Fast proton-coupled electron transfer observed for a high-fidelity structural and functional [2Fe-2S] rieske model

Article abstract of DOI:10.1021/ja412449v

Rieske cofactors have a [2Fe-2S] cluster with unique {His ₂Cys₂} ligation and distinct Fe subsites. The histidine ligands are functionally relevant, since they allow for coupling of electron and proton transfer (PCET) during quinol oxidation in respiratory and photosynthetic ET chains. Here we present the highest fidelity synthetic analogue for the Rieske [2Fe-2S] cluster reported so far. This synthetic analogue 5^x- emulates the heteroleptic {His₂Cys ₂} ligation of the [2Fe-2S] core, and it also serves as a functional model that undergoes fast concerted proton and electron transfer (CPET) upon reaction of the mixed-valent (ferrous/ferric) protonated 5H^2- with TEMPO. The thermodynamics of the PCET square scheme for 5^x- have been determined, and three species (diferric 5^2-, protonated diferric 5H^-, and mixed-valent 5^3-) have been characterized by X-ray diffraction. pK_a values for 5H^- and 5H^2- differ by about 4 units, and the reduction potential of 5H^- is shifted anodically by about +230 mV compared to that of 5^2-. While the N-H bond dissociation free energy of 5H^2- (60.2 ± 0.5 kcal mol^-1) and the free energy, ΔG _CPET, of its reaction with TEMPO (-6.3 kcal mol^-1) are similar to values recently reported for a homoleptic {N₂/N₂}-coordinated [2Fe-2S] cluster, CPET is significantly faster for 5H^2- with biomimetic {N₂/S₂} ligation (k = (9.5 ± 1.2) × 10 ⁴ M^-1 s^-1, ΔH^? = 8.7 ± 1.0 kJ mol^-1, ΔS^? = -120 ± 40 J mol^-1 K^-1, and ΔG^? = 43.8 ± 0.3 kJ mol^-1 at 293 K). These parameters, and the comparison with homoleptic analogues, provide important information and new perspectives for the mechanistic understanding of the biological Rieske cofactor.

Full text of DOI:10.1021/ja412449v

Article
pubs.acs.org/JACS
Open Access on 02/07/2015
Fast Proton-Coupled Electron Transfer Observed for a High-Fidelity
Structural and Functional [2Fe−2S] Rieske Model
Antonia Albers,† Serhiy Demeshko,† Sebastian Dechert,† Caroline T. Saouma,‡,§ James M. Mayer,‡
and Franc Meyer*,†
†Institute of Inorganic Chemistry, Georg-August-University Gottingen, Tammannstrasse 4, D-37077 Gottingen, Germany
̈
̈
‡Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195-1700, United States
S
* Supporting Information
ABSTRACT: Rieske cofactors have a [2Fe−2S] cluster with
unique {His2Cys2} ligation and distinct Fe subsites. The histidine
ligands are functionally relevant, since they allow for coupling of
electron and proton transfer (PCET) during quinol oxidation in
respiratory and photosynthetic ET chains. Here we present the
highest fidelity synthetic analogue for the Rieske [2Fe−2S] cluster
reported so far. This synthetic analogue 5x− emulates the
heteroleptic {His2Cys2} ligation of the [2Fe−2S] core, and it also
serves as a functional model that undergoes fast concerted proton and electron transfer (CPET) upon reaction of the mixed-
valent (ferrous/ferric) protonated 5H2− with TEMPO. The thermodynamics of the PCET square scheme for 5x− have been
determined, and three species (diferric 52−, protonated diferric 5H−, and mixed-valent 53−) have been characterized by X-ray
diffraction. pKa values for 5H− and 5H2− differ by about 4 units, and the reduction potential of 5H− is shifted anodically by about
+230 mV compared to that of 52−. While the N−H bond dissociation free energy of 5H2− (60.2 0.5 kcal mol−1) and the free
energy, ΔG°CPET, of its reaction with TEMPO (−6.3 kcal mol−1) are similar to values recently reported for a homoleptic {N2/
N2}-coordinated [2Fe−2S] cluster, CPET is significantly faster for 5H2− with biomimetic {N2/S2} ligation (k = (9.5 1.2) × 104
M−1 s−1, ΔH‡ = 8.7 1.0 kJ mol−1, ΔS‡ = −120 40 J mol−1 K−1, and ΔG‡ = 43.8 0.3 kJ mol−1 at 293 K). These parameters,
and the comparison with homoleptic analogues, provide important information and new perspectives for the mechanistic
understanding of the biological Rieske cofactor.
