Biochimica et Biophysica Acta 1797 (2010) 1573–1586

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Biochimica et Biophysica Acta

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a b i o

Review

Guidelines for tunneling in enzymes

Christopher C. Moser ⁎, J.L. Ross Anderson, P. Leslie Dutton
Dept. Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA

a r t i c l e i n f o a b s t r a c t

Article history: Here we extend the engineering descriptions of simple, single-electron-tunneling chains common in
Received 3 December 2009 oxidoreductases to quantify sequential oxidation–reduction rates of two-or-more electron cofactors and
Received in revised form 26 April 2010 substrates. We identify when nicotinamides may be vulnerable to radical mediated oxidation–reduction and
Accepted 28 April 2010
merge electron-tunneling expressions with the chemical rate expressions of Eyring. The work provides
Available online 10 May 2010
guidelines for the construction of new artificial oxidoreductases inspired by Nature but adopting
Keywords:
independent design and redox engineering.
Electron tunneling © 2010 Elsevier B.V. All rights reserved.
Hydride transfer
Marcus theory
Protein engineering

1. Introduction We also describe the important interplay between distance and

driving force in the design and engineering of natural oxidoreduc-
To understand the engineering of oxidoreductases and so gain insight tases when an overall exergonic series of electron transfers includes
into the proper function and sources of malfunction of natural enzymes, as one or more endergonic (uphill) steps. To some extent the increased
well as uncover guidelines for the construction of new oxidoreductase height of an uphill step can be compensated for by shrinking the
designs with desirable novel properties [1], we must consider the role of distance between donor and acceptor while still maintaining
tunneling. In the natural oxidoreductases of photosynthesis, respiration electron tunneling rates within the approximately millisecond
and metabolism, electrons commonly get from one place to another by physiologically acceptable threshold. In extended single-electron
tunneling one at a time through the insulating protein medium that transfer chains, these uphill tunneling steps may play a regulatory
separates one redox center from another. These redox centers typically role, so that overall electron transfer through the chain accelerates
link up to form chains within the enzyme that connect sites for binding only after the chain has accumulated a threshold supply of electrons.
diffusible substrates or other redox proteins. Simple electron transfer In oxidoreductases with multi-electron redox cofactors or substrates,
theory applied to the photosynthetic reactions inspired the development such as quinone, flavin, pterin or nicotinamide, oxidation and
of a practical semi-empirical set of electron tunneling rate expressions reduction may take place by sequential single-electron tunneling
that predicted tunneling rates within an order of magnitude [2,3]. The with a significantly uphill single electron transfer to a radical state.
parameters of these expressions continue to be examined experimentally The balance between tunneling distance and uphill driving force
and theoretically [4–8]. There is considerable debate as to what theoretical sketches an unexpectedly broad engineering choice in distances and
descriptions are best or useful, and what level of molecular detail must be driving forces to achieve acceptable nanosecond to milliseconds
understood to appreciate how natural selection has shaped the design of electron transfer rates.
oxidoreductases over the history of evolution. Despite the abundance of When electron-tunneling reactions are coupled to subsequent bond
models and parameters used to describe intraprotein electron tunneling, breaking/forming catalysis, the tunneling expressions can substitute for
and the intriguing suggestions that Nature might have finely tuned atomic the transmission coefficient κe of Eyring's absolute reaction rate
level redox protein structure, dynamics and quantum interference effects expressions using classic transition state theory [14,15]. We describe
[4,5,9–13], we find that the engineering of single electron transfer how to use the empirical electron tunneling expressions to identify the
reactions can be largely understood in terms of natural selection of just boundaries where sequential electron-transfer mechanisms become
two principle parameters: tunneling distances and driving forces. impossibly slow for physiological purposes. When distances are already
short and an initial uphill single-electron step is too high, as is often the
case for common oxidoreductases using nicotinamides as substrates, a
⁎ Corresponding author. Tel.: + 1 215 573 3909; fax: + 1 215 573 2235. sequential two electron transfer mechanism must yield to proton
E-mail address: [email protected] (C.C. Moser). mediated concerted two-electron, hydride transfer over van der Waals

0005-2728/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbabio.2010.04.441
1574 C.C. Moser et al. / Biochimica et Biophysica Acta 1797 (2010) 1573–1586

distances or less. The geometric constraints on these reactions and enzymatic reactions [22,23]. For exergonic electron tunneling
engineering challenges are much more severe so that significant protein (Eq. (2)), the rate is also exponentially dependent on the uphill
mobility can become essential to achieve appropriate geometries. Because driving force, which can easily slow a reaction so that it falls outside
the proton is much more massive than an electron, proton tunneling, if it is the physiological threshold. Distance can partly offset the penalty of
to have a noticeable effect, must employ much shorter donor/acceptor uphill driving force. If the electron tunneling takes place at the short
distances than the natural 4 to 14 Å range used for electron tunneling, end of the distance range, then electron tunneling rates can be faster
namely around 1 to 2 Å [16,17]. than milliseconds even when the driving force is several hundred meV
uphill. This article explores how these two parameters, distance and
2. Empirical electron tunneling rate expressions driving force, are selected to shape the design of oxidoreductase
proteins found in nature.
By virtue of their experimentally facile light activation, photosynthetic These rate expressions are intentionally simple and grounded in
reaction centers provided an early and accessible system for the study of empirical observations with the intent of providing insight into the
electron tunneling in proteins. DeVault and Chance [18] showed that the design and operation of a wide spectrum of natural proteins as well as
charge separation reactions of photosynthesis continued at cryogenic engineering guidelines for the construction of novel electron transfer
temperature and displayed the temperature independence that is a proteins. Nevertheless, the form of the expressions is related to electron
hallmark of electron tunneling. These observations spurred a host of tunneling theory. In our simplification of theory, we follow the advice of
theoretical descriptions, including that of Hopfield [19]. Bacterial reaction Hopfield: “From an experimental point of view, the essential thing about
centers not only provided access to a range of electron tunneling reactions trying to do reasonable science in these [biological] systems is to know
at different temperatures, but also proved amenable to manipulation of what facts to suppress. From the theoretical point of view, it is knowing
driving force by cofactor replacement and mutagenesis [20,21]. Hopfield's what theoretical refinements to ignore” [24]. The following sections
relatively simple quantum version of driving force dependent Marcus address the theoretical significance of the parameters and coefficients of
electron transfer theory provided a simple framework that could Eqs. (1) and (2).
encompass these observations at different distances, driving forces and
temperatures in a single set of semi-empirical expressions [2,3]. For 2.1. The importance of proximity
downhill, exergonic electron tunneling:
Although it is commonly believed that over many millions of years
ex 2 natural selection will tend to speed and optimize electron transfers
log10 ket = 13−0:6ðR−3:6Þ−3:1ðΔG + λÞ = λ ð1Þ
within a protein, we find that this is generally not the case. Electron
transfer speeds are selected to be merely fast enough. There are many
The corresponding expression for the reverse, endergonic electron examples of entirely adequate electron transfer chains that do not have
transfer is a factor of ten smaller for every 0.06 eV uphill, maintaining the smooth progression of redox midpoint potential that would support
a Boltzmann ratio between forward and reverse rates: the fastest possible individual electron transfer steps. Instead they
include a series of up and downhill reactions that are nevertheless faster
en 2
log10 ket = 13−0:6ðR−3:6Þ−3:1ð−ΔG + λÞ = λ−ΔG = 0:06 ð2Þ than the overall turnover of the enzyme because chain elements are
placed close enough together [3,25]. This sort of design provides some
In each of these expressions, the tunneling rate, ket is given in units robustness against random mutations and protein changes over time.
of s−1, the edge-to-edge distance between redox centers, R, in Å, 3.6 Å Furthermore, because the electron can tunnel in all directions, and
being van der Waals contact, the driving force, ΔG, and the because the barrier to electron tunneling in natural proteins is more or
reorganization energy, λ, in eV. Both equations express an exponential less generic in all directions, the engineering of where a tunneling
dependence of electron transfer rate on distance, the principle electron travels is primarily set by which acceptor is closest, provided
constraint on natural electron transfer protein design. Almost all that the driving force is favorable. Even with unfavorable driving force, if
productive electron transfer reactions in biology fall within the the acceptor is near and the driving force is subsequently favorable for
distance range of 4 to 14 Å (Fig. 1A) [2,3], which places these reactions stepwise electron transfer to some other center (i.e. uphill then
within the approximately millisecond threshold required to support downhill electron transfer) the tunneling electron can be drawn in
the physiologically relevant catalytic rates found in majority of this direction. This leads to the real engineering problem of avoiding

