Electrochimica Acta 294 (2019) 117e125

Contents lists available at ScienceDirect

Electrochimica Acta
journal homepage: www.elsevier.com/locate/electacta

The role of molecular crowding in long-range metalloprotein electron

transfer: Dissection into site- and scaffold-specific contributions

s Espinoza-Cara b,
Ulises A. Zitare a, 1, Jonathan Szuster a, 1, Magali F. Scocozza a, Andre
Alcides J. Leguto b, Marcos N. Morgada b, Alejandro J. Vila b, Daniel H. Murgida a, *
a
Instituto de Química Física de los Materiales, Medio Ambiente y Energía (INQUIMAE), Departamento de Química Inorga
nica, Analítica y Química Física,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires and CONICET, 1428, Buenos Aires, Argentina
b
gica, Facultad de Ciencias Bioquímicas y Farmac

Instituto de Biología Molecular y Celular de Rosario (IBR), Departamento de Química Biolo euticas,
Universidad Nacional de Rosario and CONICET, 2000, Rosario, Argentina

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

Article history: Here we report the effect of molecular crowding on long-range protein electron transfer (ET) and
Received 23 August 2018 disentangle the specific responses of the redox site and the protein milieu. To this end, we studied two
Received in revised form different one-electron redox proteins that share the cupredoxin fold but differ in the metal center, T1
9 October 2018
mononuclear blue copper and binuclear CuA, and generated chimeras with hybrid properties by incor-
Accepted 11 October 2018
porating different T1 centers within the CuA scaffold or by swapping loops between orthologous proteins
Available online 16 October 2018
from different organisms to perturb the CuA site. The heterogeneous ET kinetics of the different proteins
was studied by protein film electrochemistry at variable electronic couplings and in the presence of two
Keywords:
Metalloproteins
different crowding agents. The results reveal a strong frictional control of the ET reactions, which for 10 Å
Loop engineering tunnelling distances results in a 90% drop of the ET rate when viscosity is matched to that of the
Electron transfer mitochondrial interior (ca. 55 cP) by addition of either crowding agent. The effect is ascribed to the
Molecular crowding dynamical coupling of the metal site and the milieu, which for T1 is found to be twice stronger than for
Frictional control CuA, and the activation energy of protein-solvent motion that is dictated by the overall scaffold. This
work highlights the need of explicitly considering molecular crowding effects in protein ET.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction arrest) of nuclear modes imposed by the milieu. Indeed, dynamical

Most kinetic experimental studies of protein electron transfer
(ET) reactions are performed with close to ideal diluted solutions
[1e3] and rationalized within the framework of Marcus semi-
classical equation for long-range (nonadiabatic) ET [4,5]. This
treatment, implies a number of underlying assumptions that are
not necessarily fulfilled for proteins in real biological environments
[6], which are characterized by high macromolecular crowding
[7,8] and strong local electric fields [9]. For example, water-protein
nuclear fluctuations required to equalize donor-acceptor electronic
energies before ET, as well as the subsequent electrostatic relaxa-
tion, are assumed to be much faster than electron tunnelling. This
condition may break down due to either enhanced electron
tunnelling probability or to a slowdown (and eventually dynamical
* Corresponding author.
E-mail address: dhmurgida@qi.fcen.uba.ar (D.H. Murgida).
1
Equal contributions.
effects on ET reactions have been theoretically addressed by a
number of researchers such as Marcus [10], Weaver [11], Zusman
[12], Jortner [13], Beratan [14], Waldeck [15], Matyushov [16] and
others, employing different approximations. From the experi-
mental side examples of dynamical effects on protein ET reactions
are rather limited and viscosity or crowding studies on protein ET,
although very valuable, mainly refer to the effect of crowding
agents on the diffusional component of interprotein ET in solution,
rather than to the electron tunnelling step [17,18]. Relevant to the
present work, different authors studied metalloproteins immobi-
lized on electrodes coated with self-assembled monolayers (SAMs)
of functionalized alkanethiols, and found exponential distance-
dependencies of the measured ET rate constants (kET ) only for
thick SAMs, but distance-independent plateaus for thinner spacers
[19e24]. For the soluble metalloproteins cytochrome c [25e27] and
azurin [28e30] ET rates were found to be sensitive to the medium
viscosity. These observations were interpreted as a change of ET
regime from nonadiabatic at longer distances to frictional control in

https://doi.org/10.1016/j.electacta.2018.10.069
0013-4686/© 2018 Elsevier Ltd. All rights reserved.
118 U.A. Zitare et al. / Electrochimica Acta 294 (2019) 117e125
terms of Zusman's equations at thinner films [25,26,28,29], and to
electric field dependent protein-solvent collective motions of large
and small amplitude [31e33]. These preceding investigations
demonstrate that moderate viscosities may have an impact on long
range ET rates. The goal of the present work is to deepen our un-
derstanding of possible kinetic effects of biological molecular
crowding on protein ET and, more specifically, to dissect the con-
tributions to this outcome of the different structural and dynamical
elements of metalloproteins that are relevant to the ET reaction
coordinate. To the best of our knowledge, this crucial issue remains
to date elusive and largely unexplored.
In this context it is pertinent to highlight the structural and
dynamical complexity of proteins, which requires multidimen-
sional and hierarchical free energy landscapes to describe the
conformational substates explored at biologically relevant tem-
peratures. Time scales for such exploration are also hierarchical,
ranging from femtoseconds for bond vibrations, picoseconds to
nanoseconds for side-chain rotations and microseconds to seconds
for collective motions of larger protein domains [34]. This flexibility
at different levels proved pivotal for canonical and alternative
functions of different proteins and enzymes [35e43].
Based on notions adopted from the physical chemistry of
glasses, Frauenfelder and coworkers [44e46] formulated a model
that divides protein fluctuations into three types: a and bh and
vibrations. The a motions are associated to changes in the protein
shape and, therefore, are slaved to bulk solvent fluctuations, while
bh fluctuations refer to internal protein motions and are slaved to
the hydration shell. Hence, the surrounding milieu (solvent)
crucially assists protein function through structural and dynamical
modulation [47,48]. One should note, however, that the in vivo
milieu seriously departs from the nearly ideal behaviour of dilute
aqueous solutions typically used for in vitro and in silico studies.
Cells present a number of large structures such as membranes and
cytoskeleton, as well as dissolved macromolecules in concentra-
tions that can be as high as 450 g/l and occupy up to 40% of the
cytoplasmic volume, in addition to a large variety of small mole-
cules [7]. In these complex and highly crowded environments
fundamental physicochemical parameters, such as viscosity, diffu-
sion and activity coefficients, may diverge from ideality by several
orders of magnitude [49e52]. Moreover, crowding is expected to
reshape the free energy landscapes of proteins and, therefore, their
dynamics [53]. This may be particularly true for proteins that
partake in respiratory ET chains of all living organisms. In eukary-
otes respiratory complexes are embedded into the inner mito-
chondrial membrane (IM), while electron shuttles such as
cytochrome c are present at millimolar levels in the intermembrane
space (IMS) [41,54]. These species coexist with about 49 other
proteins present in the IMS and 481 proteins that are either integral
or peripherally bound to the IM [55]. Moreover, membrane-integral
respiratory complexes may form giant supercomplexes (respir-
osomes) of variable stoichiometry [56,57]. Such level of crowding
undoubtedly affects the structure and dynamics of water and,
severally, those of the proteins themselves, as documented for
instance for small peptides and large photosynthetic complexes
[58e60].
The intricate electrostatic description of media such as the IM
and the IMS adds another layer of complexity. The IM is essentially
an energy-transduction device that uses a cascade of downhill ET
reactions to drive proton translocation, thus building up a gradient
that energizes ATP synthesis. This proton gradient generates a
transmembrane potential that combined with the membrane sur-
face and dipolar potentials may create local electric fields of up to
0.1 V Å
1 [9], in addition to local contributions due to protein sur-
face charges. Experimental and theoretical investigations on model
systems show that electric fields of biologically meaningful
magnitude may affect hydration, structure, dynamics and reactivity
of proteins [61e64], as well as relaxation times, viscosity, hydrogen
bonding and other features of water [65e72]. Conceptually similar
considerations stand for other biological systems based on protein
ET, such as in photosynthesis and bacterial respiration.
As pointed out by Ellis [8] almost two decades ago, the potential
of macromolecular crowding to affect reactivity is obvious but often
underappreciated, also for ET reactions.
In the present work, we specifically assess the role of frictional
effects in protein ET, i.e. of the viscosity component of molecular
crowding. To this aim, we envisage an approach that involves the
engineering of different metal centers into two protein scaffolds.
Namely, we consider two different types of one-electron copper
redox proteins that share the cupredoxin fold but differ by their
redox centers: the type 1 (T1) mononuclear blue copper site and
the purple binuclear CuA center. Protein samples with hybrid
properties were generated by loop engineering to obtain chimeras
that incorporate different T1 centers within the CuA scaffold, as well
as perturbed CuA sites obtained by loop replacement without
altering the protein scaffold. The heterogeneous ET kinetics of the
different protein variants was studied by protein film electro-
chemistry at variable electronic couplings and in the presence of
two different crowding agents. The obtained results reveal metal
site- and scaffold-specific frictional control for tunnelling distances
shorter than ca. 24 Å.
2. Experimental
2.1. Protein preparation
WT and mutant CuA-soluble fragments from subunit II of the
cytochrome ba3 from T. thermophilus were produced as described
previously [73e76] and stored in 100 mM phosphate buffer (pH
6.0; 100 mM KCl). Azurin from Pseudomonas aeruginosa was pur-
chased from Sigma-Aldrich. Before use, protein samples were
buffer exchanged to the desired final condition by thorough filtra-
tion with Amicon Ultracel-5K filters employing a refrigerated
centrifuge at 4000 rpm and 4  C (Hermile Z326K).
2.2. Protein film voltammetry
Cyclic voltammetry (CV) experiments were performed with
either a Gamry REF600 or a PAR263A potentiostat using a water-
jacketed non-isothermal cell. The cell was placed inside a Faraday
cage (Vista Shield) and equipped with a homemade polycrystalline
gold bead working electrode, a Pt wire auxiliary electrode and an
Ag/AgCl (3 M KCl) reference electrode. All potentials quoted here
are referred to NHE. Prior to use Au electrodes were oxidized in 10%
HClO4 applying a 3 V potential for 2 min, sonicated in 10% HCl for
15 min, rinsed with water and subsequently treated with a 1:3 v/v
H2O2: H2SO4 mixture at 120  C. The electrodes were then subjected
to repetitive voltage cycling between
0.2 and 1.6 V in 10% HClO4.
After thorough rinsing with water and ethanol, Au electrodes were
coated with self-assembled monolayers (SAMs) by overnight in-
cubation in ethanolic solutions containing 2 mM HS-(CH2)n-CH3
and 3 mM HS-(CH2)n-CH2OH. Before protein adsorption SAM-
coated electrodes were routinely cycled at 0.1 V s
1 within the
potential window required for each protein in 10 mM acetate
buffer, pH 4.6, containing 250 mM KNO3. Electrodes used for sub-
sequent experiments were those that showed a well behaved and
stable capacitive response only, with currents lower than 5 nA
measured at 0.1 V s
1 for n ¼ 15 and lower than 300 nA measured at
10 V s
1 for n ¼ 5. Effective areas of the working electrodes were
obtained by CV before SAM-coating using 20 mM Fe(CN)3
/4- 6 in
 U.A. Zitare et al. / Electrochimica Acta 294 (2019) 117e125 119