For many years, synthetic analogues have contributed
significantly to elucidating the properties and electronic
structures of biological iron−sulfur cofactors,10 but models
for the Rieske cluster have remained elusive until recently. In
2008 we reported the first (and so far only) synthetic [2Fe−2S]
cluster that emulates the heteroleptic {N2/S2} ligation
characteristic for the biological site, (NEt4)21 (Figure 1,
left).11 While (NEt4)21 is an excellent structural and
INTRODUCTION
■
Rieske-type [2Fe−2S] clusters are unique biological electron
transfer (ET) cofactors that feature a heteroleptic ligand
environment distinct from that of common [2Fe−2S]
ferredoxins, with one of the Fe atoms ligated by two cysteine
thiolates and the other by two histidine imidazoles.1 Rieske
clusters serve as structural gates in bacterial oxygenase enzymes
that catalyze oxidative hydroxylation of aromatic compounds,
and they play an important role in the bifurcated Q-cycle of the
quinol-oxidizing cytochrome complexes in respiratory and
photosynthetic ET chains.2,3 Histidine ligation is functionally
relevant, since it enables coupling of electron and proton
transfer upon reaction of the diferric Rieske cluster with
hydroquinone substrates, producing the mixed-valent cluster
that is protonated at the His ligands.3,4 Redox potentials of
Rieske [2Fe−2S] clusters in bc-type proteins have indeed been
found to be pH-dependent and coupled to the protonation
state of the Fe-bound histidines.5 NMR investigations on a 15N-
labeled Rieske protein have revealed a change of the histidines’
pKa values from around 12.5 in the reduced mixed-valent
cluster to around 7.4/9.1 in the oxidized diferric cluster.6
However, mechanistic details of the hydroquinone oxidation
mediated by Rieske proteins, such as the sequence or
synchronism of proton and electron transfer, have remained a
topic of debate.6−9
Figure 1. The first (and so far only) structural Rieske model 1 (left)
and functional homoleptic Rieske models 2, 3, and 4 offering the
possibility of protonation at the backside of the N-ligand (right).
Received: December 12, 2013
Published: February 7, 2014
© 2014 American Chemical Society
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spectroscopic Rieske model in both the diferric and mixed-
properties. Diferric (NEt4)25 was synthesized via a stepwise
ligand exchange reaction starting from the tetrachloro-
coordinated [2Fe−2S] cluster (NEt4)2[Cl2FeS2FeCl2], in
close analogy to the synthesis of the first structural Rieske
model, (NEt4)21.11 To this end, phenylbis(benzimidazolyl)-
methane was first deprotonated with KH and then added to a
solution of (NEt4)2[Cl2FeS2FeCl2] in MeCN at −30 °C to
furnish the {N2} cap. 1,1′-Biphenyl-2,2′-dithiolate, after
deprotonation with KH, was subsequently attached as the
{S2} capping ligand. The integrity of 52− in solution has been
supported by ESI-MS, and no ligand scrambling was observed
(see the Supporting Information, Figure S1). Diffusion of
diethyl ether into a solution of diferric (NEt4)25 in MeCN led
to growth of crystals, but of rather low quality. Better quality
crystals suitable for X-ray analysis could be obtained by
diffusion of diethyl ether into a solution of (CoCp*2)25 in
MeCN at 4 °C; the molecular structure of the diferric cluster
anion is shown in Figure 3.
valent states (Mossbauer and EPR, respectively), the reduced
̈
mixed-valence species proved quite unstable and the lack of
peripheral N atoms at the bis(indole) ligand in (NEt4)21
precluded any functional studies toward proton-coupled
electron transfer (PCET).
Several robust diferric [2Fe−2S] complexes, 22− to 42−, with
homoleptic bis(benzimidazolate) ligation have been pub-
lished.12−15 In two cases these have allowed, just recently and
for the first time, structural characterization of synthetic [2Fe−
2S] analogues in their mixed-valent state (33−, 43−),13−16 and
even in the super-reduced diferrous state (44−).16 For mixed-
valent 23− and 43− significant valence delocalization was
inferred from Mossbauer and EPR spectroscopy (about 20%,
̈
class II according to Robin and Day),13,15 whereas the EPR
spectrum of mixed-valent 13− did not reflect delocalization to
such an extent. Thus, the unique Rieske-type heteroleptic
coordination of 13− seems to promote enhanced valence
localization. Homoleptic analogues 22− to 42− furthermore
provide peripheral N atoms akin to the histidine ligands in
Rieske cofactors, and their reversible protonation and their
redox activity toward PCET have thus been investigated during
the past two years. The twice-protonated neutral diferric cluster
4H2 could even be isolated and structurally characterized,15 and
for both systems 3x− and 4x− thermodynamic parameters of the
PCET square scheme have been elucidated.14,15 The
protonated mixed-valent species, upon reaction with TEMPO,
were found to undergo concerted proton−electron transfer
with rather similar rate constants on the order of 103 M−1 s−1
under pseudo-first-order conditions at 20 °C.
Figure 3. Left: schematic view of diferric cluster 52−. Right: molecular
structure of the anion of (CoCp*2)25 in the crystal (thermal
displacement ellipsoids set at 30% probability). For clarity all hydrogen
atoms have been omitted.
An important open question now remains regarding the
effect of the heteroleptic {N2/S2} ligation on the PCET
reaction. Here we present the first synthetic Rieske model, 52−,
that comprises all beneficial features of 12− to 42−, namely, the
characteristic {N2/S2} donor set that leads to enhanced valence
localization in the one-electron-reduced state and a potential
protonation site at the {N2} ligand backbone that is similar to
the His-ligated subunit in the natural archetype. 52− thus
represents the highest fidelity Rieske model so far and allows
for the effect of the electronic structure on the PCET reaction
to be evaluated. The thermodynamic square scheme (Figure 2)
is fully established, and three of the four species involved are
characterized by single-crystal X-ray diffraction.