Fig. 1. (A) Edge-to-edge distances for natural single-electron transfers are nearly evenly distributed over 4 to 14 Å. (B) Short distances dominate separations between NADH/NADPH
and substrate. (C) In protein crystal structures with both nicotinamide and flavin, most distances are equally short, but some flavins are more remote from the nicotinamide.
 C.C. Moser et al. / Biochimica et Biophysica Acta 1797 (2010) 1573–1586 1575

disadvantageous electron transfer to unwanted acceptors, which diverts

the reducing power of the electron, wastes energy in short-circuit
reactions, or produces destructive chemically reactive radical states.
It appears that natural proteins deal with these dangers by burying
most redox centers sufficiently far inside the electrically insulating
protein medium that adventitious electron transfers to unwanted redox
partners are distant and slow. This is especially important for clusters of
multi-electron centers near catalytic sites, since there are many more
potential two-electron reactants in the cell than one-electron reactants,
and two-electron transfer can be fast even if the first single electron
transfer is uphill. Burial of natural redox centers in insulating protein to
keep them away from most substrates helps achieve redox selectivity.
However, there are some proteins that by design include clusters of
large numbers of redox centers, such as the periplasmic proteins that
include 16 or more c type hemes [26]. We expect that these proteins can
Fig. 2. Log of free energy optimized rates as a function of edge-to-edge distance in
promiscuously catalyze the oxidation or reduction of a large range of natural and modified electron transfer proteins [2,10,32,37–62]. Symbols and distance
reactants that can approach within a ∼10 Å range of these centers. definitions are as described in text.
Perhaps these designs are tolerated in the periplasmic space, where
unspecific electron transfer is less of a danger than in the interior of
the cell.
The Qo quinone oxidation site of Complex III in respiration resolve empirically because of the limited number of examples of
represents a special distance engineering challenge in that produc- different orientations between cofactors in resolved crystal structures
tive energy conservation requires the two electrons of the quinone for which electron tunneling rates and driving forces have been
to be delivered to two different and diverging redox chains. The measured. In the absence of an extensive data set, our practical
nearest elements of the two chains must be close enough to the definition of the edge of a cofactor includes all atoms making up the
quinone substrate to allow sub-millisecond electron tunneling, but aromatic/conjugated systems of porphyrins, chlorins, flavins, pterins,
far enough apart to avoid direct short circuit electron transfer from amino acid radicals and quinones, including oxygens attached to the
the low to high potential chain. Natural selection has found a quinone rings. With non-conjugated metal containing redox centers,
solution by placing the nearest components of the two chains about we include within the definition of the cofactor edge both the metal
23 Å from one another, which slows the innate short-circuit time to atoms and the atoms directly ligated to two or more metal atoms.
many seconds. When the reduced quinone substrate arrives at the Photosynthetic bacteria provide an example of how a relatively
site nearly in line between these two centers, and binds in such a subtle and fine-scale empirical determination of the appropriate “edge”
way that proton transfer can be appropriately managed, then two of a cofactor might be performed. Rb. sphaeroides has a type-a BChl
smaller electron-transfer gaps of less than ∼ 10 Å are created. where the outer carbons of ring II and IV are not conjugated, while the
Electron transfer at these smaller distances can proceed faster than carbonyl of ring V appears to have some conjugated quality [28]. Blc.
the millisecond physiological turnover window. Yet more engineer- viridis has a type-b BChl with an ethylidene group on ring II that makes
ing seems to be required, however, to avoid short-circuits that this ring conjugated. Support for the view that the conjugated atoms do
involves unproductive electron transfer that exploits a semiquinone appropriately define the cofactor edge comes from the observation that
intermediate state in between the two chains. It appears to be by the rate of electron transfer is slower in BChl a containing Rb.
design that the semiquinone at Qo exists at unusually low levels due sphaeroides. While the distances between the non-conjugated atoms
to the protein environment conferring an unusually small quinone of heme c and BChl in Rb. sphaeroides and Blc. viridis photosynthetic
stability constant [27]. reaction centers are the same, the conjugated rings are 1.9 Å further
apart in the slower BChl a containing species [29].
2.2. Meaning of distance R Further support for this edge definition comes from employing
Eq. (1) to estimate the electron tunneling rate of heme to BChl electron
Distance between redox cofactors is clearly the most important transfer using the crystal distance of the reaction center/cytochrome c2
parameter that sets the order of magnitude for the rate of electron complex and the reorganization energy of 500 meV derived by changing
tunneling in proteins. There are two popular means to measure this the driving force through a series of mutations in the BChl dimer
distance: edge-to-edge or center-to-center. For redox centers contain- environment [21]. The calculated rate is within a factor of 2 of that seen
ing metals, where the bulk of the change in electron density upon in crystals [30], supporting the edge estimate as appropriate.
oxidation/reduction is frequently concentrated at the metal, the center- A complication arises when the reorganization energy is estimated
to-center distance may be the same as the metal-to-metal distance. For not from the direct driving force variation but from the experimentally
metal-free cofactors the edge can be estimated in terms of the volume easier temperature variation of the reaction rate [31]. It is often assumed
that includes the large majority of the wave function associated with the that a classical, non-quantum Marcus theory description of the
tunneling electron. Beyond this perimeter, the wave function tails off activation energy is appropriate. Under this assumption, the resulting
roughly exponentially and of course forms the basis for the overall almost two-fold larger reorganization energy leads to estimated optimal
exponential decrease in electron tunneling rate with distance seen, for rates more than an order of magnitude slower, which would argue for an
example, in Fig. 2. edge definition almost 2 Å smaller. However, as described below, the
The empirical resolution of this edge is best achieved by comparing quantum theory behind Eq. (1) leads to a more modest temperature
the electron transfer reactions between similar cofactors in many dependency than classical Marcus theory or a smaller reorganization
different proteins with different electron transfer distances and energy. Using driving force estimates of reorganization energy rather
geometries. The conjugated macrocycle seems the appropriate edge than temperature dependence estimates avoids this classic/quantum
for metal-free photosynthetic cofactors of pheophytins and bacter- mismatch problem.
iopheophytins as well as the Mg containing chlorophylls and There are two opinions as to the best distance metric for non-
bacteriochlorophylls. Whether or not any substituent on the perim- photosynthetic porphyrins such as hemes: either the conjugated ring
eter of the ring should be included within this edge is harder to extending around the macrocycle [2,19,32,33] or the central iron at
1576 C.C. Moser et al. / Biochimica et Biophysica Acta 1797 (2010) 1573–1586

