspondence should be addressed. E-mail:
bridge (hAb, hbB), the coupling between places an upper limit on the time scale hbgray@caltech.edu.
adjacent bridge elements (hbb), and the (⬍10⫺9 s) and, therefore, the tunneling © 2005 by The National Academy of Sciences of the USA
Gray and Winkler PNAS 兩 March 8, 2005 兩 vol. 102 兩 no. 10 兩 3535
smaller subunits linked by covalent bonds, time for protein ET (具典) is on the order
hydrogen bonds, or through-space jumps. of 200 ns. We emphasize, however, that
More elaborate computational protocols eight tunneling times measured for four
also have shed light on the factors that different Ru-proteins are ⬍200 ns
determine distant couplings in proteins (Fig. 3).
(42–46). Indeed, in this issue of PNAS, Bixon and Jortner (53, 54) have noted
Beratan and coworkers (47) present a de- that there are several examples of ET
tailed analysis, including the effects of rates exceeding the solvent-controlled adi-
protein dynamics on the distant couplings abatic limit; model calculations suggest a
of six Ru-azurins (Fig. 2). possible explanation. Reactions at low
driving force (⫺⌬G° ⬍ ) require sub-
Medium Dynamics stantial reorganization along solvent coor-
The nonadiabatic ET model embodied in dinates and rates are predicted to exhibit
Eq. 2 rests on the assumption that the a pronounced dependence on relaxation
electronic transition from the reactant dynamics. The calculations suggest, how-
potential energy surface (D ⫹ A) to the ever, that the rates of activationless
product surface (D⫹ ⫹ A⫺) is much (⫺⌬G° ⬇ ) and inverted (⫺⌬G° ⬎ )
slower than the frequency of nuclear mo- reactions will be nearly independent of
tion on these surfaces. Both theoreticians and, hence, the dynamics of medium re-
and experimentalists have long been inter- laxation, as we have found in the case of
Fig. 3. Tunneling timetable for intraprotein ET in ested in charge-transfer processes that are Ru(diimine)-protein ET reactions (7).
Ru-modified azurin (blue circles), cyt c (red circles), not well described by this model (48–54).
myoglobin (yellow triangles), cyt b562 (green Protein–Protein Reactions
squares), HiPIP (orange diamonds), and for inter- NA At least three elementary steps are re-
protein ET Fe:Zn-cyt c crystals (fuchsia triangles). ⫽ [5] quired to complete a redox reaction be-
The solid lines illustrate the tunneling-pathway
1⫹
tween soluble proteins: (i) formation of an
predictions for coupling along -strands ( ⫽ 1.0
Å⫺1) and ␣-helices ( ⫽ 1.3 Å⫺1); the dashed line 4HA2
B具 典
active donor–acceptor complex; (ii) elec-
⫽ . [6] tron tunneling within the donor–acceptor
illustrates a 1.1-Å⫺1 distance decay. Distance decay –h
for electron tunneling through water is shown as a
o complex; and (iii) dissociation of the oxi-
cyan wedge. Estimated distance dependence for dized and reduced products. Because the
Theory suggests that, under certain cir-
tunneling through vacuum is shown as the black dynamics of the first and third steps ob-
cumstances, the time scales for reorienta-
wedge. scure the electron tunneling reaction,
tion of solvent molecules can be slower experimental studies often focus on ET
than the reactant-product transition fre- reactions within protein–protein com-
ated holes in Ru(bpy)2(im)(HisX)3⫹ com- quency. In this solvent-controlled adia- plexes that form at low ionic strength. It
plexes remain localized on the metal batic limit, reactions are limited by the has been difficult to interpret the results,
center. The energy gap between the dynamics of solvent relaxation. Bixon and however, as neither the donor–acceptor
Ru(III) hole and oxidized bridge states Jortner (53, 54) developed a generalized docking geometries nor the conformations