(CoCp*2)25 crystallizes in the monoclinic space group P21/c.
Selected metric parameters are listed in Table 1, together with
selected data for three different biological Rieske clusters for
comparison.17−19 The Fe···Fe distance in 52− (2.687 Å) is
slightly shorter than in diferric 12− (2.703 Å) and the biological
systems (2.71−2.72 Å), but overall geometric parameters are in
good agreement. Further discussion is provided below.
III
Protonated Diferric Cluster H−FeNIIIFeS . To investigate
the left part of the PCET square scheme (Figure 2),
protonation and subsequent deprotonation experiments with
diferric (NEt4)25 and varying acids and bases (see the
Supporting Information for an overview) were followed by
UV−vis spectroscopy. Addition of 1 equiv of 2,6-DMPH(BF4)
(2,6-DMP = 2,6-dimethylpyridine) to a solution of (NEt4)25 in
MeCN at −20 °C led to evolution of a prominent absorption
band at 385 nm (4.7 × 104 M−1 cm−1) (Figure 4, left), in
analogy to what has been observed upon protonation of the
related homoleptic [2Fe−2S] cluster (NEt4)24.15 This band has
been found to indicate a tautomerism process at the particular
bis(benzimidazolate) ligand, where initial protonation at one of
the benzimidazolate N atoms induces migration of the
bridgehead methine proton to the other peripheral benzimida-
zolate N atom. As a consequence, the ligand backbone becomes
roughly planar and both peripheral N atoms of the
benzimidazolate groups finally carry a proton (Figure 5). A
similar tautomerism has previously been described for simple
bis(imidazolium) compounds.20 Furthermore, the band at 541
nm (6300 M−1 cm−1) for 52− is shifted to 568 nm (6100 M−1
cm−1) in 5H−, whereas the band at 450 nm (10000 M−1 cm−1)
is shifted to lower wavelengths at 433 nm (11000 M−1 cm−1).
Clean conversion is indicated by four isosbestic points at 556,
RESULTS AND DISCUSSION
■
Diferric Cluster FeNIIIFeS . Rieske model 52− was designed
III
with the same bis(benzimdazolate) capping ligand as was used
for homoleptic cluster 42−, because this ligand proved
advantageous with respect to solubility and crystallization
Figure 2. Square scheme of protonation and reduction reactions
involving [2Fe−2S] Rieske model 5x−. The subscripts denote the
{N2}- and {S2]-ligated Fe sites.
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Table 1. Selected Bond Lengths (Å) and Angles (deg) of Diferric Clusters (CoCp*2)25 and (NEt4)25H, Mixed-Valent (NEt4)35,
a
,
and Biological Rieske Clusters17−19
b
(CoCp*2)25
(NEt4)5H
2.694(1)
(NEt4)35
2.682(1)
SOFX17
2.719
RIE18
2.71
RFS19
2.72
d(Fe···Fe)
2.687(1)
d(FeN−μ-S)
d(FeS−μ-S)
d(FeN−N)
2.191(1)/2.205(1)
2.200(2)/2.206(2)
1.984(4)/1.988(4)
2.189(1)/2.194(1)
2.200(1)/2.216(1)
1.985(2)/1.988(2)
2.297(1)/2.298(1)
92.61(10)
2.241(2)/2.248(1)
2.210(2)/2.212(2)
2.057(4)/2.074(4)
2.335(2)/2.345 (2)
86.65(17)
2.258/2.259
2.267/2.263
2.100/2.083
2.348/2.332
92.12
2.23/2.25
2.24/2.25
2.13/2.16
2.22/2.29
90.78
2.28/2.31
2.35/2.34
2.19/2.23
2.24/2.31
90.52
b
d(FeS−S)
2.22(2)/2.44(2), 2.37(2)/2.17(2)
91.65(16)
∠(N−FeN−N)
∠(S−FeS−S)
∠(μS−FeN−μ-S)
∠(μS−FeS−μ-S)
b
102.8(6)/106.8(5)
104.45(3)
102.23(6)
109.73
105.61
110.19
104.88(6)
104.57(6)
104.90(3)
104.68(6)
106.24
105.62
109.14
103.80(3)
106.95(6)
105.81
105.64
105.70
a
SOFX = Rieske protein II from Sulfolobus acidocaldarius, RIE = soluble domain of Rieske protein from bovine mitochondrial bc1 complex, and RFS
b
= soluble domain of Rieske protein from spinach chloroplast b6 f complex. Disordered {S2} ligand (see the Supporting Information for details); the
true FeS−S bond length likely is an average value, 2.30 Å.
idine) to a solution of (NEt4)25 in DMF did not lead to full
conversion to 5H−, but 4 equiv is needed. We conclude that the
pKa of 2,2,6,6-TMPH(BF4) in DMF (≥19) is in the same range
as the pKa of 5H− and thus is too low to achieve full
protonation. Interestingly, λmax of the absorption characteristic
for the tautomerized ligand is shifted by about 5 nm if the
reaction is carried out in DMF instead of MeCN, suggesting the
involvement of solvent molecules in H-bonding. This, as well as
the tautomerism discussed above, has been confirmed by the X-
ray diffraction analysis of singly protonated diferric cluster
Figure 4. Left: addition of 0.5 (dark blue) and 1.0 (blue) equiv of 2,6-
(NEt4)5H, which could be crystallized via DMF/Et2O diffusion
DMPH(BF4) to 52− (black) in MeCN at −20 °C, generating 5H−.