which the redox orbitals are concentrated [13,34]. The direct electron 1013 s−1 at van der Waals contact, which matches the Eyring pre-
tunneling distance measured from the conjugate heme macrocycle edge exponential kBT/h value in absolute reaction rate theory [14,15].
can be up to 4.8 Å longer than that measured from the heme center. In a
†
typical protein, this distance change amounts to nearly a thousand-fold ket = ðκ kB T = hÞ Expð−ΔG = kB TÞ ð3Þ
change in the expected tunneling rate. Pathway calculations that
estimate the decay of the wave function along a series of covalent, In this equation, κ is the transmission coefficient, the Boltzmann/
hydrogen bonded and through space connections between donor and Planck term kBT/h is about 1013 s−1, and ΔG† is the free energy of
acceptor have used both edge definitions, using either the iron [35] or activation. This frequency corresponds to the time of a typical
the conjugated rings of the heme [36] as the pathway termini. vibrational motion of nuclei that distort reactant geometries into
We find that for natural electron transfer reactions, the conjugated product geometries and may be viewed as the frequency at which
heme edge gives a distance metric consistent with observed rates using typical chemical systems approach the activation energy barrier. It is
Eq. (1). In the photosynthetic cases the heme edge orients towards the also close to a typical time in which two molecules approaching one
BChls so there is a significant difference in distance estimates between the another are in near contact of less than ∼0.5 Å and can be viewed as the
conjugated edge vs. central metal atom. The rather spare set of natural approximate lifetime of a transition state activated complex. In an
heme electron transfer reactions in which tunneling rates, crystal activationless system, when the electron transfer reaction has no
structure distances and estimates of reorganization energies are all activation energy due to electron tunneling (because the driving force
available is enhanced by using heme proteins that have been chemically matches the reorganization energy so that κ in Eq. (3) or κe in Eyring's
modified by attaching a light-activated ruthenium label. Here again it notation is ∼1), and when there is also no chemical reaction barrier to
appears that Eq. (1) gives rates consistent with observation when using be crossed, it seems plausible that the reaction would proceed at the
the ruthenium to heme conjugated edge distance, especially when taking 1013 s−1 vibrational rate as described by Eyring [14]. If a metal-to-metal
into account the mobility of certain of these ruthenium labels [32,37]. distance metric is used instead [10,13], the intercept at contact distance
Fig. 2 shows that the free energy optimized rates for electron tunneling for becomes orders of magnitude larger and this simple interpretation no
myoglobin (circles) [10,38,39], cytochrome c (diamonds) [37,38,40–46], longer applies. Some pathway analyses adjust down the large intercept,
cytochrome b5 (star) [47,48] and cytochrome b562 (squares) [37] fall in partly to compensate for the tendency of pathway methods to
line with analogous rates found in the photosynthetic reaction centers overestimate through space jump decays [36].
(open circles) [2] when using a heme conjugated edge definition. This is
the case even when the ruthenium center geometry places it either 2.4. Significance of 0.6 coefficient of exponential distance dependence
approximately in the same plane as the heme, where the metal to
conjugated edge distance is much different from the metal to metal The empirical electron tunneling rate expression of Eqs. (1) and (2)
distance, or approximately normal to the plane, where there metal to follows the exponential decay of the tunneling rate with distance
metal and metal to conjugated edge distances are similar. expected for a protein medium that presents a typical insulating barrier
In contrast, in a pathway perspective using the center-to-center Ru for electron tunneling. It does not appear that many, if any,
to Fe metric, the same data set is interpreted to reveal that rates are physiologically relevant intraprotein electron transfers adjust the
faster or slower for a given metal-to-metal distance when approaching protein medium to modulate the height of the electron tunneling
the heme edge on vs. face [13]. From the metal-centered point of view, barrier, for example by significantly imposing gaps and spaces between
coupling in the heme face direction appears better. A possible amino acids to slow down electron tunneling, or by increasing the
explanation is that face-on pathways have a single dominant pathway bonded quality of the amino acid medium to speed electron transfer.
that has opportunity to contribute much better or much worse than Non-biological synthetic media show these barrier modulations [2], but
average coupling, while the edge-on direction offers multiple interfering in both natural productive charge separating reactions and unproduc-
paths that average out the coupling [13]. It could be a coincidence that tive charge recombination or short-circuit reactions the intervening
the face-on examples in Fig. 2 each provide better than average protein medium seems to have similar insulating properties. There are
coupling. It remains to be seen if the pathway interference explanation variations in the bonded quality and packing (ρ) of protein medium
receives experimental support by the discovery of a face-on pathway between natural redox cofactors (Fig. 3) [3]. The average density within
with unusually poor coupling for the metal-to-metal distance. The the united atom radius is about 0.76 with a standard deviation of about
simple edge-to-edge perspective suggests no such example will be 0.10, which might be expected to contribute to lowering or raising the
found, as the tunneling distance will always be shorter and the rate tunneling barrier. When atoms are omitted from published PDB x-ray
faster for a given metal-to-metal distance when the heme edge turns crystal structures, large enough voids are filled with one or more water
towards the electron transfer partner. molecules for the sake of calculation. If the volume between cofactors
Turning to other examples, Fig. 2 includes natural heme within the atomic radius corresponds to a barrier similar to covalently
reactions with more poorly defined reorganization energies and linked redox centers with a β (base e exponential decay) of 0.9 Å−1
consequently larger uncertainties in the free energy optimized rate and the volume outside this radius to a vacuum-like barrier with a β of
(crosses) [49–59]. Despite the larger uncertainty in tunneling 2.8 Å−1, then at the risk of ignoring Hopfield's advice and introducing
parameters, once again the heme conjugated edge metric seems another parameter that complicates the analysis, the 0.6 average
appropriate. Fig. 2 also includes the modified non-heme proteins coefficient can be replaced with the term (1.2–0.8ρ):
with copper centers (triangles) [10,60,61] and iron-sulfur centers
ex 2
(hexagons) [62] where the preferred distance metric includes only log10 ket = 13−ð1:2−0:8ρÞðR−3:6Þ−3:1ðΔG + λÞ = λ ð4Þ
the metal centers and atoms between two or more metal centers.
Rate estimates are better if metal ligands that are only bonded to a For the clear majority of proteins, it appears that the packing method
single metal are not included, although the issue is not yet entirely makes similar adjustments in the expected rate of electron tunneling as
resolved as the differences between the distance metrics are alternate methods, such as the pathways method [4]; better or worse
relatively small compared to the experimental scatter. than average pathway connectivity correlates well with analogous
deviations from average packing density. Fig. 3 presents a detailed look
2.3. Significance of 1013 s−1 at van der Waals contact at the effect of variable packing between redox centers in natural and
modified proteins. In total, the effects of packing on natural electron
Using the edge-to-edge distances defined above, the collection of free transfer reactions are smaller than the uncertainties in the measure-
energy optimized electron tunneling rates in natural proteins approaches ments of rates and tunneling parameters such as reorganization energy
 C.C. Moser et al. / Biochimica et Biophysica Acta 1797 (2010) 1573–1586 1577