0.25 M KNO3 as redox probe. The obtained values ranged from 2.6 Raman microscope equipped with a CCD detector. Prior to mea-
to 7.9 mm2, with an average value of 6 mm2. The SAM-modified surement ca. 10 mL protein samples were placed and frozen at 77 K
electrodes were finally incubated in 0.1e0.5 mM protein solutions in a Linkam THMS 300 thermostat. RR spectra were acquired at
during 2 h for protein adsorption, and then transferred to the 0.5 cm
1 resolution. UVevis and RR determinations were per-
electrochemical cell. Measurements were performed in 10 mM formed in 10 mM acetate buffer, pH 4.6, containing 250 mM KNO3,
acetate buffer, pH 4.6, containing 250 mM KNO3. The solution vis- with and without addition of crowding agent.
cosity was adjusted by dissolving variable amounts of either su-
crose or polyethylene glycol 4000 (PEG4000) in the same buffer/ 3. Results and discussion
electrolyte mixture, thus maintaining constant ionic strength
throughout all the experiments. The temperature of the jacketed The present work aims to gain a deeper understanding of mo-
cell was varied employing a coupled circulation thermostat (Lauda lecular crowding effects on long-range protein ET and, specifically,
Alpha RA8) and continuously monitored with a thermocouple to dissect the responses of the redox site and the protein milieu to
(Fluke 51 II). CVs were typically acquired at scan rates between 50 viscous media. The proteins selected for this study are: (i) wild type
and 500 mV s
1 for the thicker SAMs, and 1e60 V s
1 for the azurin (Azu WT) from P. aeruginosa as a prototypical mononuclear
thinner films. All the CVs display the shape characteristic of T1 blue copper protein, (ii) the CuA-containing soluble domain of
surface-confined redox species and linear variations of the anodic the ba3 O2-reductase from T. thermophilus (Tt-CuA) as a prototypical
and cathodic currents with the scan rate. Protein films were quite binuclear purple copper protein and (iii) three chimeric proteins
stable for about 100 voltammetry cycles. For longer cycling we constructed by loop engineering of Tt-CuA where the sequences of
observe a small gradual loss of the CV signals without changes in the three loops that surround the metal site are replaced by those
peak positions and FWHM, which suggest slow desorption of the corresponding to other organisms to create novel T1 and CuA var-
protein film. CV measurements used throughout this work corre- iants (Fig. 1). The structure and spectroscopy of the WT and
spond to stable signals. Electrodes were replaced by freshly pre- chimeric proteins has been reported elsewhere [43,73e76,79,80].
pared ones as soon as a small drop of intensity was detected. Rate Remarkable features relevant to the present work are: (i) Tt-CuA
constants were obtained using Laviron's formalism [77], and acti- and WT Azu share the cupredoxin motif, but with some differences
vation parameters were estimated from Arrhenius plots in a tem-
perature range from ca. 5  Ce40  C.
Control kinetic experiments were performed using Creager's
method [78] based on alternating current voltammetry (ACV). ACVs
were acquired in stepped mode every 20 mV in a potential window
of 0.5 V centred on the reduction potential of each sample using a
rms amplitude of 10 mV. The range of frequencies was
1 Hze100 kHz for SAMs with n ¼ 3, 5 and 7, 0.3 Hz to 30 kHz for
n ¼ 10 and 0.03 Hze30 Hz for n ¼ 15.
Uncompensated resistance (Ru) was routinely determined using
the optimized impedance routine included in the Framework Data
Acquisition Software from Gamry (Version 6.33). For Au electrodes
coated with SAMs with n ¼ 5, i.e. the thinnest SAMs employed for
investigating viscosity effects, Ru values were typically 10 U in the
absence of thickening agent and 20 U at 5 cP. After protein
adsorption Ru slightly increases, reaching values of up to 40 and
50 U for viscosities of 1 and 5 cP, respectively, for the highest pro-
tein surface concentrations employed here of ca. 8 pmol cm
2,
which are attained with azurine. The highest currents obtained in
CV experiments that are used for subsequent quantitative treat-
ment were achieved for azurine films on SAMs with n ¼ 5 and scan
rates of 60 V s
1. These maximum currents were about 40 mA and
independent of the addition of thickening agents. Based on these
numbers, we can establish an upper limit for ohmic losses that is
2 mV for the most demanding conditions employed here, and
typically one order of magnitude lower or less.
CV measurements of bulk protein solutions were performed
using a home-made water jacketed non-isothermal three electrode
cell that requires ca. 40 mL samples with concentrations around
100 mM (10 mM buffer acetate, pH 4.6, 250 mM KNO3). Gold
working electrodes were coated with HS-(CH2)6-OH to prevent
protein adsorption.