at 4 °C. The solid-state structure clearly shows hydrogen bonds
Right: further addition of 0.5 (purple) and 1.0 (red) equiv of 2,6-
DMPH(BF4) to 5H− (blue), generating 5H2.
between the protonated benzimidazolate N and DMF solvent
molecules included in the crystal lattice (Figure 5, bottom
right). After crystallographic characterization of twice-proto-
nated 4H2,16 this represents the second synthetic [2Fe−2S]
cluster that could be isolated in protonated form and the first
that also emulates the protonated Rieske cluster with its
heteroleptic ligation. Geometric parameters of the cluster core
remain almost unchanged upon protonation (see Table 1),
which will be discussed below.
Addition of a second equivalent of 2,6-DMPH(BF4) (Figure
4, right) led to disappearance of the band at 385 nm
characteristic for 5H− that had emerged during the first
protonation event. This observation led us to conclude that
binding of a second proton to give 5H2 is possible and restores
the original situation at the C atom bridging the two
benzimidazolate moieties, disrupting conjugation within the
{N2} capping ligand just as in 52− (Figure 5). Clean conversion
is indicated by four isosbestic points at 537, 447, 283, and 258
nm. Shifts of the other absorptions are relatively minor.
However, the product 5H2 seemed to be unstable in MeCN at
−20 °C, and upon addition of DBU minor decomposition was
detected by the appearance of a new band at 424 nm
concomitant with broadening of all bands, especially in the
region at about 540 nm (see Figure S6 in the Supporting
Information).
Figure 5. Protonation of diferric 52− leading to the reversible
formation of 5H− and 5H2. Lower right: molecular structure of the
anion of (NEt4)5H·2DMF·Et2O in the crystal (thermal displacement
ellipsoids set at 30% probability). For clarity all hydrogen atoms except
the N−H atoms, which are hydrogen bonded to the two DMF
molecules, have been omitted.
442, 293, and 265 nm. Protonation to give 5H− proved to be
reversible, since the original spectrum of 52− was restored upon
addition of base (either diazabicycloundecane (DBU) or
phosphazene base 1-tert-butyl-2,2,4,4,4-pentakis-
(dimethylamino)-2λ5,4λ5-catenadiphosphazene (t-BuP2); see
the Supporting Information for formulas and abbreviations
and Figure S5 in the Supporting Information for the back-
titration).
1
Following the protonation events by H NMR in MeCN-d3
at room temperature showed the formation of a new signal at
about 14 ppm which has been attributed to the formation of an
NH group. Furthermore, the double set of signals for the {N2}
capping ligand turned into a single set, clearly revealing the
flattening of the bis(benzimidazolate) scaffold with resulting 2-
fold symmetry in 5H− (see Figures S3 and S4 in the Supporting
Information for NMR spectra). The process is almost reversible
upon addition of t-BuP2, though formation of minor amounts
In contrast to 2,6-DMPH(BF4), addition of 1 equiv of
2,2,6,6-TMPH(BF4) (2,2,6,6-TMP = 2,2,6,6-tetramethylpiper-
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of free ligand and an unknown paramagnetic species evidence
the limited stability of 5H− in solution at room temperature.
Addition of a second equivalent of 4-DMAPH(OTf) (4-DMAP
= 4-(dimethylamino)pyridine) did not lead to spectral changes
at −30 °C, suggesting that 4-DMAPH+ (pKa = 17.95)21 is a
weaker acid than 5H2, in contrast to 2,6-DMPH+ (pKa =
14.13).21
The pKa value of diferric 5H−, relevant for establishing the
square scheme in Figure 2, has been determined by protonation
of 52− with 1 equiv of 2,6-DMPH(BF4) and back-titration with
DBU (pKa = 24.34)21 in MeCN followed by UV−vis
spectroscopy under inert conditions at room temperature.
According to mass balance, a pKa of 23.6 0.3 was thus derived
(see the Supporting Information for details).
Mixed-Valent Cluster FeN FeS . Electrochemical proper-
ties of 52− were studied by cyclic voltammetry in MeCN/0.1 M
NBu4PF6 at various scan rates and at room temperature (Figure
6, left). The cluster undergoes two cathodic redox processes:
Figure 7. EPR spectrum of 53− in MeCN/0.1 M Bu4NPF6 measured
as frozen glass at 20 K. The red line is a powder simulation with g =
2.017, 1.934, and 1.854 and Gaussian line widths Γ = 8.5, 14, and 26
G.
II
III
spectrum gave g values of 2.017, 1.934, and 1.854, with an
average value gav = 1.935.
In accordance with the UV−vis results, the wide g anisotropy
of the EPR spectrum indicates that the unpaired electron in 53−
is largely localized at the {N2}-coordinated iron atom,22 in
analogy to what has been observed for reduced Rieske
cofactors. Table 2 compares g values for a series of natural
Table 2. EPR Data of Model Complexes 53−, 13−, and 43−
a
,
and Selected Rieske Proteins25−27
Figure 6. Left: cyclic voltammogram of 52− (c = 1.0 mM) in MeCN/
0.1 M Bu4NPF6 at rt vs Fc/Fc+. E1 = −1.43 V and E2 = −2.19 V at
various scan rates (v = 100, 200, 300, 500, and 1000 mV s−1). Right:
electrochemical reduction of 52− in MeCN/0.1 M Bu4NPF6 at rt at a
potential of −1.6 V. The course of reduction was followed by UV−vis
spectroscopy.