Fig. 4. Packing densities in random regions of proteins that are predominantly alpha
helical (gray) or beta sheet (black) compared to densities between cofactors in
physiologically productive (black dashed) or unproductive (gray dashed) electron
transfers. Mean packing densities for these four classes are similar: 0.78 ± 0.08, 0.75 ±
Fig. 3. Experimental vs. calculated free energy optimized rates [2,10,32,37–48,60–62] 0.09, 0.74 ± 0.14 and 0.76 ± 0.06, respectively.
for physiologically productive and unproductive electron transfer reactions in two
species of bacterial photosynthetic reaction centers (thick and thin open circles,
respectively) and a range of light activated ruthenium modified proteins (filled
symbols) using Eq. (1) with an average protein insulating barrier (gray) or a variable
barrier estimated from the packing density rho, (black). Error bars reflect estimates of dependence of rate as a function of driving force. Electron transfer
uncertainties in distances, rates and reorganization energies in determining the free- rates typically speed up as the driving force increases, until the driving
energy optimized rates.
force matches the negative of the reorganization energy, and electron
transfer rates are maximal for that given distance. Overdriving the
reaction with larger driving forces causes the electron transfer rate to
slow down once again. The most reliable way to measure the
which often lead to an order of magnitude uncertainty in calculated vs. reorganization energy is to vary the driving force of a reaction while
measured tunneling rates [63]. A rate estimate error of less than a factor keeping the other parameters relatively constant. An experimentally
of 8 is typically made by considering or ignoring packing density; much easier method measures the temperature dependence of the
packing density adjustments apparently slightly improve the accuracy reaction and converts the apparent activation energy into a
of rate estimates in natural proteins and slightly decrease the accuracy in reorganization energy. This will lead to inappropriate values,
ruthenated proteins. especially if the reaction is not in fact limited by electron tunneling,
Rather than adjusting the insulating properties of the protein or if parameters such as driving force or reorganization energy are
medium itself, it appears that natural selection controls the direction themselves affected by temperature. Furthermore, the relationship
and speed of electron tunneling principally by adjusting the distance between activation energy and reorganization energy will be different
between redox cofactors. In principle, proteins composed of beta depending upon whether nuclear vibrational quantum effects are
sheets have the ability to lower the electron tunneling barrier for a minor, so that classical Marcus theory applies (as is frequently
given distance compared to proteins composed of alpha helices, assumed), or vibrational quantum effects are significant and a model
provided the cofactors are rather carefully positioned so that the including quantum effects is more appropriate.
region between cofactors falls right along the beta sheet backbone, Eq. (1) is based on Hopfield theory, which has a Gaussian
leading to relatively high packing or pathway connectivity. They have dependence of driving force on free energy and uses a relatively
been arranged this way by design in some ruthenated azurins [60,64]. simple trigonometric function to approximate the progression
However, there are so far no known examples of this structure being between classical behavior matching Marcus theory at high tempera-
exploited in unmodified natural proteins. In the volumes between tures and non-classical behavior at low temperatures involving a
N120 natural redox partners, we found roughly 70% have no protein characteristic quantum frequency coupled to electron transfer. When
backbone of any sort, often because the distance between cofactors is this characteristic quantum frequency is larger than ambient
too short to accommodate one. Of the remaining volumes including temperature thermal energy, kBT, quantum effects become obvious
backbones, most are part of loose, irregular coils. Beta sheets and the electron-tunneling rate will have a smaller apparent
backbones intrude between cofactors only about 3% of the time and activation energy and more modest temperature dependence than
there does not appear to be any case in which the sheet aligns with the classical models using the same driving force and reorganization
direction of electron transfer. In the absence of this special cofactor energy. While it is challenging to perform a genuine free energy
alignment, alpha helix and beta sheet proteins have unremarkably dependence of the reaction rate to obtain the reorganization energy,
similar distribution of packing densities (Fig. 4) [63]. Despite and much more convenient to measure the temperature dependence
declarations that beta sheet proteins are best for long distance of a reaction and assume classical Marcus theory, the activation
electron transfer [64], there is no evidence that nature actually favors energies and reorganization energies estimated by this classical
beta sheet designs for this purpose. temperature dependence will be higher, typically by about a factor
of 50% to 60% near room temperature. There is the additional danger
2.5. Measuring reorganization energy that the temperature dependence will be sensitive to activation
energies not associated with the tunneling event itself and to changes
Reorganization energies are defined as the driving force that leads in the molecular motions and geometries associated with the electron
to the maximal electron-tunneling rate in a roughly Gaussian transfer reaction.
1578 C.C. Moser et al. / Biochimica et Biophysica Acta 1797 (2010) 1573–1586

While reorganization energy is a significant factor in determining vibration ω, nk is an expression of the temperature dependent
the electron transfer rate, it is usually not as important as the distance population of higher vibrational levels, kB is Boltzmann's constant and
or driving force. When reorganization energies are entirely unknown, T the temperature. If the electron transfer were coupled only to a
we prefer to fall back on rough estimates of small, medium and large single high-energy vibration, then the electron transfer would take
values, around 0.7, 1.0 and 1.4 eV, respectively, which correspond to place only at discrete driving forces corresponding to multiples of the
typical protein environments and cofactors which are relatively non- vibrational quantum energy. This is a precise but rather unrealistic
polar, moderately polar or polar. Reorganization energies larger than description of natural electron transfer systems, because there will be
these tend to be associated with adiabatic bond breaking systems, many types of vibrations that can be coupled to different degrees to
although Warshel and Parson [65] have estimated the reorganization the reorganization upon electron transfer. This has the effect of
energy of hydride transfer as low as 1.4 eV in lactate dehydrogenase. softening out the free energy dependence of the rate. With more
These small, medium and large estimates are useful in the design and complex expressions that allow a single high energy vibration and a
construction of novel protein electron transfer systems where the host of low energy vibrations, the free energy dependence of the
polarity of the cofactor environment can be reasonably predicted. tunneling rate is expected to have regular dips and wiggles [69,70].