therefore must be ⬎75 meV (3 kBT at expression for ET rates that explicitly ac- of these complexes are known with cer-
295 K). Our finding that the Cu(I) 3 counts for solvent relaxation dynamics tainty. With the aid of rapid triggering
Ru(III) ET rate in Ru(bpy)2(im)(HisX)- (Eqs. 5 and 6). The factor is a solvent methods, it has been possible to measure
azurin does not decrease in going from adiabaticity factor and kNA is the nonadia- rates of long-range ET between redox
300 to 240 K and actually increases batic ET rate given by Eq. 2. Small values sites in protein–protein complexes. In
slightly at 160 K demonstrates that hop- of correspond to the nonadiabatic limit; many complexes, there are multiple bind-
ping does not occur in this case, as a reac- large values result in solvent-controlled ing sites and it is not uncommon to find
tion with an endergonic step would be adiabatic processes. Typical solvent relax- that the ET kinetics often are regulated
highly disfavored at low temperature. We ation times (具典) are ⱕ10⫺11 s, so that sol- by the dynamics of conformational
conclude that the Ru-azurin timetable vent relaxation dynamics are expected to changes in the complex (60, 61). The
(Fig. 1) provides a benchmark for super- become important only in relatively usual interpretation is that surface diffu-
exchange-mediated electron tunneling strongly coupled systems. An adiabaticity sion of the two proteins produces a tran-
through proteins. factor of 1 in a solvent with a 1-ps relax- sient complex with enhanced electronic
The rates of high driving-force ET reac- ation time, for example, corresponds to a coupling and faster ET. Consequently,
tions have been measured for ⬎30 Ru(dii- coupling matrix element of ⬇50 cm⫺1 rates depend strongly on solvent viscosity
mine)-labeled metalloproteins (7, 29, 30, (o ⫽ 0.5 eV). rather than intrinsic ET parameters. A
38). Driving-force-optimized values are Our studies of ET in Ru-proteins indi- further complication associated with stud-
scattered around the Ru-azurin 1.1-Å⫺1 cate that a large part of the contribution ies of protein–protein ET in solution is
exponential distance decay. Rates at a to o comes from reorientation of the that binding sites and, hence, locations of
single distance can differ by as much as a polypeptide matrix (34). The dynamics of redox cofactors, often are unknown.
factor of 103, and D兾A distances that large-scale nuclear motions in polypep- Crystals containing photoactivatable
differ by as much as 5 Å can produce tides are expected to be substantially donors and acceptors at specific lattice
identical rates (Fig. 3). In seminal work, slower than those of most solvents. Relax- sites are ideal media for investigating tun-
Beratan, Onuchic, and coworkers (39–41) ation times ranging from picoseconds to neling between proteins. In crystal lattices
developed a generalization of the McCon- microseconds have been reported for the of tuna cyt c, chains of protein molecules
nell superexchange coupling model that heme pocket of myoglobin (55–58). In- form helices with a 24.1-Å separation be-
accounts for rate scatter attributable to deed, electrochemical measurements by tween neighboring metal centers (62).
protein structural complexity. In this tun- Waldeck and coworkers (59) using cyt c By doping Zn-cyt c into this lattice, inter-
neling-pathway model, the medium be- adsorbed onto self-assembled monolayers protein ET between triplet-excited Zn-
tween D and A is decomposed into suggest that the characteristic relaxation porphyrin and a neighboring Fe(III)-cyt c
Gray and Winkler PNAS 兩 March 8, 2005 兩 vol. 102 兩 no. 10 兩 3537
ping rates for any set of driving-force,
temperature, and distance parameters.