26
53−
13−
43−
Tt25
ISP bc1
Cyt b6f27
g1
g2
g3
gav
2.017
1.934
1.854
1.935
2.015
1.936
1.803
1.918
2.015
1.937
1.900
1.951
2.02
1.90
1.80
1.91
2.024
1.89
1.79
1.90
2.03
1.90
1.76
1.90
a
Tt = Rieske protein of Thermus thermophilus, ISP bc1 = bovine
mitochondrial Cyt bc1, and Cyt b6 f = cytochrome b6 f complex from
spinach.
the first chemically reversible reduction occurs at E1/21 = −1.43
V and the second quasi reversible reduction at E1/22 = −2.19 V
(vs Fc/Fc+). From the separation of the two redox waves a
conproportionation constant Kc = 7.1 × 1012 can be derived.
While Kc is 4 orders of magnitude smaller than the value
calculated for 43−,13 the stability of mixed-valent 53− against
disproportion is still relatively high.
and synthetic [2Fe−2S] clusters. Biological Rieske clusters
usually show an average value gav of 1.90−1.91,23 whereas
higher values are observed for common all-cysteinato-ligated
[2Fe−2S] ferredoxins (gav = 1.945−1.975).23 As Mouesca has
pointed out, electronic delocalization in [2Fe−2S] clusters
Mixed-valent 53− was thus generated by bulk electrolysis
starting from diferric 52− in MeCN/0.1 M Bu4NPF6 at room
temperature at an applied potential of −1.6 V. The course of
reduction was followed by UV−vis spectroscopy (Figure 6,
right), and clean conversion is reflected by an isosbestic point at
353 nm. Reduction led to an overall decrease of intensity in the
visible region of the spectrum, the band at 543 nm dropping to
4900 M−1 cm−1. Only a band at 330 nm assigned to a ligand to
metal charge transfer (LMCT) deriving from the {S2} ligand
increased in intensity (16300 M−1 cm−1). Both bands at 374
and 447 nm, assigned to CT transitions from the {N2} ligand
by comparison with homoleptic 52−,13,15 almost vanished,
suggesting that the reduction is localized at the {N2}-ligated
iron atom.
In another experiment after 50% of the reduction was
completed (to ensure that side products had not been formed
yet), a sample was taken and investigated by EPR spectroscopy
(Figure 7). The total spin of the mixed-valent species ST = 1/2
caused by antiferromagnetic coupling of FeIII and FeII gives rise
to a characteristic rhombic EPR spectrum. Simulation of the
tends to increase the average g value, gav = /3∑gi, toward the
1
free electron value (g = 2.0023).24 Therefore, the value gav
=
1.935 reflects increased valence localization in 53− compared to
the synthetic homoleptic {N2/N2} analogues 23− (gav = 1.940 in
DMF/0.25 M n-BuNClO4, 77 K)12 and 43− (gav = 1.951 in
DMF, 6 K),13 but electron localization is less pronounced than
in 13− (gav = 1.918).11
Increased valence localization in the new Rieske model 53−, if
compared to previous homoleptic Rieske models 23− and 43−
with two bis(benzimidazolate) capping ligands in their mixed-
valent states, reflects the reduced symmetry of the complex and
the heteroleptic {S2/N2} ligation, which leads to site preference
of the unpaired electron. Furthermore, the negatively charged
thiolate ligand is a σ- and π-donor, which stabilizes the higher
oxidation state (FeIII). Hence, the new model 53− more closely
emulates the electronic situation of the biological antetype than
previous models 23− and 43−.
The mixed-valent cluster was generated chemically by
reduction of (NEt4)25 with CoCp*2 in DMF at −20 °C,
giving microcrystalline (CoCp*2)(NEt4)25. Crystalline material
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of mixed-valent (NEt4)35 could be obtained from DMF after
addition of 1 equiv of NEt4Br and subsequent slow diffusion of
Et2O at 4 °C into the solution. (NEt4)35 crystallizes in the
monoclinic space group P21/n and represents the first exact
Rieske model with heteroleptic {N2/S2} ligation that has been
characterized by X-ray diffraction in the reduced state. This
now allows a unique comparison of the molecular structures of
a high-fidelity synthetic analogue for the Rieske cluster in three
relevant forms, namely, in the diferric 52−, the diferric
protonated 5H−, and the mixed-valent 53− states. Inspection
of the central [2Fe−2S] core shows that only minor changes
occur upon reduction or protonation (Figure 8), with the Fe···
Figure 9. Zero-field Mossbauer spectra of solid (NEt ) 5 (left) and
̈
4
2
(CoCp*)(NEt4)25 (right) at 80 K. Simulation of the data gave the
following parameters: (left) δ1 = 0.26 mm s−1 and ΔEQ1 = 0.52 mm
s
−1 (red), δ2 = 0.28 mm s−1 and ΔEQ2 = 1.16 mm s−1 (blue); (right) δ1
= 0.35 mm s−1 and ΔEQ1 = 1.26 mm s−1 (red), δ2 = 0.69 mm s−1 and
ΔEQ2 = 3.23 mm s−1 (blue).
distribution. Overall the Mossbauer data show good agreement
̈
with parameters found for (NEt4)21 and biological Rieske
clusters (see Table S2 in the Supporting Information).