2.6. Significance of 3.1 coefficient of free energy term  

2π 2 exp½−Sk ð2nk + 1Þ
+ ∞
ðnk + 1Þ m = 2 pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ket = jHj pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ∑ Im ð2Sk nk ðnk + 1ÞÞ ð8Þ
h ω
2
4πλS kB T n = −∞ nk
The free-energy dependent Franck–Condon factors in both classical
Marcus theory [66] (Eq. (5)) and Hopfield theory [19] (Eq. (6)) both ðλS mhω−ΔEÞ2
exp½− 
4λS kB T
have a Gaussian dependence of the electron-tunneling rate on the
driving force (ΔG).
Here the symbols are the same as Eq. (7), but ΔE is an expression of
1 2 the redox potential difference between the donor and acceptor, which
FCMarcus = pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Exp½−ðΔG + λÞ = 4λkB T ð5Þ
4πλkB T allows the expression to be a continuous function of the driving force.
This analysis has been applied to photosynthetic reaction centers [67].
1 2 We find that within the uncertainty of rate, distance and driving
FCHopfield = pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Exp½−ðΔG + λÞ = 2λhωCoth½hω = 2kB T
2πλhωCoth½hω = 2kB T force measurements in biological systems, there is no evidence that a
ð6Þ specific set of vibrations leads to dips and wiggles in the driving force
dependence of the reaction rate. Indeed, given the experimental
Hopfield introduced the characteristic frequency coupled to challenges in defining the parameters, it seems unlikely that any real
electron transfer (ω) to capture the transition seen in early biological system will clearly identify these vibrational subtleties.
photosynthetic reaction center experiments by DeVault and Chance Overall, the broad Gaussian of Eq. (1) adequately describes driving
[18] from a quantum mechanical temperature insensitive rate of force behavior.
electron transfer at cryogenic temperatures, to increasingly classical Because of the typical uncertainties in studying the rates of
temperature sensitive behavior at higher temperatures. The hyper- electron tunneling in protein (uncertainties in measuring the rates
bolic cotangent function (Coth) modulates the width of the Gaussian themselves, and the challenge of varying driving force while
(variance) from a temperature insensitive quantum 2λhω at the low maintaining other parameters such as distance and reorganization
temperature limit to a temperature sensitive classical 2λkBT at the energy constant), it is even difficult to determine if the ∼ 60 meV
high temperature limit. characteristic frequency suggested by the broad, relatively tempera-
Extensive work in photosynthetic reaction centers over a wide ture insensitive driving force dependence in photosynthetic reaction
range of driving force and temperatures shows rather clearly that a centers, is general for all natural protein electron tunneling reactions
classical dependence is inappropriate [67], presumably because the and reflects the typical electron-transfer-coupled vibrational spec-
atomic reorganization that takes place upon electron transfer in trum of protein and organic cofactors as materials. On the other hand,
proteins is typically coupled to vibrations that are noticeably larger in there is as yet no evidence for dramatic variation in this characteristic
energy than ambient temperature Boltzmann thermal energy kBT. The frequency that would conspicuously broaden or narrow the width of
net effect of introducing quantum nuclear tunneling into the classic the Gaussian free energy dependence of the reaction rate for any given
Marcus picture is to broaden the driving force dependence of the rate. reorganization energy.
In the common log rate expression of Eq. (1), the coefficient in front of
the free energy term that seems to fit photosynthetic reaction centers 2.7. Approximations for endergonic electron transfer
and other protein systems is closer to 3.1 than the classic Marcus 4.2.
This coefficient corresponds to a characteristic quantum of vibration Although the driving force dependence of the electron-tunneling
coupled to electron transfer of about 60 meV. rate described by Hopfield is Gaussian-like for exergonic reactions, the
Hopfield's expression is not the only way to introduce quantum Gaussian behavior does not simply extend into the endergonic region,
terms into the free-energy dependence of the electron tunneling. An as an examination of exact quantum mechanical harmonic oscillator
exact quantum mechanical oscillator description has a considerably descriptions makes clear. There is a noticeable transition in these
more complex form than the simple Gaussian free energy dependence more complex equations as the driving force becomes uphill, with an
of Eq. (1) and uses modified Bessel functions (Im) [68]. increasingly deep fall in the electron transfer rate as the reaction
  becomes more and more uphill. At cryogenic temperatures, the break
2π ðnk + 1Þ m = 2 pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi between endergonic and exergonic behavior becomes even more
= jH j exp½−Sk ð2nk Im ð2Sk nk ðnk + 1ÞÞ
2
ket = + 1Þ
h2 ω nk conspicuous. A quite simple, and moderately accurate way to model
ð7Þ this endergonic quantum behavior is to use Eq. (1) to calculate
biological electron tunneling for exergonic conditions, and to calculate
where nk = 1/(exp[hω/kBT]-1) the endergonic rate of the reverse reaction by requiring that the ratio
Here ω is the same characteristic frequency of the vibration of the forward and reverse reaction rates obeys a Boltzmann
coupled to electron transfer used by Hopfield, H is the overlap of the equilibrium term (Eq. (2)) [2,3]. Fig. 5 shows that this combination
donor and acceptor electronic wave function, Sk is the reorganization of equations for the exergonic and endergonic regions follows the
energy in units of hω, m is the change in the quantum number of the general trend of the exact multi-vibration quantum harmonic
 C.C. Moser et al. / Biochimica et Biophysica Acta 1797 (2010) 1573–1586 1579

Fig. 5. Electron tunneling rates vs. driving force for classical Marcus description (dashed
line, Eq. (5)) [66]; and exact quantum simple harmonic oscillator model with one
frequency of 70 meV (black circles, Eq. (7)) [68]; a harmonic oscillator model with
multiple vibrations both quantum and classical (thin line, Eq. (8)) [69,70]; and the
simple empirical approximation using Eqs. (1) and (2) presented here (thick line).

oscillator equations rather well, and with considerably fewer para-

Fig. 6. In bacterial photosynthetic reaction centers, quinone/bacteriochlorophyll dimer
meters and a much simpler equation that can be evaluated in your
charge recombination takes place via thermally activated, uphill electron transfer when
head. Because this pair of equations by definition obeys a Boltzmann the native ubiquinone at the QA site is replaced with lower redox midpoint potential
equilibrium expression, there is neither danger of circumventing the quinones [20,72]. The activation energy for the reaction directly tracks the drop in the
second law of thermodynamics nor any algebraic error, despite midpoint potential (lower panel) and the thermally activated rate increases by a factor
of 10 for every 0.06 eV drop in potential (upper panel), the behavior expected for uphill
suggestions to the contrary [71]. Any attempt to single-mindedly use
electron tunneling according to Eq. (2).
only Eq. (1) for both the endergonic and exergonic regions, or only
Eq. (2) in both the exergonic and endergonic regions is doomed to
paradox and failure.
Support for the use of Eq. (2) for endergonic electron transfer is
provided by classic low temperature experiments on endergonic transfers in photosynthetic reaction centers and the presence or lack
intraprotein electron tunneling [20]. Fig. 6 is adapted from the thesis of temperature dependence are the original basis for the protein
of M. R. Gunner [72] and shows the driving force dependent behavior tunneling expressions of Eqs. (1) and (2).
of the uphill charge recombination reaction after light-induced charge
separation in bacterial photosynthetic reaction centers in which the 2.8. What matters to biology
native ubiquinone at the QA site has been replaced with a variety of
exotic quinones of different redox midpoint potentials. These Of the various parameters that could be manipulated by natural
midpoint potentials were measured in situ by examining the initial selection to engineer and control electron transfer in proteins, it is
amplitude of the delayed fluorescence reaction as the excited clear that distance is the most important. Mutagenic selection of
bacteriochlorophyll dimer (P*) thermally repopulated from the amino acids that allow or disallow redox cofactors to covalently or
charge-separated state P+Q-A. As the redox midpoint potential of the non-covalently bind at certain locations within an existing protein
exotic QA quinones became more negative, the recombination to the P scaffold or to refold the protein to bring redox elements closer or
ground state moved from a mostly temperature insensitive hundred further apart, is the principle element of natural design. This principle
millisecond timescale to a temperature sensitive and much faster allows even a low-resolution structural view of a protein, with at least
millisecond to microsecond timescale (Fig. 6 top). The rate of some discrimination of individual redox centers, to be highly useful in
recombination accelerated by a factor of 10 for every approximately understanding its overall electron transfer design and operation. The
60 mV drop in quinone redox midpoint potential. At the same time, influence of distance is magnified for electrons coupled to protons in
the activation energy associated with this temperature sensitive hydride transfer, especially if the proton must tunnel.
reaction fell in direct proportion to the downward shift in midpoint The next most accessible handle upon which natural selection acts
potential and associated electron transfer driving force is the driving force of the electron transfers. Driving force is especially
(Fig. 6 bottom). This is powerful evidence that the charge recombi- important in engineering uphill electron transfers and in suppressing
nation takes place via an uphill electron transfer, presumably physiologically unproductive electron transfers, where a relatively
returning the electron to bacteriopheophytin (which originally modest 0.06 eV change in driving force can slow electron transfer
relayed the electron from the light excited bacteriochlorophyll reactions by an order of magnitude, equivalent to an increase in
dimer to the quinone) followed by downhill electron transfer from electron tunneling distance of 1.7 Å. This principle is active in safe-
the bacteriopheophytin to the dimer ground state. The slopes of Fig. 6 guarding the reactivity of two-electron redox centers with interme-
indicate that this uphill electron transfer is related to the reverse diate semiquinone redox states, such as ubiquinone, because non-
downhill electron transfer back to the quinone by a 60 mV per decade specific electron transfer with adventitious redox centers will often
Boltzmann equilibrium. The driving force dependent rates of both the involve an initial electron transfer that is significantly uphill. Driving
forward, downhill electron transfers and the reverse, uphill electron force is not as influential in controlling downhill electron transfers,
1580 C.C. Moser et al. / Biochimica et Biophysica Acta 1797 (2010) 1573–1586