Consider the two-step tunneling reaction
defined in Eq. 7 (reactants, R ⫽ D-I-A;
redox intermediate, H ⫽ D⫹-I⫺-A or
D-I⫹-A⫺; products, P ⫽ D⫹-I-A⫺) The
general solution to the rate law for this
process calls for biexponential production
of P, although under some circumstances
the appearance of P can be approximated
by a single exponential function. Taking a
value of ⫽ 0.8 eV for both tunneling
reactions (i.e., R 3 H and H 3 P) and a
distance decay constant of 1.1 Å⫺1, we
can calculate the time dependence of the
populations of all three reacting species
for various values of ⌬GRH ° , ⌬GHP
° , rRH,
and rHP (7). Results for the particular case
in which ⌬GRH ° ⫽ ⫺⌬GHP ° and rRH ⫽ rHP
are illustrated in Fig. 5. This model ap-
proximates a biological electron-transport
Fig. 4. Tunneling-time (1兾kobs) contours as functions of donor-acceptor distance ( ⫽ 1.1 Å⫺1) and driving
chain (⌬GRP ° ⫽ 0) reaction with a single
force [in units of ; kBT兾 ⫽ kB(295 K)兾(0.8 eV) ⫽ 0.318].
endergonic step. Transport across 20 Å is
104 times faster than a single tunneling
Based on a tunneling currents analysis, cases deliver electrons or holes rapidly step at this distance and submillisecond
Stuchebrukhov and colleagues (96) sug- to very distant sites (7, 30, 99). Require- transfers can be realized. For D-A separa-
gested a slightly different CuA-to-cyt a ments for functional hopping include tions ⬍20 Å, endergonic intermediate
coupling route through His-204. It is likely optimal positioning of redox centers and steps as large as 0.4–0.5 eV will afford
that, owing to strong Cu-S(Cys) electronic fine-tuning of reaction driving forces. submillisecond transport times. An impor-
interactions, pathways involving the bridg- Modeling the kinetics of electron hop- tant conclusion is that hopping can facili-
ing Cys residues are important for mediat- ping is a straightforward problem that tate electron flow over distances ⬎20 Å in
ing coupling even though they involve can be solved analytically without using cases where the free-energy changes for
more bonds than the His-204 route. The simplifying approximations (7). endergonic intermediate steps are no
current view is that the sequence Cys-200兾 more than 0.2 eV.
Ile-199兾Arg-439兾heme-propionate (cyt a) k RH k HP
is the dominant CuA to cyt a electron tun- -0 H |
R| -0 P [7] Concluding Remarks
neling pathway (92, 96). k HR k PH More than 75 years have passed since the
Both Regan et al. (94) and Stuchebruk- Gamow (1) and Gurney-Condon (2) pa-
hov and colleagues (96) have identified Using the well defined properties of ET pers appeared. Activity in the electron
pathways between cyt a and cyt a3. In- reactions (Eq. 2), and the average dis- tunneling field over the last 20 years has
cluded among these routes is a direct tance dependence defined by Ru-protein been intense, most especially on the ex-
covalent pathway from the heme-a axial tunneling timetables, we can predict hop- perimental side, where investigators have
ligand His-378 through Phe-377 to the a3
His-376. Importantly, although the CuA-
cyt a3 distance (22.4 Å) is similar to that
of CuA to cyt a, neither Regan et al. (94)
nor Stuchebrukhov and colleagues (96)
found a coupling pathway that would fa-
cilitate electron flow to a3 in a single step
from the binuclear copper center.
Hopping
Electron tunneling times must be in the
millisecond to microsecond range for bio-
logical redox machines to function properly.
As a result, the maximum center-to-center
distance for single-step tunneling through
proteins can be no more than ⬇20 Å (Fig.
4). The structures of several redox enzyme
assemblies, however, suggest that charge
transport may occur over distances that
far exceed this single-step limit (7, 97, 98).
How can charge transport in proteins
cover distances well over 20 Å? One pos- Fig. 5. Distance dependences of the rates of single-step and two-step electron tunneling reactions. Solid
sibility is by hopping, as it can be shown line indicates theoretical distance dependence for a single-step, ergoneutral (⌬GRP ⫽ 0) tunneling process
that coupled tunneling reactions, particularly ( ⫽ 1.1 Å⫺1). Dashed lines indicate distance dependence calculated for two-step ergoneutral tunneling
with endergonic steps, can in favorable (R%H%P) with the indicated free-energy changes for the R%H step.
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