Variable-temperature zero-field Mossbauer spectra of mixed-
̈
valent (CoCp*2)(NEt4)25 show two doublets at a ratio of
about 1:1 in the range from 6 to 200 K, as expected for a mixed-
valent [2Fe−2S] cluster with heteroleptic terminal coordina-
tion (spectrum at 80 K shown in Figure 9 (right), spectra at 6
and 200 K shown in the Supporting Information; see also Table
S3 in the Supporting Information). The doublets can be
assigned to the {N2}-coordinated FeN atom with an isomeric
shift typical for FeII and a large quadrupole splitting (δ2 = 0.69
mm s−1, ΔEQ2 = 3.23 mm s−1, 80 K) and to the {S2}-
coordinated FeIII atom featuring a smaller isomeric shift and
smaller splitting (δ1 = 0.35 mm s−1, ΔEQ1 = 1.26 mm s−1, 80
K). These values are in good agreement with data for biological
Rieske cofactors, though quadrupole splittings ΔEQ are
somewhat smaller for the latter (Table 3). In contrast to
Figure 8. Overlay of the molecular structures of diferric 52− (red),
mixed-valent 53− (blue), and protonated diferric 5H− (yellow).
Fe distance showing negligible variations (<0.02 Å). This
reflects the low reorganization energies of [2Fe−2S] clusters
that make them favorable electron transfer cofactors in biology.
Close comparison of the subtle structural changes upon
reduction (52− versus 53−) is interesting, however, because it
reveals that changes mainly occur around the {N2}-coordinated
iron atom: bonds between FeN and the μ-S elongate by ∼0.05
Å upon reduction, while bonds between FeS and the μ-S remain
essentially unchanged (<0.01 Å). Interestingly, in the case of
homoleptic {N2}-capped 32−/33− and 42−/43−, the bonds
between both Fe atoms and the μ-S lengthen by ∼0.03 Å upon
reduction, showing that the unpaired electron is delocalized in
43− (on the crystallographic time scale), but is largely localized
at the single {N2}-coordinated iron site in 53−. In line with
these considerations reduction of 52− leads to a more
pronounced lengthening of the bonds between FeN and the
{N2} capping ligand (0.08 Å) than for the bonds between FeS
and the {S2} capping ligand (0.04 Å). Fe−N bonds in the
homoleptic {N2}-capped clusters show averaged elongations of
0.07 Å (32−/33−) or 0.05 Å (42−/43−). The most prominent
structural difference was found for the N−FeN−N angle, which
shrinks by around 5° in mixed-valent 53− compared to diferric
52− and 5H−. All these crystallographic findings, though subtle,
corroborate that reduction of the Rieske model occurs at the
Table 3. Mossbauer Parameters (mm s−1) of
̈
(CoCp*2)(NEt4)25 and Biological Rieske Clusters in the
a
Reduced State
(CoCp*2)(NEt4)25,
Tt,25
4.2 K
ISP,29
4.2 K
T4MOC,30
4.2 K
6 K
δ1
0.34
0.70
1.29
3.24
0.36
0.31
0.74
0.63
3.05
0.43
0.25
0.73
0.70
2.95
0.48
0.30
0.72
0.71
3.07
0.42
δ2
ΔEQ1
ΔEQ2
Δδ
a
Tt = Rieske protein of Thermus thermophilus, ISP = Rieske protein of
Cyt bf complex from spinach, and T4MOC = Rieske protein from
Pseudomonas mendocina in Escherichia coli.
mixed-valent (NEt4)32 and (NEt4)34, in which cases the two
quadrupole doublets collapsed to a single quadrupole doublet
Fe site in accordance with EPR and Mossbauer spectroscopy
(see below). Selected geometric parameters are compiled in
Table 1.
at 200 K, electron hopping on the Mossbauer time scale cannot
be observed for (CoCp*2)(NEt4)25.
̈
̈
N
An empirical correlation for δ and the oxidation number (x)
of FeS4 units δ(x) = (1.43 − 0.40x) mm s−1 has been
reported.28 This would predict values of δ(III) = 0.23 mm s−1
and δ(II) = 0.63 mm s−1 and hence a difference of 0.4 mm s−1
for fully localized ferric and ferrous sites a and b. While for
homoleptic mixed-valent 43− only half as much (Δδ = 0.22 mm
s−1 at 4 K)13,15 was observed, the present heteroleptic Rieske
model 53− gives Δδ = 0.36 mm s−1 at 6 K (0.34 mm s−1 at 80
K), close to the expected value. Though this is still less than Δδ
in the range 0.42−0.48 mm s−1 observed for biological Rieske
The zero-field Mossbauer spectrum of diferric (NEt ) 5
̈
4
2
shows two quadrupole doublets at a ratio of 1:1 with isomeric
shifts δ1 = 0.26 mm s−1 and δ2 = 0.28 mm s−1, as expected for
two distinct ferric sites (Figure 9, left). Differences in
quadrupole splitting allow an assignment to the all-sulfur-
coordinated FeS (ΔEQ1 = 0.52 mm s−1) and the {N2}-capped
FeN (ΔEQ2 = 1.16 mm s−1); the larger quadrupole splitting in
the case of FeN reflects the increased electric field gradient
resulting from the higher asymmetry of electronic charge
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sites featuring full valence localization (Table 3), it reflects the
increased valence localization in 53− compared to previous
homoleptic models, in accordance with EPR data.