because the quantum broadening of the free energy dependence of tunneling does not contribute significantly to enzyme catalysis, as
the tunneling rate means that relatively large changes in driving force compared to the uncatalyzed reaction in water [74], is consistent with
of 0.2 to 0.5 eV are required to execute an order of magnitude change recent acknowledgments that enzymes have not evolved to accelerate
in tunneling rate. Driving force is an expensive and often inefficient tunneling per se [16,75]. In any event, the hydrogen isotope effects
way to engineer exergonic electron transfer rates. often used to expose nuclear tunneling are expected to generally fade
Reorganization energy can be used to control electron transfer as catalysis becomes faster and more effective.
rates, as it appears that changes from 0.7 to 1.4 eV can take place by At the largest nicotinamide/substrate distances of Fig. 1B, proton
introducing polar amino acids and water molecules into an otherwise tunneling would be impossibly slow. In such molecules we may
non-polar site. However, even such a dramatic manipulation of the expect either that hydrogen transfer distances shrink through large-
reorganization energy will likely change the electron tunneling rate scale rearrangement, or that electron and proton transfer occur over
by less than two orders of magnitude, a change that can be effected different distances to separate acceptors. Protein crystal structures
through a 3.3-Å change in distance; this is about the effect of including both nicotinamides and flavins show a similar clustering of
interposing or removing the width of one amino acid between distances between 3 and 4 Å (Fig. 1C) consistent with small scale
cofactor binding sites. Nevertheless, some natural selection of motion leading to hydride transfer and proton tunneling. Beyond this
reorganization energy has probably taken place; the unusually low cluster there is nearly flat distance distribution out to about 18 Å,
reorganization energies associated with the initial charge separation where direct hydride transfer is highly unlikely. While in some cases
in photosynthesis improve the quantum yield and provide a likely electron transfer between NADH and flavin is simply mediated by
selection target. other redox centers, in other cases, large-scale motion could facilitate
Changes in the bonded quality or packing density of the protein close approach or electron and proton transfer could be managed with
medium could significantly influence electron tunneling rates, separate acceptors.
especially if the bonding or packing change is extensive along a It should not be forgotten that significant motion is sometimes
relatively long distance between redox centers. At short distances critical for simple single-electron tunneling. Although extensive
however, changes in packing density will have relatively little effect, molecular motion is not essential for electron transfer over distances
since the rate depends on the packing term multiplied by the distance. of up to 14 Å, because the electron's wavelength is so much longer than
For example, at a distance of 5 Å, a change in packing density of a full 4 proton's, molecular motion and diffusion can be an important to allow
standard deviations will only have about a 3-fold effect on the donors and acceptors to approach within this electron tunneling range
tunneling rate, equivalent to a distance change of less than an [76]. For example, tethered diffusion of the FeS subunit of Complex III in
Angstrom. This does not mean that packing changes could not be the mitochondrial respiratory chain brings the FeS cluster alternately in
selected in principle for some particular case, only that there is no and out of electron tunneling distance with its quinone or heme c1 redox
clear evidence that the packing deviations seen in natural electron partners [77]; motion in sulfite reductase greatly reduces the electron
transfer reactions are not merely random. transfer 20 Å distance seen in the crystal structure [78]. Molecular
One last parameter, the characteristic frequency of vibration coupled motion can also modulate other parameters coupled to electron
to electron transfer, could also be modified by natural selection to speed tunneling, such as driving force and reorganization energy [65,79,80]
or slow any particular electron transfer. This is one of the hardest although these typically have less dramatic effects.
parameters to measure and the one with the most uncertain value; Next to distance, driving force has a principle influence of the
indeed, the range of natural variation of this parameter is not at all clear, kinetics of electron transfer in two-electron transfer. The energetics of
let alone the influence of natural selection. However, as it is also one of the ubiquitous biological two electron redox centers nicotinamides,
the weakest parameters in terms of its effect on the electron-tunneling flavins and quinones can be described in terms of the redox midpoint
rate, it is one of the least likely parameters for continued natural potentials of the one electron redox couples (in the various
selection. protonated or deprotonated states, see Fig. 7). For NADH, the average
The overall picture of what matters most to biological electron potential of the two redox couples is around −0.32 V. The extent of
tunneling is location, location, location. Furthermore, proximity the split between the two redox couples gives a measure of how much
becomes even more important when there is a significant uphill of the half-reduced or semiquinone species can be seen under
electron transfer barrier to be surmounted, as is often the case in multi- equilibrium conditions. To use NADH as an example, the stability
electron transfer systems involving quinone, flavin, pterin, nicotina- constant (Kstab) near room temperature is
mides or bond-breaking catalysis.
2 þ ðEmNADH=NAD•EmNAD•=NADþÞ=:06V
Kstab = ½NAD• = ½NADH½NAD  = 10
3. Electron versus hydride transfer
ð9Þ
Oxidoreductases represent nearly a third of named natural
enzymes and within this group by far the most common substrates When the redox midpoint potential (Em) values of the two redox
are the nicotinamides NADH and NADPH. Fig. 1B shows the couples are the same, then the stability constant is 1 and an equilibrium
distribution of distances between the nicotinamides and bound redox titration will have a maximum concentration of the singly
substrate from a sampling of the PDB protein structure database. reduced species equal to 33% at the average redox midpoint potential. If
Unlike single electron transfer distances in Fig. 1A, there is a clear the Em of the first reduction is more positive than the second reduction,
clustering around 3.4 ± 0.2 Å, i.e. near van der Waals contact; outside as is the case sometimes with flavins and quinones, then the Kstab is
this range distances trail out to 7 Å. At the closest distances, molecular greater than one (log Kstab is positive) and we say the semiquinone
motion can foster ∼ 2 Å close approaches required for tunneling of the species is stable and observable, for example as an unpaired spin by EPR.
hydrogen nucleus between donor and acceptor in biological systems This property allows flavin to act as a transducer between obligate two-
[16,17], just as in non-biological systems. Indeed, by studying light- electron redox centers and obligate one-electron redox centers in an
activated steps of chlorophyll synthesis in protochlorophyllide electron transfer chain. If the Em of the first reduction is more negative
oxidoreductase, it has been possible to trigger the conformational than the second, the Kstab is less than one (log Kstab is negative) and the
changes that enhance hydride transfer and follow their infrared single reduced species is considered unstable and never reaches a high
vibrational signatures [73]. concentration at redox equilibrium. For NADH in neutral aqueous
It is not immediately clear if motion in protein has been naturally solution the redox midpoint of the NAD•/NAD+ couple has been
selected to optimize tunneling effects. Warshel's thesis, where nuclear reported to be a strongly reducing −0.92 V [81] with leads to a stability
 C.C. Moser et al. / Biochimica et Biophysica Acta 1797 (2010) 1573–1586 1581

Fig. 7. The common multi-electron redox substrates engage in a matrix of redox and protonation states. The values of redox midpoint potentials and pKs are those expected for
cofactors in solution: (a)[105] (b) [106] (c) [107] (d) [108] (e) [109] (f) [81] (g) [82] (h) [110].