2.3. Spectroscopic determinations

UVevis absorption spectra were acquired at 25  C with a
Thermo Scientific Evolution Array spectrophotometer employing
1 cm or 0.1 cm path length as required, placed into a jacketed cell-
holder for temperature control through a circulation thermostat
(Fisherbrand FBC620). Raman spectra were acquired in backscat-
tering geometry with 532 nm excitation using a Dilor XY800
Fig. 1. Top: Crystal structure of the CuA-containing soluble domain of the ba3 O2-
reductase from Thermus thermophilus (Tt-CuA; pdb 2CUA) and of the metal sites of the
Tt-CuA and Ami-CuA variants (pdb 2CUA and 5U7N, respectively). Bottom: sequences of
the three engineered loops. Letters in light blue denote first sphere ligands of the
copper ions. The last column indicates the organism from which the loop sequence was
adopted for the chimeras: Thermus thermophilus (Tt), Homo sapiens (Hs), Paracoccus
denitrificans (Pc) and Pseudomonas aeruginosa (Pa). (For interpretation of the references
to colour in this figure legend, the reader is referred to the Web version of this article.)
120 U.A. Zitare et al. / Electrochimica Acta 294 (2019) 117e125
that elicit a higher thermal stability in Tt-CuA [80e83]; (ii) Azu WT
is a canonical T1 blue copper center [3,84]; (iii) Tt-CuA is a canonical
CuA center, while Tt-3L is a slightly distorted CuA site that preserves
the mixed valence character and the typical purple colour [43,75];
(iv) Ami-CuA and Azu-CuA are distorted and greenish mononuclear
T1 sites [76].
The heterogeneous ET reactions of the five protein variants were
investigated by protein film electrochemistry using Au electrodes
coated with self-assembled monolayers (SAMs) of SH-(CH2)n-CH3/
SH-(CH2)n-CH2OH mixtures in 4/6 proportion and variable length
(n ¼ 3, 5, 7, 10 and 15). Proteins were adsorbed on the SAM-coated
electrodes and measured at pH 4.6 in 10 mM acetate buffer con-
taining 250 mM KNO3.
This combination of coating and electrolyte composition was
adopted from previous reports, which demonstrate that these
conditions optimize adsorption without altering the redox copper
centers [20,73,74,79]. Cyclic voltammetry (CV) experiments afford
quasi reversible responses in all cases (Figs. S1eS4) with FWHM
values close to ideal that, in average, yield charge transfer co-
efficients between 0.45 and 0.55. Moreover, the reduction poten-
tials are very similar to those obtained in solution under
comparable conditions (Table S1), thus confirming the integrity of
the adsorbed proteins. Surface-enhanced RR spectra of the SAM-
coated electrodes recorded before and after protein adsorption do
not exhibit changes in the intensity ratio of the DnC-S bands char-
acteristic of the gauche and trans conformations of the alkanethiols
found at 634 and 702 cm
1, respectively (data not shown) [85]. The
intensity ratio of the DnC-S bands is a sensitive marker of order in
SAMs and, therefore, its invariance strongly suggests that the SAM-
protein interactions are relatively weak and non-perturbative, in
agreement with the preservation of the reduction potentials of the
adsorbed proteins. From the integration of the voltammetric peaks
we obtain protein coverages that are typically below 4 pmol cm
2
for Azu WT and below 2 pmol cm
2 for the other proteins, which
represent less than 1/3 and 1/6 of full coverage, respectively, as
estimated using a crystallographic diameter of 40 Å for both
scaffolds.
Since Chidsey's seminal work [86], SAM-coated electrodes are
broadly used for kinetic studies because they allow the systematic
variation of protein-electrode electronic coupling through the
chain length of the thiols. Hence, we measured the ET rate constant
kET , for the different protein variants as a function of the SAM
thickness employing two different and independent experimental
approaches: Laviron's formalism [77] based on the peak separation
of CVs obtained at variable scan rates and Creager's method [78]
based on variable frequency ac voltammetry. Figs. S5 and S6 show
typical Laviron's working curves and trumpet plots, respectively,
while a representative Creager's plot is presented in Fig. S7. The two
methods yield almost identical kET values with a nearly 1:1 corre-
spondence (Figs. S8 and S9).
The results are summarized in Fig. 2 and Fig. S10, and are
characterized by an exponential distance dependence of kET at
thicker SAMs (n  10) and a softer dependency at thinner films that
tends to a plateau. Qualitatively similar results have been reported
by other authors for Azu WT and Tt-CuA, although with slightly
lower rate constants ascribable to the different experimental con-
ditions [20,28,30,87e89]. The long distance exponential decay,
with tunnelling decay factor b z 1 per methylene group, is the
fingerprint of a nonadiabatic ET mechanism [90]. The softer varia-
tion at shorter distances, on the other hand, suggests a change of
regime associated to the stronger electronic coupling.
Waldeck et al. observed a similar distance dependence for cy-
tochrome c coordinatively bound to pyridinyl-terminated alka-
nethiols and for Azu WT adsorbed on hydrophobic SAMs, and
Fig. 2. Normalized ET rate constants as a function of the SAM thickness. Each data
point is the average of at least three independent experiments performed at 25  C in
10 mM acetate buffer, pH 4.6, containing 0.25 M KNO3. Absolute kET values, as obtained
by Laviron's method, are displayed in Fig. S10.
ascribed it to a friction-controlled ET mechanism at shorter dis-
tances [25,26,91]. Under the working hypothesis that Waldeck's
proposal is correct, we reason out that the construction of chimeras
as those summarized in Fig. 1 offers a unique opportunity to
advance for the first time in assessing and dissecting biologically
relevant crowding effects on the ET reaction coordinate at different
levels of the hierarchical protein architecture and dynamics. To this
end, we first investigated the impact on the ET kinetics of adding
variable amounts of sucrose and PEG4000 (Fig. 3, Figs. S11 and S12).
These two chemicals were selected because they are commonly
used as crowding agents in biological studies due to their ability to
emulate in-cell high viscosities while minimizing specific in-
teractions [8]. The concentrations of the crowding agents were
adjusted to produce viscosities up to 4 cP, i.e. a typical cytosolic
value, and control experiments were performed up to 60 cP, which
correspond to the typical intramitochondrial viscosity [90e92].
In all these experiments electrolyte and buffer composition
were kept constant, thus fixing the ionic strength at I ¼ 250 mM
that, as shown in Fig. S13, is sufficiently high to warrant I-inde-
pendent kET values, even at high viscosities.
For all the protein variants we observe a drop of kET upon
addition of either crowding agent. When plotted against the solu-
tion viscosity h, we obtain a power law dependence of the form
kET fh
g (Fig. 3) which, for a given protein at a given SAM thickness,
is nearly identical for the two crowding agents, thus pointing out
that the kinetic effect most likely arises from the increase of the
bulk viscosity rather than due to specific interactions of sucrose and
PEG4000 with the different components of the SAM/protein sys-
tems. Similar results are obtained in control experiments with
extended viscosity range of up to ca. 60 cP (Fig. S14).
It is important to point out that additions of large amounts of
sucrose or PEG4000 do not affect the active sites, as resonance
Raman and electronic absorption spectra remain unaffected for all
the protein variants studied here (Figs. S15 and S16). In agreement
with these observations, CV responses are not affected by the
crowding agents aside from the increased peak separations
(Figs. S11 and S12). Indeed, reduction potentials remain largely
 U.A. Zitare et al. / Electrochimica Acta 294 (2019) 117e125
Fig. 3. Normalized ET rate constant as a function of the relative viscosity for proteins
adsorbed on SAMs with n ¼ 5 (25  C; pH 4.6, 0.25 M KNO3). (A) Mononuclear T1
centers. (B) Binuclear CuA sites. (C) Comparison of Azu WT and Tt-CuA for experiments
performed with sucrose (empty symbols) or PEG4000 (filled symbols). The lines are
fittings to kETðhÞ ¼ kETðh0 Þ h
g .
constant over a broad concentration range of crowding agents, with
only some minor variations for some of the protein variants and no
changes for the others (Fig. S17), which may reflect slightly
different sensitivity to changes in the dielectric constant. Note that
the addition of both crowding agents not only rises the viscosity but
also decreases the static dielectric constant (εs ) and increases the
optical dielectric constant (εop ) of the solution, which results in a
reduction of the Pekar factor ε
1
1
op
εs (Fig. S18). In terms of clas-
sical Marcus theory this would imply lower outer sphere reorga-
nization energies and, therefore, slightly faster ET. The results
shown in Fig. 3 suggest that the accelerating effect of a lower Pekar
factor is overcompensated by the slowing down effect of the higher
viscosity, thus pointing out to a friction-controlled ET reaction.
Interestingly, ET rates are sensitive to the solution viscosity not
only in the plateau region of the kET vs distance curves but,
essentially for all chain lengths with n < 15, which approximately
corresponds to tunnelling distances < 23 Å [95]. As shown in Fig. 4
for two representative examples, the g factors exhibit a sigmoidal
distance dependence that denotes the interplay between through-
SAM electron tunnelling times and friction-controlled protein
dynamics.
Note that at the sub-monolayer coverages employed here, kET
121
Fig. 4. Frictional parameter g as a function of the SAM thickness for Azu WT and Tt-
CuA using sucrose as crowding agent. All measurements were performed at 25  C in
10 mM acetate buffer (pH 4.6, 0.25 M KNO3).
and activation free energy values are insensitive to protein surface
concentration (Fig. S19), thereby confirming that the observed ki-
netic effects are ascribable to the external crowding agent. For
SAMs with n ¼ 5, which roughly corresponds to tunnelling dis-
tances of ca 10 Å, we observe a 30e45% drop of kET at h ¼ 4 cP, and
more than 90% drop at 55 cP. Interestingly, in-cell local micro-
viscosities have been reported to vary between 1 and 400 cP [92],
with values of around 35e63 cP in healthy mitochondria and one
order of magnitude higher values under apoptotic conditions
[93,94].
The ET rate is given by the product of two terms: a
FranckeCondon factor, which accounts for the probability of
achieving donor-acceptor energetic degeneracy through thermal
fluctuations, and the electronic coupling, which decays exponen-
tially with distance and represents the probability of electron
tunnelling between degenerate states. Both terms may be affected
by molecular crowding through the increased viscosity. As dis-
cussed in detail by Matyushov and coworkers [96,97], the first term
is sensitive to the ratio between the reactant-product relaxation
time (trelax ) and the reaction time (tr ). Marcus theory works under
the assumption that trelax ≪tr , which may not be valid in slowly
relaxing media, thus leading to a reduced phase sub-space acces-
sible to thermal exploration within the reaction time scale. This
may result in a decrease of the apparent Gibbs energy barrier and
reorganization energy with respect to the Marcus equilibrium
values. Moreover, Matyushov et al. [98]. concluded that for the
electrochemical ET of cytochrome c the pre-exponential term of the
ET rate constant is not affected by solvent dynamics. In this sce-
nario, one should expect an increase of kET upon raising the solvent
viscosity, as a result of lowering the activation barrier. The experi-
mental results obtained for the copper proteins studied here show
the opposite trend, which strongly suggests that the system does
not reach the limiting case of trelax ≪tr but, instead, the reaction
proceeds in an intermediate regime where the viscosity effect is
dominated by the pre-exponential factor. These experimental
conditions are better described by Zusman equation for electro-
chemical reduction at zero overpotential of adsorbed species
[91,99]:
 !
rHDA j2 p3 kB T