Protonation of Mixed-Valent Cluster FeN FeS . Proto-
nation of the mixed-valent cluster proved to be challenging
because of low stability of the resulting species. Instantaneous
degradation could be observed upon protonation at room
temperature. At −30 °C in MeCN the addition of 1 equiv of
2,6-DMPH(BF4) could be followed by UV−vis spectroscopy
(see Figure S7 in the Supporting Information), and only minor
5H−). However, protonation obviously is not complete upon
addition of 1 equiv of 4-DMAPH(OTf), since the original peak
c
at Ep = −1.50 V is still discernible. Addition of a second
equivalent of 4-DMAPH(OTf) caused the peak at Epc = −1.50
II
III
V to disappear, concomitant with a broadening of the peak at
Ep = −1.27 V assigned to 5H− and emergence of a third redox
c
c
event characterized by a peak at Ep = −1.02 V, shifted
anodically by +480 mV compared to 52− (Epc = −1.50 V). The
additional peak presumably reflects the presence of some twice-
protonated species 5H2. When base was then added, the
original redox wave characteristic of 52− (Epc = −1.50 V, E1/2
=
absorption changes were detected: The band at 328 nm (2.4 ×
−1
104 M−1 cm−1) drops by about 3000
M
cm−1, whereas
−1.43 V) reappeared, confirming chemical reversibility of the
protonation events (Figure 10).
absorption in the region between 350 and 650 nm rises by
about 2000 M−1 cm−1. The maximum at 550 nm (4800 M−1
cm−1) broadens and experiences a slight red shift (5500 M−1
cm−1). If handled at −30 °C throughout, addition of t-BuP2 did
lead to nearly full conversion back to the original spectrum of
53−. Since no intense band around 385 nm evolved upon
protonation of 53−, it can be assumed that mixed-valent 5H2−
does not undergo any tautomerism that was observed for
diferric 5H− .
The anodic shift of about +480 mV upon 2-fold protonation
of 52− is more than twice as large as the shift observed for
homoleptic clusters 32− and 42− after binding of two protons
(+240 and +200 mV, respectively).14,15 The difference reflects
that the two protons are bound to the same bis(benzimidazole)
ligand at the unique FeN site in 5H2, in close analogy to the
situation in Rieske proteins, while the two protons are bound to
two bis(benzimidazole) ligands at different Fe sites in
homoleptic 3H2 and 4H2. Indeed, a shift of +300 to +440
mV upon going from very low to very high pH (hence going
from the twice-protonated to the fully deprotonated form) has
been reported for biological Rieske clusters depending on the
type of protein;31 redox potentials for the intermediate singly
protonated forms have not yet been reported. Hence, the
electrochemical response to protonation for the synthetic
analogue 52− nicely emulates the properties of the biological
antetype.
EPR spectra of the singly protonated species 5H2− showed a
rhombic spectrum with g values of 1.994, 1.938, and 1.875 (gav
= 1.937), but with rather large line widths (60, 33, and 55 G)
even when spectra were recorded at low temperatures (10 K;
see the Supporting Information). The reasons for this are
unclear; a more detailed investigation and comparative analysis
of the EPR spectra of 43− and 53− and their protonated forms is
currently in progress.
The effect of protonation on the redox potentials was
investigated by cyclic voltammetry, focusing on the first
reduction wave that appears at E1/2 = −1.43 V vs Fc/Fc+ for
To establish a thermodynamic square scheme for the new
Rieske model, not only redox potentials but also the pKa value
of mixed-valent 5H2− was determined (Figure 11). To this end
5
2− (Figure 10). Addition of 1 equiv of 4-DMAPH(OTf) led to
the emergence of a new cathodic peak at Epc = −1.27 V, which
is anodically shifted by +230 mV compared to the cathodic
peak potential of the reversible couple for 52− (Ep = −1.50 V).
c
This leads to an estimated redox potential of −1.20 V for
protonated 5H− (assuming a similar shift Ep − E1/2 for 52− and
c
Figure 11. Square scheme summarizing thermodynamic parameters
for the second-generation Rieske model cluster in MeCN with
potentials referenced against Fc/Fc+.