constant around 10−20. This means the equilibrium concentration of 103 s−1 physiological window, even over distances of more than 14 Å
NAD• will be less than one part in 1010 at best. The Em of the NADH/NAD• (rates along the x-axis of panel B). When the net driving force for the
couple is moderately oxidizing, at around +0.28 V. The Em of the overall two-electron transfer reaction is favorable (moving up panel B),
protonated couple NADH/NADH+• in aqueous solution has also been the reaction can proceed even faster.
estimated at +0.93 V [82]. With the much smaller stability constant of 10−20 typical of
Although it is commonly believed that NADH is exclusively a NADH or NADPH (panel C), where the equilibrium population of the
two-electron, hydride transfer agent, there are clear indications of half-reduced, radical form will be less than one in a billion, the
electron and proton transfer with obligate one-electron redox distance between donor and acceptor must be significantly smaller,
centers that do not involve transfer of a hydride [83]. Excellent or the overall driving force significantly more exergonic, in order for
examples are provided by ferricyanide [84] and ferrocenium ions a two-step electron tunneling reaction to be within the typical
[82,85]. There is even good evidence that two-electron redox centers physiological window of faster than 103 s−1. Yet with a net driving
such as quinones can also participate in single electron transfer with force of a few hundred meV, donor and acceptor need only to
NADH [86,87]. Indeed, it is often profitable to view many classical approach within 10 Å of each other in order to allow the pair of
organic chemical reactions, traditionally regarded as obligatory two- single electron tunneling reactions to proceed within a millisecond.
electron transfer reactions, in terms of the passage of electrons one This means that a large amount of flexibility of donor/acceptor
by one [88,89]. Single-electron oxidation of NADH becomes more binding site geometries could be tolerated, and that NADH
facile as the redox couple of the oxidant approaches or becomes oxidoreductase enzymes have the potential to proceed by non-
more positive than the NADH/NAD• couple at 0.28 V if proton adjacent, non-hydride, sequential electron transfer mechanisms with
release is not rate limiting, or 0.93 V if proton release is rate limiting. some robustness, provided that the environment provides sufficient
Under these conditions it makes sense to test the extension of the resources for proton management. This is a promising prospect for
electron-tunneling rate expressions developed for biological single- engineering de novo, non-natural protein environments for multi-
electron-transfer reactions [2], to a sequence of two one-electron- electron catalysis.
transfer reactions between a pair of two electron donors/acceptors, If both the donor and the acceptor have very low stability constants
such as NADH, flavin or quinone (Fig. 8C–F) and compare these to so that the stability constant product is around 10−40 (panel D), as in
observed rates. NADPH/NADH transhydrogenase [90], there are virtually no conditions
Fig. 8 A shows that with a typical 1 eV reorganization energy, in which a sequential electron tunneling reaction can proceed on the
expected electron tunneling reactions exceed the typical 103 s−1 of millisecond timescale, and it is certain that electron transfer proceeds by
many enzymes when the reaction is exergonic and the tunneling an alternate mechanism such as hydride transfer or, if conditions are
distance is 14 Å or less (green and blue regions in Fig. 8A). This speed for sufficiently polar, simultaneous two-electron transfer [91]. Eqs. (1) and
endergonic electron tunneling means that if redox centers are relatively (2) will not be of much use in predicting the rate of this reaction.
closely placed, then a sequence of two single-electron transfer reactions,
one uphill (for example to a relatively unfavorable semiquinone state) 4. Merging electron tunneling and chemical barrier expressions
and one down hill (for example to a favorable fully reduced state) can
occur in a physiologically acceptable timescale. Panels B–F of Fig. 8 show The electron tunneling expressions of Eqs. (1) and (2), and hence the
several examples. rates shown in Fig. 8, are based on non-adiabatic electron transfer
Flavin FMN in aqueous solution has a stability constant of ∼10−3, so theory, which presumes that electron donor and acceptor are clearly
the maximum semiquinone at equilibrium reaches modest concentra- identifiable and separate. As donor and acceptor come in near contact,
tions of a few percent. With a product of the stability constant of donor interactions between donor and acceptor strengthen and the simple
and acceptor of ∼10−3 (panel B), electron tunneling uphill for the first common diabatic Marcus-like view of parabolic energy surfaces with
electron, followed by an equally downhill reaction for the second occasional hops from donor to acceptor surface starts to fail. A reaction
electron for an over all ΔG = 0 reaction, can easily take place within the proceeding over a single adiabatic energy surface through transition
1582 C.C. Moser et al. / Biochimica et Biophysica Acta 1797 (2010) 1573–1586

Fig. 8. (A) Expected single electron tunneling rates as a function of distance and driving force. Contour lines show log rate in seconds. 1 eV reorganization energy. (B) The activation
free energy barrier at a catalytic site that can be scaled on a physiological timescale depends. (C–F) Electron tunneling rates expected for two sequential electron transfers from a
two-electron donor to acceptor as a function of distance, average driving force and the product of the stability constants of the two redox centers. Typically, the first electron transfer
is uphill and slow while the second is down hill and faster. Physiologically acceptable rates are shown in greens and blues. The reorganization energy is a generic 1 eV.

state region becomes a better description. The latter adiabatic view is for the electron on the donor, passing many times through the
also more appropriate if there is significant bond making and breaking intersection with the parabolic surface for the electron on the acceptor
taking place during the electron transfer reaction. As with classical (dotted line), and then finally jumping onto this new surface (show as a
chemical reactions, a simple Eyring rate expression based on transition sharp bend in the line). Proton transfer is shown as a smooth line with
state theory (Eq. (3)) is often used in these circumstances [14]. the thermally activated path reaching the low-lying pass of an adiabatic
Besides a purely adiabatic description, it is also possible that
electron transfer coupled to some other reaction can be both non-
diabatic for long distance electron transfer and adiabatic for the
coupled reaction. The overall progress from reactant to product may
be sequential, with an electron transfer first and the bond changes
second, or the bond changes first and the electron transfer second, or
concerted, with both occurring at the same time.
Classical and well-described examples of this behavior are found in
coupled proton and electron transfer [92,93]. Fig. 9 illustrates a
simplified picture of coupled proton and electron transfer with parabolic
potential energy surfaces along the reaction coordinates for electron and
proton transfer. An analogous reaction surface may apply to any other
reaction coupled to electron transfer, such as a protein conformational
change that performs useful work. The arrows in Fig. 9 show possible
paths for each of the reaction sequences, sequential ETPT, PTET or
concerted. Which mechanism is favored will depend on the activation
energy barrier of the proton transfer before or after electron transfer, the
activation energy of the electron transfer before or after proton transfer,
and the κe or transmission coefficient for electron tunneling through the
long distance between electron donor and acceptor.
Fig. 9. Schematic illustration of possible energy surfaces of a non-adiabatic electron
tunneling reaction coupled to an adiabatic chemical reaction, here proton transfer.
Log κe = 13−0:6ðR−3:6Þ ð10Þ Coupled electron and proton transfer can take place sequentially in either order (ETPT
for electron first, PTET for proton first) or concertedly, depending on reaction activation
energies and transmission coefficients. Arrows show possible paths along the energy
surfaces. In the direction of electron transfer, energy surfaces intersect, but because the
This κe, which is much less than one for long-distance electron transmission coefficient κ is much less than one, the reaction path moves repeatedly
transfer, is illustrated as a wiggling back on forth on a parabolic surface past the intersection and only rarely jumps to the new surface.
 C.C. Moser et al. / Biochimica et Biophysica Acta 1797 (2010) 1573–1586 1583