1=2 
l l 

4k
kET ¼ e BT ln (1)
p3 t2s kB T lE
where l is the classical reorganization energy, ts is the solvent
relaxation time, kB is Boltzmann constant, HDA is the electronic
coupling matrix element, r is the density of states at the electrode
surface and - is the reduced Planck constant. In a usual first order
122 U.A. Zitare et al. / Electrochimica Acta 294 (2019) 117e125

approximation, ts can be expressed in terms of the longitudinal

relaxation time tL and, thus, as a function of the solvent viscosity h
and molar volume Vm :

εop 3hVm
ts ¼ tL ¼ (2)
εs RT
Assuming a simple Debye solvent model, the viscosity can be
described in terms of Andrade's empirical equation:


DG#
h ¼ A exp s
(3)
RT

where A is an empirical pre-exponential parameter and DG#s is the

activation free energy for the solvent viscous flow.
As proposed by Waldeck and co-workers, the empirical power
law kET fh
g, together with equations (1)e(3) leads to the
following approximated expression for the electrochemical ET rate
constant at zero driving force [25,26,91]:
rffiffiffiffiffiffiffiffi
#
εs RT l DG#
ET þ gDGs
kET ¼ exp
(4)
3Ag Vm εop p3 RT

where DG# ET ¼ l=4 is the classical Marcus activation free energy for
ET. This equation predicts that for a friction-controlled ET reaction
the overall apparent activation free energy DG#app, contains a first
term that represents the intrinsic ET reorganization energy of the
system and a second term, gDG#
s ; which accounts for the temper-
ature dependence of the medium relaxation dynamics. DG#
app can
be estimated from the temperature dependence of kET . Arrhenius
and Eyring treatments of these data yield essentially identical re-
sults, in agreement with previous observations for similar copper
proteins [20,74,87] that the entropic contribution is negligibly
small and, therefore, DG# #
app zDH app . Arrhenius plots obtained in the Fig. 5. Empirical frictional parameter (g) and activation free energy for the milieu
absence of crowding agents for the five protein variants adsorbed frictional motion (DG#s ) for the copper proteins and wired Cyt c. Blue, green, red and
orange bars are data obtained in this work at 25  C in 10 mM acetate buffer (pH 4.6,
on SAMs with n ¼ 5 and n ¼ 15 are shown in Fig. S20, and the re- 0.25 M KNO3) and using HS-(CH2)5-CH3/HS-(CH2)5- CH2OH SAMs. Gray bars are data
sults are summarized in Table S1. Note that for all the protein var- taken from literature for WT azurin on HS-(CH2)5-CH3 SAMs (Azu WT (CH3)) and cy-
iants kET is independent of solvent viscosity when measured using tochrome c coordinated to a pyridinyl-terminated SAM (Cyt c (Py-SAM)). (For inter-
the thickest SAM, i.e. g ¼ 0 for n ¼ 15, hence we obtain pretation of the references to colour in this figure legend, the reader is referred to the
# # Web version of this article.)
DH#
app zDGET ¼ l=4 in these cases. DGs values can then be ob-
tained by subtracting DG#
ET measured at the thickest SAM from the
parameter as a measure of the dynamic coupling between the
DG#app values measured with thin films in the absence of crowding
redox metal site and the protein/solvent milieu. This observation is
agents, and dividing by the corresponding g factors obtained from
consistent with the structural rigidity of the Cu2S2 diamond core in
viscosity experiments with the thin SAMs. The two relevant fric-
CuA sites, which includes a CueCu covalent bond, compared to the
tional parameters g and DG# s obtained for SAMs with n ¼ 5 are more easily distorted T1 sites [84].
plotted in Fig. 5. Interestingly, the value of g seems to be a property
On the other hand, DG# s is similar, within experimental error, for
of the metal site, with an average value of 0.54 for the different
all the protein variants that share the Tt-CuA fold, with an average
native and non-native T1 centers, and an average value of 0.29 for
value of 0.037 eV. This value rises to 0.120 eV for Azu WT. While
Tt-CuA and Tt-3L. DG# s , in contrast, seems to be a specific property both types of proteins are characterized by a high rigidity that is
of the protein scaffold, with an average value of 0.037 eV for the Tt- regarded crucial to ensure efficient ET [80,81], the lower melting
CuA fold independently of the metal center incorporated, and a temperatures of T1 proteins and backbone mobility studies by NMR
value of 0.120 eV for the native azurin fold. reveal lower rigidity compared to CuA [80,82,83]. At first sight this
The parameter g is a measure of the degree of frictional control, suggests the counterintuitive idea that the activation parameter
i.e. of the influence of solvent/protein relaxation on the ET rate
DG#
s is higher for more flexible proteins, which is reinforced by the
[25,26]. It is also an empirical correction that accounts for the
even larger value reported in literature for the highly flexible cy-
distribution of relaxation times not contemplated in the Debye
solvent model [13]. Limiting values of 0 and 1 correspond to the tochrome c (Fig. 5) [26]. Our proposal is that DG#
s is not reporting

fully nonadiabatic and adiabatic regimes, respectively, while values
in between denote an intermediate regime with overdamped sol-
vent/protein dynamics, which is consistent with the observed
distance dependence of g (Fig. 4). The site specificity of g revealed
in the present work suggests a more distinct interpretation for this
on the overall protein flexibility. Instead, this scaffold-specific
parameter is a measure of the temperature-dependence of
protein-solvent motions that are relevant to the ET reaction
coordinate.
Given that the long-range ET rates for the systems studied here
 U.A. Zitare et al. / Electrochimica Acta 294 (2019) 117e125
are within a range of ca. 1e8000 Hz (Fig. 2 and Fig. S10), low fre-
quency protein-solvent motions may be critical for the ET reaction.
Moreover, protein-solvent dynamics may be affected by local
electric fields at the SAM/protein interface [61,100], which are
similar in magnitude to those estimated for biological membranes
[101,102], thus possibly increasing DG#s under in vivo conditions
with respect to diluted protein solution.
4. Conclusions
The present results highlight the need of explicitly considering
molecular crowding effects in protein ET reactions. Highly crowded
environments, such as the mitochondrial intermembrane space, are
characterized by microviscosities that are one to two orders of
magnitude higher than for diluted aqueous solutions, and by the
presence of high local electric fields imposed by the membrane
potentials that may affect protein-solvent dynamics. Under these
conditions nuclear motions relevant to the ET reaction coordinate
are overdamped, thus breaking down some of the underlying as-
sumptions of Marcus theory, and leading to a friction-controlled
mechanism for electron tunnelling distances shorter than ca.
24 Å. The degree of frictional control is determined by two pa-
rameters: (i) the frictional activation barrier, which we show to be
specific for the protein scaffold, and (ii) the dynamical coupling
between the redox site and the surrounding protein-solvent milieu,
which reports on the electronic structure and the functional fea-
tures of the electron transfer site. The first and second parameters
resemble Frauenfelder's a and bh fluctuations [44e46], respec-
tively, whose characteristic time scales are modulated by the local
electric fields and by the high viscosity prevailing in biological
electron-proton energy transduction. Overall, our strategy allows a
dissection of the effects that impact on ET under crowding condi-
tions, as well as it opens new possibilities of studying in detail the
impact of these empirical parameters in a central biological phe-
nomenon under physiological conditions.
Acknowledgements
Financial support from ANPCyT (PICT2015-0133) and UBACyT is
gratefully acknowledged. UAZ and JS are recipients of CONICET
fellowships. AJV and DHM are CONICET members.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.electacta.2018.10.069.
References
[1] J.R. Winkler, H.B. Gray, Electron flow through metalloproteins, Chem. Rev.
114 (2014) 3369e3380, https://doi.org/10.1021/cr4004715.
[2] C.C. Moser, M.M. Sheehan, N.M. Ennist, G. Kodali, C. Bialas, M.T. Englander,
B.M. Discher, P.L. Dutton, De Novo construction of redox active proteins, in:
Methods in Enzymology, Elsevier, 2016, pp. 365e388, https://doi.org/
10.1016/bs.mie.2016.05.048.
[3] J. Liu, S. Chakraborty, P. Hosseinzadeh, Y. Yu, S. Tian, I. Petrik, A. Bhagi, Y. Lu,
Metalloproteins containing cytochrome, ironesulfur, or copper redox cen-
ters, Chem. Rev. 114 (2014) 4366e4469, https://doi.org/10.1021/cr400479b.
[4] R.A. Marcus, On the theory of electron-transfer reactions. VI. Unified treat-
ment for homogeneous and electrode reactions, J. Chem. Phys. 43 (1965)
679e701, https://doi.org/10.1063/1.1696792.
[5] R.A. Marcus, N. Sutin, Electron transfers in chemistry and biology, Biochim.
Biophys. Acta Rev. Bioenergies (BBA) 811 (1985) 265e322, https://doi.org/
10.1016/0304-4173(85)90014-X.
[6] A. De la Lande, F. Cailliez, D. Salahub, Electron transfer reaction in enzymes:
vanilla Marcus theory and how to fix them if they do, in: Simulating Enzyme
Reactivity: Computational Methods in Enzyme Catalysis, 2017, pp. 89e149.
[7] R.J. Ellis, A.P. Minton, Join the crowd: cell biology, Nature 425 (2003) 27e28,
https://doi.org/10.1038/425027a.
123
[8] R.J. Ellis, Macromolecular crowding: obvious but underappreciated, Trends
Biochem. Sci. 26 (2001) 597e604, https://doi.org/10.1016/S0968-0004(01)
01938-7.
[9] R.J. Clarke, The dipole potential of phospholipid membranes and methods for
its detection, Adv. Colloid Interface Sci. 89e90 (2001) 263e281, https://
doi.org/10.1016/S0001-8686(00)00061-0.
[10] H. Sumi, R.A. Marcus, Dynamical effects in electron transfer reactions,
J. Chem. Phys. 84 (1986) 4894e4914, https://doi.org/10.1063/1.449978.
[11] M.J. Weaver, Dynamical solvent effects on activated electron-transfer re-
actions: principles, pitfalls, and progress, Chem. Rev. 92 (1992) 463e480,
https://doi.org/10.1021/cr00011a006.
[12] L.D. Zusman, Dynamical solvent effects in electron transfer reactions, Z. Phys.
Chem. 186 (1994) 1e29, https://doi.org/10.1524/zpch.1994.186.Part_1.001.
[13] I. Rips, J. Jortner, Dynamic solvent effects on outer-sphere electron transfer,
J. Chem. Phys. 87 (1987) 2090e2104, https://doi.org/10.1063/1.453184.
[14] D.N. Beratan, C. Liu, A. Migliore, N.F. Polizzi, S.S. Skourtis, P. Zhang, Y. Zhang,
Charge transfer in dynamical biosystems, or the treachery of (static) images,
Acc. Chem. Res. 48 (2015) 474e481, https://doi.org/10.1021/ar500271d.
[15] A.K. Mishra, D.H. Waldeck, A unified model for the electrochemical rate
constant that incorporates solvent dynamics, J. Phys. Chem. C 113 (2009)
17904e17914, https://doi.org/10.1021/jp9052659.
[16] D.V. Matyushov, Protein electron transfer: is biology (thermo)dynamic?
J. Phys. Condens. Matter 27 (2015), 473001 https://doi.org/10.1088/0953-
8984/27/47/473001.
[17] M. Herv
as, J.A. Navarro, Effect of crowding on the electron transfer process
from plastocyanin and cytochrome c6 to photosystem I: a comparative study
from cyanobacteria to green algae, Photosynth. Res. 107 (2011) 279e286,
https://doi.org/10.1007/s11120-011-9637-1.
[18] B.G. Schlarb-Ridley, H. Mi, W.D. Teale, V.S. Meyer, C.J. Howe, D.S. Bendall,
Implications of the effects of viscosity, macromolecular crowding, and
temperature for the transient interaction between cytochrome f and plas-
tocyanin from the cyanobacterium Phormidium laminosum, Biochemistry 44
(2005) 6232e6238, https://doi.org/10.1021/bi047322q.