transition state between two parabolic surfaces and crossing over more electron from QA is expected to be more complex with protonation and
or less directly. finally release of the fully reduced quinone. The rate for this reaction is a
The path marked “concerted” can be described by Eq. (1), although much slower ∼103 s−1. Eq. (11) suggests a coupled, non-tunneling
the proton transfer (or other chemical) component may lead to a energetic barrier of about ∼4 kcal/mole, which matches the measured
reorganization energy that is larger than a typical intraprotein activation free energy for this reaction [94,95].
electron transfer, and the concerted reaction may be more likely to Cytochrome c oxidase provides another example of how multi-
freeze out at lower temperatures compared to simpler electron electron tunneling and coupling of electron and proton transfer might
tunneling reactions. Because the reorganization energy for a reaction work in practice, with a single step electron transfer coupled to an
of this sort can be difficult to estimate a priori, Eq. (1) may be useful energetic barrier that does the work of proton pumping [93,96]. X-ray
only in providing an upper bound of the electron tunneling rate. crystal structures [97,98] show that heme a is 7 Å away from heme a3
The path marked PTET could be rate limited by uphill proton and that heme a3 and CuB centers are less than 5 Å away from the bound
transfer, followed by rapid electron transfer. In this case, the reaction O2 substrate. The tight clustering of heme a3, CuB and O2 means that
will generally not be sensitive to the electron tunneling parameters, electron tunneling to deliver two electrons to O2 can be faster than the
R, ΔG or λ and we are forced to fall back on the simple Eyring 5 × 103 s−1 turnover of the enzyme [99] even if the first electron transfer
expression (Eq. (3)) with a difficult to predict activation energy and were 0.6 eV uphill to a redox center as low as −0.3 V. The redox
little ability to predict rates a priori. However, when the electron potential of the O2 bound to heme a3 is uncertain, but is not likely to be
transfer is not too short, κe can be small enough that electron as hard to reduce as this. Even without the benefit of being bound to the
tunneling can be slower than the proton transfer step. In this case, heme iron, the redox potential of the oxygen/superoxide anion couple
Eq. (1) applies, but a term needs to be added to reflect the in solution is only −0.14 V [100].
unfavorable equilibrium constant between the forward and reverse It appears the electron transfer that sets up the reduction of
proton transfer reaction, −ΔGchem/.06 (Eq. (11)). Generally, there oxygen to water in cytochrome oxidase, namely electron transfer
will also be an increase in the driving force for the electron transfer between heme a and a3, couples to the work of proton pumping by
step compared to the difference in the redox potentials of donor and the enzyme [101]. CO photolysis experiments with the mixed-
acceptor measured at equilibrium with protons. However, the effect valence state of oxidase (starting with heme a3 reduced and CO
of a change in driving force in the electron tunneling (ΔG + λ)2/λ bound, heme a oxidized) show an inherent time of electron
term will usually be much smaller than the effect of the −ΔGchem/ tunneling between heme a3 and a of about 1 ns [102]. This is the
0.06 term—uphill driving forces once again dominate rates. If the rate expected from Eqs. (1) and (2) given the close distance between
overall rate of the reaction, distance and driving force are these heme groups and typical reorganization energies [96]. Yet the
moderately well known, then the energetic penalty of the uphill physiological electron transfer between heme a and a3 is measured
reaction ΔGchem can be estimated. at 30 μs [103]. While this reaction may be rate limited by proton
transfer, Eq. (11) suggests this 30,000 fold slowing of the electron
ex 2
log10 ket = 13−0:6ðR−3:6Þ−3:1ðΔG + λÞ = λ−ΔGchem = 0:06 ð11Þ transfer could be correlated with an uphill step coupled to the
electron transfer. Just how far uphill depends on the reorganization
In Fig. 9, the last path marked ETPT could also be rate limited by energy of the electron tunneling reaction, which is not known. With
proton transfer, but if the electron transfer distance is long enough, a 1 eV reorganization energy, a 0.3 eV uphill step would be required.
it is likely to be rate limited by electron transfer. In this case, uphill If water molecules in this region contributed to a larger reorgani-
electron transfer is followed by rapid downhill proton transfer, and zation energy of around 1.6 eV, a 0.15 eV uphill step could be
the reaction path could be described by Eq. (2). Generally, the redox involved. The temperature dependence of the Hopfield relation
potential of this uphill electron transfer intermediate will be underlying Eq. (11) gives a temperature dependent rate which is not
difficult to measure. However, if the overall rate of the reaction, far from that which is observed for the rate of electron transfer
and distance are moderately well known, then uphill driving force between the O2 bound state and the heterolytically cleaved Pr state
of the electron tunneling reaction might be estimated using this [104]. Eq. (11) leads to an apparent activation energy of 0.39 eV, in
equation. From a redox protein design perspective, when it is between the experimentally estimated activation energy is 0.2 eV
difficult to influence the redox midpoint potentials of an uphill and the estimated activation free energy of 0.45 eV [104], and
single-electron transfer intermediate in a reaction, placing redox indicates that an uphill free energy step connected to the work of
centers as close as possible is a reasonable means to attempt to proton pumping coupled to the electron tunneling reaction is
speed these reactions. Indeed, Fig. 8A shows that close distances plausible.
should permit submillisecond electron transfers several hundred
meV uphill. 5. Natural design
Fig. 8B provides a graphic example of the size of unfavorable
equilibrium-constant/energy-barrier surmountable in a given time at a Distance and driving force control of electron transfer reactivity is
given tunneling distance. These rates are provided for a tunneling ΔG° critical for life, which is essentially the management of many quasi-
near 0 eV and an average λ of 1 eV. Fig. 8B shows that at 4 Å such stable compounds, with pairs of electron locked up in chemical bonds
tunneling reactions can in principle thermally surmount barriers up to that must be teased into rearrangement during catalysis or energy
10 to 15 kcal/mol in the millisecond to second timescale of many conversion. A rush of rapid electron transfers leading to equilibrium
oxidoreductases. Thus, the observed catalytic rate, if rate limiting and set would mean death for any organism. To forbid unproductive electron
by the adiabatic chemical barrier established in the site [14], will be transfers, single electron donors and acceptors must be excluded from
sensitive to any modulation of electronic and nuclear terms of electron within the approximately 14 Å boundary, while appropriate single
tunneling at the interface [66]. electron donors and acceptors must be brought within this range in
It is rare that experimental situations fortuitously allow direct order to achieve physiologically acceptable speeds. The principal role
examination under similar conditions of both a tunneling rate between played by protein structure is to secure redox cofactors and binding
two centers and a catalytic rate coupled to the electron transfer. In an sites within this distance and to provide bulk insulation to exclude
example from photosynthetic reaction centers, the relatively long unproductive electron transfer partners. Specificity is gained by
distance, 13 Å reduction of QB site quinone by a single electron from the natural selection of amino acids that modulate binding affinity at
QA site of 4×105 s−1 is quite close to tunneling rate of ∼106 s−1 expected substrate binding pockets and hence the time spent at closer
from Eq. (1). However, reduction of the semiquinone QB by a second distances. Multi-electron donors and acceptors without readily
1584 C.C. Moser et al. / Biochimica et Biophysica Acta 1797 (2010) 1573–1586

accessible singly oxidized or reduced states are much less reactive, [21] X. Lin, J.C. Williams, J.P. Allen, P. Mathis, Relationship between rate and free-
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