[19] Q. Chi, J. Zhang, J.E.T. Andersen, J. Ulstrup, Ordered assembly and controlled
electron transfer of the blue copper protein azurin at gold (111) single-
crystal substrates, J. Phys. Chem. B 105 (2001) 4669e4679, https://doi.org/
10.1021/jp0105589.
[20] K. Fujita, N. Nakamura, H. Ohno, B.S. Leigh, K. Niki, H.B. Gray, J.H. Richards,
Mimicking Protein
Protein electron Transfer: voltammetry of Pseudomonas
aeruginosa azurin and the Thermus thermophilus CuA domain at u-deriv-
atized self-assembled-monolayer gold electrodes, J. Am. Chem. Soc. 126
(2004) 13954e13961, https://doi.org/10.1021/ja047875o.
[21] D.H. Murgida, P. Hildebrandt, The heterogeneous electron transfer of cyto-
chrome c adsorbed on Ag electrodes coated with u-carboxyl alkanethiols. A
surface enhanced resonance Raman spectroscopic study, J. Mol. Struct.
565e566 (2001) 97e100, https://doi.org/10.1016/S0022-2860(00)00781-X.
[22] A. Kranich, H. Naumann, F.P. Molina-Heredia, H.J. Moore, T.R. Lee, S. Lecomte,
L.R. De, P. Hildebrandt, D.H. Murgida, Gated electron transfer of cytochrome
c6 at biomimetic interfaces: a time-resolved SERR study, Phys. Chem. Chem.
Phys. 11 (2009) 7390e7397, https://doi.org/10.1039/b904434e.
[23] P. Zuo, T. Albrecht, P.D. Barker, D.H. Murgida, P. Hildebrandt, Interfacial redox
processes of cytochrome b562, Phys. Chem. Chem. Phys. 11 (2009)
7430e7436, https://doi.org/10.1039/b904926f.
[24] D.A. Capdevila, W.A. Marmisolle
, F.J. Williams, D.H. Murgida, Phosphate
mediated adsorption and electron transfer of cytochrome c. A time-resolved
SERR spectroelectrochemical study, Phys. Chem. Chem. Phys. 15 (2013)
5386e5394, https://doi.org/10.1039/c2cp42044a.
[25] J. Wei, H. Liu, D.E. Khoshtariya, H. Yamamoto, A. Dick, D.H. Waldeck, Elec-
tron-transfer dynamics of cytochrome C: a change in the reaction mecha-
nism with distance, Angew. Chem. Int. Ed. 41 (2002) 4700e4703, https://
doi.org/10.1002/anie.200290021.
[26] H. Yue, D. Khoshtariya, D.H. Waldeck, J. Grochol, P. Hildebrandt,
D.H. Murgida, On the electron transfer mechanism between cytochrome c
and metal electrodes. Evidence for dynamic control at short distances,
J. Phys. Chem. B 110 (2006) 19906e19913, https://doi.org/10.1021/
jp0620670.
[27] A. Avila, B.W. Gregory, K. Niki, T.M. Cotton, An electrochemical approach to
investigate gated electron transfer using a physiological model system: cy-
tochrome c immobilized on carboxylic acid-terminated alkanethiol self-
assembled monolayers on gold electrodes, J. Phys. Chem. B 104 (2000)
2759e2766, https://doi.org/10.1021/jp992591p.
[28] D.E. Khoshtariya, T.D. Dolidze, M. Shushanyan, K.L. Davis, D.H. Waldeck,
R. van Eldik, Fundamental signatures of short- and long-range electron
transfer for the blue copper protein azurin at Au/SAM junctions, Proc. Natl.
Acad. Sci. Unit. States Am. 107 (2010) 2757e2762, https://doi.org/10.1073/
pnas.0910837107.
[29] D.E. Khoshtariya, T.D. Dolidze, T. Tretyakova, D.H. Waldeck, R. van Eldik,
Electron transfer with azurin at AueSAM junctions in contact with a protic
ionic melt: impact of glassy dynamics, Phys. Chem. Chem. Phys. 15 (2013)
16515, https://doi.org/10.1039/c3cp51896e.
[30] L.J.C. Jeuken, J.P. McEvoy, F.A. Armstrong, Insights into gated electron-
transfer kinetics at the Electrode
Protein interface: a square wave voltam-
metry study of the blue copper protein azurin, J. Phys. Chem. B 106 (2002)
2304e2313, https://doi.org/10.1021/jp0134291.
[31] H.K. Ly, M.A. Marti, D.F. Martin, D. Alvarez-Paggi, W. Meister, A. Kranich,
124 U.A. Zitare et al. / Electrochimica Acta 294 (2019) 117e125
I.M. Weidinger, P. Hildebrandt, D.H. Murgida, Thermal fluctuations deter-
mine the electron-transfer rates of cytochrome c in electrostatic and cova-
lent complexes, ChemPhysChem 11 (2010) 1225e1235, https://doi.org/
10.1002/cphc.200900966.
[32] A. Kranich, H.K. Ly, P. Hildebrandt, D.H. Murgida, Direct observation of the
gating step in protein electron transfer: electric-field-controlled protein
dynamics, J. Am. Chem. Soc. 130 (2008) 9844e9848, https://doi.org/10.1021/
ja8016895.
[33] D. Alvarez-Paggi, W. Meister, U. Kuhlmann, I. Weidinger, K. Tenger,
L. Zim
anyi, G. Ra
khely, P. Hildebrandt, D.H. Murgida, Disentangling electron
tunneling and protein dynamics of cytochrome c through a rationally
designed surface mutation, J. Phys. Chem. B 117 (2013) 6061e6068, https://
doi.org/10.1021/jp400832m.
[34] K. Henzler-Wildman, D. Kern, Dynamic personalities of proteins, Nature 450
(2007) 964e972, https://doi.org/10.1038/nature06522.
[35] V.C. Nashine, S. Hammes-Schiffer, S.J. Benkovic, Coupled motions in enzyme
catalysis, Curr. Opin. Chem. Biol. 14 (2010) 644e651, https://doi.org/
10.1016/j.cbpa.2010.07.020.
[36] G. Wei, W. Xi, R. Nussinov, B. Ma, Protein ensembles: how does nature
harness thermodynamic fluctuations for life? The diverse functional roles of
conformational ensembles in the cell, Chem. Rev. 116 (2016) 6516e6551,
https://doi.org/10.1021/acs.chemrev.5b00562.
[37] X.J. Jordanides, M.J. Lang, X. Song, G.R. Fleming, Solvation dynamics in pro-
tein environments studied by photon echo spectroscopy, J. Phys. Chem. B
103 (1999) 7995e8005, https://doi.org/10.1021/jp9910993.
[38] J.T. King, K.J. Kubarych, Site-specific coupling of hydration water and protein
flexibility studied in solution with ultrafast 2d-IR spectroscopy, J. Am. Chem.
Soc. 134 (2012) 18705e18712, https://doi.org/10.1021/ja307401r.
[39] D. Laage, T. Elsaesser, J.T. Hynes, Water dynamics in the hydration shells of
biomolecules, Chem. Rev. 117 (2017) 10694e10725, https://doi.org/10.1021/
acs.chemrev.6b00765.
[40] S. Lampa-Pastirk, W.F. Beck, Polar solvation dynamics in Zn(II)-Substituted
cytochrome c : diffusive sampling of the energy landscape in the hydro-
phobic core and solvent-contact layer, J. Phys. Chem. B 108 (2004)
16288e16294, https://doi.org/10.1021/jp0488113.
[41] D. Alvarez-Paggi, L. Hannibal, M.A. Castro, S. Oviedo-Rouco, V. Demicheli,
V. To
rtora, F. Tomasina, R. Radi, D.H. Murgida, Multifunctional cytochrome c:
learning new tricks from an old dog, Chem. Rev. 117 (2017) 13382e13460,
https://doi.org/10.1021/acs.chemrev.7b00257.
[42] L. Hannibal, F. Tomasina, D.A. Capdevila, V. Demicheli, V. To
rtora, D. Alvarez-
Paggi, R. Jemmerson, D.H. Murgida, R. Radi, Alternative conformations of
cytochrome c: structure, function, and detection, Biochemistry 55 (2016)
407e428, https://doi.org/10.1021/acs.biochem.5b01385.
[43] D. Alvarez-Paggi, U.A. Zitare, J. Szuster, M.N. Morgada, A.J. Leguto, A.J. Vila,
D.H. Murgida, Tuning of enthalpic/entropic parameters of a protein redox
center through manipulation of the electronic partition function, J. Am.
Chem. Soc. 139 (2017) 9803e9806, https://doi.org/10.1021/jacs.7b05199.
[44] H. Frauenfelder, S. Sligar, P. Wolynes, The energy landscapes and motions of
proteins, Science 254 (1991) 1598e1603, https://doi.org/10.1126/
science.1749933.
[45] H. Frauenfelder, G. Chen, J. Berendzen, P.W. Fenimore, H. Jansson,
B.H. McMahon, I.R. Stroe, J. Swenson, R.D. Young, A unified model of protein
dynamics, Proc. Natl. Acad. Sci. Unit. States Am. 106 (2009) 5129e5134,
https://doi.org/10.1073/pnas.0900336106.
[46] P.W. Fenimore, H. Frauenfelder, S. Magazù, B.H. McMahon, F. Mezei,
F. Migliardo, R.D. Young, I. Stroe, Concepts and problems in protein dy-
namics, Chem. Phys. 424 (2013) 2e6, https://doi.org/10.1016/
j.chemphys.2013.06.023.
[47] B.H. McMahon, H. Frauenfelder, P.W. Fenimore, The role of continuous and
discrete water structures in protein function, Eur. Phys. J. Spec. Top. 223
(2014) 915e926, https://doi.org/10.1140/epjst/e2014-02125-y.
[48] M.C. Bellissent-Funel, A. Hassanali, M. Havenith, R. Henchman, P. Pohl,
F. Sterpone, D. van der Spoel, Y. Xu, A.E. Garcia, Water determines the
structure and dynamics of proteins, Chem. Rev. 116 (2016) 7673e7697,
https://doi.org/10.1021/acs.chemrev.5b00664.
[49] M. Gao, C. Held, S. Patra, L. Arns, G. Sadowski, R. Winter, Crowders and
cosolvents-major contributors to the cellular milieu and efficient means to
counteract environmental stresses, ChemPhysChem 18 (2017) 2951e2972,
https://doi.org/10.1002/cphc.201700762.
[50] J. van den Berg, A.J. Boersma, B. Poolman, Microorganisms maintain
crowding homeostasis, Nat. Rev. Microbiol. 15 (2017) 309e318, https://
doi.org/10.1038/nrmicro.2017.17.
[51] G. Rivas, A.P. Minton, Macromolecular crowding in vitro , in vivo , and in
between, Trends Biochem. Sci. 41 (2016) 970e981, https://doi.org/10.1016/
j.tibs.2016.08.013.
[52] M.K. Kuimova, Mapping viscosity in cells using molecular rotors, Phys. Chem.
Chem. Phys. 14 (2012) 12671e12686, https://doi.org/10.1039/C2CP41674C.
[53] M. Feig, I. Yu, P. Wang, G. Nawrocki, Y. Sugita, Crowding in cellular envi-
ronments at an atomistic level from computer simulations, J. Phys. Chem. B
121 (2017) 8009e8025, https://doi.org/10.1021/acs.jpcb.7b03570.
[54] J.M. Herrmann, J. Riemer, The intermembrane space of mitochondria, Anti-
oxidants Redox Signal. 13 (2010) 1341e1358, https://doi.org/10.1089/
ars.2009.3063.
[55] F.-N. Vo € gtle, J.M. Burkhart, H. Gonczarowska-Jorge, C. Kücükko€ se, A.A. Taskin,
D. Kopczynski, R. Ahrends, D. Mossmann, A. Sickmann, R.P. Zahedi,
C. Meisinger, Landscape of submitochondrial protein distribution, Nat.
Commun. 8 (2017), https://doi.org/10.1038/s41467-017-00359-0.
[56] J. Gu, M. Wu, R. Guo, K. Yan, J. Lei, N. Gao, M. Yang, The architecture of the
mammalian respirasome, Nature 537 (2016) 639e643, https://doi.org/
10.1038/nature19359.
[57] R. Guo, S. Zong, M. Wu, J. Gu, M. Yang, Architecture of human mitochondrial
respiratory megacomplex I 2 III 2 IV 2, Cell 170 (2017) 1247e1257, https://
doi.org/10.1016/j.cell.2017.07.050, e12.
[58] C. Lu, D. Prada-Gracia, F. Rao, Structure and dynamics of water in crowded
environments slows down peptide conformational changes, J. Chem. Phys.
141 (2014), 045101, https://doi.org/10.1063/1.4891465.
[59] R. Harada, Y. Sugita, M. Feig, Protein crowding affects hydration structure
and dynamics, J. Am. Chem. Soc. 134 (2012) 4842e4849, https://doi.org/
10.1021/ja211115q.
[60] M. Malferrari, F. Francia, G. Venturoli, Retardation of protein dynamics by
trehalose in dehydrated systems of photosynthetic reaction centers. Insights
from electron transfer and thermal denaturation kinetics, J. Phys. Chem. B
119 (2015) 13600e13618, https://doi.org/10.1021/acs.jpcb.5b02986.
[61] B. De, D.A. Paggi, F. Doctorovich, P. Hildebrandt, D.A. Estrin, D.H. Murgida,
M.A. Marti, Molecular basis for the electric field modulation of cytochrome c
structure and function, J. Am. Chem. Soc. 131 (2009) 16248e16256, https://
doi.org/10.1021/ja906726n.
[62] I. Zoi, D. Antoniou, S.D. Schwartz, Electric fields and fast protein dynamics in
enzymes, J. Phys. Chem. Lett. 8 (2017) 6165e6170, https://doi.org/10.1021/
acs.jpclett.7b02989.
[63] S.D. Fried, S.G. Boxer, Electric fields and enzyme catalysis, Annu. Rev. Bio-
chem. 86 (2017) 387e415, https://doi.org/10.1146/annurev-biochem-
061516-044432.
[64] P.K. Nandi, Z. Futera, N.J. English, Perturbation of hydration layer in solvated
proteins by external electric and electromagnetic fields: insights from non-
equilibrium molecular dynamics, J. Chem. Phys. 145 (2016), 205101,
https://doi.org/10.1063/1.4967774.
[65] I. Danielewicz-Ferchmin, A.R. Ferchmin, Review: water at ions, biomolecules
and charged surfaces, Phys. Chem. Liq. 42 (2004) 1e36, https://doi.org/
10.1080/0031910031000120621.
[66] M. Druchok, M. Holovko, Structural changes in water exposed to electric
fields: a molecular dynamics study, J. Mol. Liq. 212 (2015) 969e975, https://
doi.org/10.1016/j.molliq.2015.03.032.
[67] R. Richert, Relaxation time and excess entropy in viscous liquids: electric
field versus temperature as control parameter, J. Chem. Phys. 146 (2017),
064501, https://doi.org/10.1063/1.4975389.
[68] C. Rønne, L. Thrane, P.O. Åstrand, A. Wallqvist, K.V. Mikkelsen, S.R. Keiding,
Investigation of the temperature dependence of dielectric relaxation in
liquid water by THz reflection spectroscopy and molecular dynamics simu-
lation, J. Chem. Phys. 107 (1997) 5319e5331, https://doi.org/10.1063/
1.474242.
[69] A.M. Saitta, F. Saija, P.V. Giaquinta, Ab initio molecular dynamics study of
dissociation of water under an electric field, Phys. Rev. Lett. 108 (2012),
https://doi.org/10.1103/PhysRevLett.108.207801.
[70] J. Sýkora, P. Kapusta, V. Fidler, M. Hof, On what time scale does solvent
relaxation in phospholipid bilayers happen? Langmuir 18 (2002) 571e574,
https://doi.org/10.1021/la011337x.
[71] A. Vegiri, Reorientational relaxation and rotationaletranslational coupling in
water clusters in a d.c. external electric field, J. Mol. Liq. 110 (2004) 155e168,
https://doi.org/10.1016/j.molliq.2003.09.011.
[72] D. Zong, H. Hu, Y. Duan, Y. Sun, Viscosity of water under electric field:
anisotropy induced by redistribution of hydrogen bonds, J. Phys. Chem. B 120
(2016) 4818e4827, https://doi.org/10.1021/acs.jpcb.6b01686.
[73] G.N. Ledesma, D.H. Murgida, K.L. Hoang, H. Wackerbarth, J. Ulstrup,
A.J. Costa-Filho, A.J. Vila, The met axial ligand determines the redox potential
in CuA sites, J. Am. Chem. Soc. 129 (2007) 11884e11885, https://doi.org/
10.1021/ja0731221.
[74]

L.A. Abriata, D. Alvarez-Paggi, G.N. Ledesma, N.J. Blackburn, A.J. Vila,
D.H. Murgida, Alternative ground states enable pathway switching in bio-
logical electron transfer, Proc. Natl. Acad. Sci. U. S. A 109 (2012)
17348e17353, https://doi.org/10.1073/pnas.1204251109.
[75] M.N. Morgada, L.A. Abriata, U. Zitare, D. Alvarez-Paggi, D.H. Murgida, A.J. Vila,
Control of the electronic ground state on an electron-transfer copper site by
second-sphere perturbations, Angew. Chem. Int. Ed. 53 (2014) 6188e6192,
https://doi.org/10.1002/anie.201402083.
[76]

A. Espinoza-Cara, U.A. Zitare, D. Alvarez-Paggi, D.H. Murgida, A.J. Vila,
Biosynthesis of type 1 copper centers with unusual electronic and functional
features by loop engineering, Chem. Sci. 9 (2018) 6692e6702, https://
doi.org/10.1039/C85C01444B.
[77] E. Laviron, General expression of the linear potential sweep voltammogram
in the case of diffusionless electrochemical systems, J. Electroanal. Chem.
Interfacial Electrochem. 101 (1979) 19e28, https://doi.org/10.1016/S0022-
0728(79)80075-3.
[78] S.E. Creager, T.T. Wooster, A new way of using ac voltammetry to study
redox kinetics in electroactive monolayers, Anal. Chem. 70 (1998)
4257e4263, https://doi.org/10.1021/ac980482l.
[79] U. Zitare, D. Alvarez-Paggi, M.N. Morgada, L.A. Abriata, A.J. Vila, D.H. Murgida,
Reversible switching of redox-active molecular orbitals and electron transfer
pathways in CuA sites of cytochrome c oxidase, Angew. Chem. Int. Ed. 54
(2015) 9555e9559, https://doi.org/10.1002/anie.201504188.
 U.A. Zitare et al. / Electrochimica Acta 294 (2019) 117e125
[80] M.E. Zaballa, L.A. Abriata, A. Donaire, A.J. Vila, Flexibility of the metal-binding
region in apo-cupredoxins, Proc. Natl. Acad. Sci. Unit. States Am. 109 (2012)
9254e9259, https://doi.org/10.1073/pnas.1119460109.
[81] S.A. Pe
rez-Henarejos, L.A. Alcaraz, A. Donaire, Blue Copper Proteins: a rigid
machine for efficient electron transfer, a flexible device for metal uptake,
Arch. Biochem. Biophys. 584 (2015) 134e148, https://doi.org/10.1016/
j.abb.2015.08.020.
[82] J. Chaboy, S. Díaz-Moreno, I. Díaz-Moreno, M.A. De la Rosa, A. Díaz-Quintana,
How the local geometry of the Cu-binding site determines the thermal
stability of blue copper proteins, Chem. Biol. 18 (2011) 25e31, https://
doi.org/10.1016/j.chembiol.2010.12.006.
[83] P. Wittung-Stafshede, B.G. Malmstro € m, D. Sanders, J.A. Fee, J.R. Winkler,
H.B. Gray, Effect of redox state on the folding free energy of a thermostable
electron-transfer metalloprotein: the Cu a domain of cytochrome oxidase
from Thermus thermophilus y, Biochemistry 37 (1998) 3172e3177, https://
doi.org/10.1021/bi972901z.
[84] E.I. Solomon, R.G. Hadt, Recent advances in understanding blue copper
proteins, Coord. Chem. Rev. 255 (2011) 774e789, https://doi.org/10.1016/
j.ccr.2010.12.008.
[85] W.A. Marmisolle
, D.A. Capdevila, L.L. De, F.J. Williams, D.H. Murgida, Self-
assembled monolayers of NH2-terminated thiolates: order, pKa, and specific
adsorption, Langmuir 29 (2013) 5351e5359, https://doi.org/10.1021/
la304730q.
[86] C.E.D. Chidsey, Free energy and temperature dependence of electron transfer
at the metal-electrolyte interface, Science 251 (1991) 919e922, https://
doi.org/10.1126/science.251.4996.919.
[87] S. Monari, G. Battistuzzi, C.A. Bortolotti, S. Yanagisawa, K. Sato, C. Li, I. Salard,
D. Kostrz, M. Borsari, A. Ranieri, C. Dennison, M. Sola, Understanding the
mechanism of short-range electron transfer using an immobilized cupre-
doxin, J. Am. Chem. Soc. 134 (2012) 11848e11851, https://doi.org/10.1021/
ja303425b.
[88] K. Yokoyama, B.S. Leigh, Y. Sheng, K. Niki, N. Nakamura, H. Ohno,
J.R. Winkler, H.B. Gray, J.H. Richards, Electron tunneling through Pseudo-
monas aeruginosa azurins on SAM gold electrodes, Inorg. Chim. Acta. 361
(2008) 1095e1099, https://doi.org/10.1016/j.ica.2007.08.022.
[89] Q. Chi, O. Farver, J. Ulstrup, Long-range protein electron transfer observed at
the single-molecule level: in situ mapping of redox-gated tunneling reso-
nance, Proc. Natl. Acad. Sci. Unit. States Am. 102 (2005) 16203e16208,
https://doi.org/10.1073/pnas.0508257102.
[90] D.H. Murgida, P. Hildebrandt, Redox and redox-coupled processes of heme
proteins and enzymes at electrochemical interfaces, Phys. Chem. Chem. Phys.
125
7 (2005) 3773e3784, https://doi.org/10.1039/b507989f.
[91] D.E. Khoshtariya, T.D. Dolidze, L.D. Zusman, D.H. Waldeck, Observation of the
turnover between the solvent friction (overdamped) and tunneling
(nonadiabatic) charge-transfer mechanisms for a Au/Fe(CN)63-/4- electrode
process and evidence for a freezing out of the Marcus barrier, J. Phys. Chem.
A 105 (2001) 1818e1829, https://doi.org/10.1021/jp0041095.
[92] T. Liu, X. Liu, D.R. Spring, X. Qian, J. Cui, Z. Xu, Quantitatively mapping cellular
viscosity with detailed organelle information via a designed PET fluorescent
probe, Sci. Rep. 4 (2014) 5418, https://doi.org/10.1038/srep05418.
[93] Z. Yang, Y. He, J.-H. Lee, N. Park, M. Suh, W.-S. Chae, J. Cao, X. Peng, H. Jung,
C. Kang, J.S. Kim, A self-calibrating bipartite viscosity sensor for mitochon-
dria, J. Am. Chem. Soc. 135 (2013) 9181e9185, https://doi.org/10.1021/
ja403851p.
[94] N. Jiang, J. Fan, S. Zhang, T. Wu, J. Wang, P. Gao, J. Qu, F. Zhou, X. Peng, Dual
mode monitoring probe for mitochondrial viscosity in single cell, Sensor.
Actuator. B Chem. 190 (2014) 685e693, https://doi.org/10.1016/
j.snb.2013.09.062.
[95] D.H. Murgida, P. Hildebrandt, Heterogeneous electron transfer of cytochrome
c on coated silver electrodes. Electric field effects on structure and redox
potential, J. Phys. Chem. B 105 (2001) 1578e1586, https://doi.org/10.1021/
jp003742n.
[96] D.V. Matyushov, Protein electron transfer: dynamics and statistics, J. Chem.
Phys. 139 (2013), 025102, https://doi.org/10.1063/1.4812788.
[97] D.V. Matyushov, M.D. Newton, Electrode reactions in slowly relaxing media,
J. Chem. Phys. 147 (2017), 194506, https://doi.org/10.1063/1.5003022.
[98] S.S. Seyedi, M.M. Waskasi, D.V. Matyushov, Theory and electrochemistry of
cytochrome c, J. Phys. Chem. B 121 (2017) 4958e4967, https://doi.org/
10.1021/acs.jpcb.7b00917.
[99] L.D. Zusman, Outer-sphere electron transfer reactions at an electrode, Chem.
Phys. 112 (1987) 53e59, https://doi.org/10.1016/0301-0104(87)85021-8.
[100] L. Khoa, N. Wisitruangsakul, M. Sezer, J.J. Feng, A. Kranich, I.M. Weidinger,
I. Zebger, D.H. Murgida, P. Hildebrandt, Electric-field effects on the interfacial
electron transfer and protein dynamics of cytochrome c, J. Electroanal. Chem.
660 (2011) 367e376, https://doi.org/10.1016/j.jelechem.2010.12.020.
[101] R.J. Clarke, The dipole potential of phospholipid membranes and methods for
its detection, Adv. Colloid Interface Sci. 89e90 (2001) 263e281, https://
doi.org/10.1016/S0001-8686(00)00061-0.
[102] J.K. Staffa, L. Lorenz, M. Stolarski, D.H. Murgida, I. Zebger, T. Utesch, J. Kozuch,
P. Hildebrandt, Determination of the local electric field at Au/SAM interfaces
using the vibrational Stark effect, J. Phys. Chem. C 121 (2017) 22274e22285,
https://doi.org/10.1021/acs.jpcc.7b08434.