5
3− was protonated with 1 equiv of 4-DMAPH(BF4) in MeCN-
d3 at low temperatures, and back-titration with phosphazene
base (tert-butylimino)tris(1-pyrrolidinyl)phosphorane (t-BuP1-
1
(pyrr)) (pKa = 28.42) was followed by H NMR at room
temperature (see the Supporting Information for details,
Figures S11 and S12). A pKa of 27.9 0.2 was thus determined
for 5H2− in MeCN. In comparison to the homoleptic model
Figure 10. Cyclic voltammogram of (NEt4)25 (c = 1.0 mM) in
MeCN/0.1 M Bu4NPF6 at rt vs Fc/Fc+ at a scan rate of 500 mV/s
(top). The redox potential is shifted upon addition of acid (second and
third pictures from top). Subsequent addition of t-BuP2 proves the
reversibility of the process (bottom).
complex 33− (pKa = 24.7
0.4), the present heteroleptic
Rieske-type cluster 53− is more basic, probably due to the
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increased valence localization and the more pronounced ferrous
character at the protonation site. pKa values of reduced Rieske
proteins (12.3−13.3; see above) are much lower than those of
53− and 33−. The comparability is limited, however, since those
values for Rieske proteins have been determined in aqueous
solution, and they are tuned by interactions with the
surrounding protein environment comprising, for instance,
several hydrogen bonds.32
The bond dissociation free energy (BDFE) of the N−H
bond of the protonated cluster 5H2− can be calculated from the
available pKa and E1/2 data (see the Supporting Information),33
giving BDFE = 60.2 0.5 kcal mol−1 (252 2 kJ mol−1). This
BDFE for the NH bond is close to the value found for the
related homoleptic model complex 3H2− (60.5 kcal mol−1),14
but about 10 kcal mol−1 less than that of Rieske protein RsRp
under basic conditions (71.5 kcal mol−1) and even 15 kcal
mol−1 less than the value obtained for the protonated form of
RsRp under acidic conditions (75.1 kcal mol−1).5e,33 The lower
BDFE, hence the weaker N−H bond in the model complexes,
is likely related to the differently charged ligands in the mixed-
valent state, namely, neutral histidines versus monoanionic
bis(benzimidazolate). The very similar BDFE values for 3H2−
and 5H2− evidence that heteroleptic ligation of the [2Fe−2S]
core does not play any role in this respect.
Since ΔG°PT = 184 kJ mol−1 and ΔG°ET = 72.4 kJ mol−1 are
larger than the activation energy for the concerted pathway
(ΔG‡ = 43.8 kJ mol−1), the stepwise pathways can be excluded.
ΔG‡ for 5H2−is about 10 kJ mol−1 smaller compared to the
value for cluster 3H2−;14 therefore, the rate constant at room
temperature is more than 1 order of magnitude higher for
heteroleptic 5H2− than for homoleptic 3H2−. ΔH‡ is of the
same order of magnitude (about 2 kJ mol−1 larger), while ΔS‡
for 5H2− is less negative than for 3H2−.14 The transition-state
parameter ΔH‡ obtained for homoleptic 4H2− is at least twice
as large as the value found for 5H2− (Table 4),15 because the
second-order rate constant is about 2 orders of magnitude
lower than the one derived for 5H2−.
Table 4. Thermodynamic and Kinetic Parameters for the
Reactions of TEMPO with 3H2−, 4H2−, and 5H2−
15
14
4H2−
3H2−
(in DMF)
(in MeCN)
5H2− (in MeCN)
ΔH⧧ (kJ mol−1
)
17.6 0.6
6.7 1.3
8.7 1.0
ΔS⧧ (J mol−1 K−1
)
−130
2
−159 10
2200 350
−120
5
a
k at 20 °C (M−1 s−1
ΔG⧧ at 20 °C
)
722 14
95000 12000
43.8 0.3
55.7 1.1
54.0 1.2
(kJ mol−1
)
BDFE(Fe−H)
253
4
252
2
(kJ mol−1
)
With those values at hand, a pKa of about 23.8−24.2 could be
calculated for diferric 5H− according to Hess’s law (see the
Supporting Information). This is in good agreement with the
value determined experimentally by UV−vis titration of 5H−
with DBU, 23.6 0.3 as described above. The difference in pKa
for mixed-valent 5H2− and diferric 5H− of about 4 units is in
accord with the change of the histidines’ pKa values from
around 12.5 in the reduced mixed-valence forms to around 7.4/
9.1 in the oxidized diferric forms of Rieske proteins.5
ΔG°CPET (kJ mol−1
)
−25.1
−26.4
a
At 25 °C.
The differences in activation parameters and in rate constant
k for the closely related complexes 3H2− and 4H2−, both with
homoleptic bis(benzimidazolate) ligation, can likely be
attributed to the different solvents used in those studies.14,15
Polar solvents such as DMF can interact with both reaction
partners, presumably decelerating the reaction; H-bonding
interaction between DMF and the N−H units of 4H2 had
indeed been detected by X-ray crystallography and by IR
spectroscopy,15 similar to what is seen here for the structure of
5H− in the solid state (vide supra). The significantly different
rate constants k for 3H2− and 5H2−, both measured in MeCN
solution, may appear counterintuitive at first sight, since
ΔG°CPET = −6.0 kcal mol−1 (−25.1 kJ mol−1) found for
3H2− is quite similar to the value determined for 5H2−. The
much lower activation energy ΔG‡ that leads to accelerated
PCET in the case of 5H2−, however, might be an effect of
increased localization of electron density in the Rieske-type
[2Fe−2S] core with its heteroleptic {N2/S2} ligation. It should
be noted that Fe−N bonds, upon reduction, elongate almost
equally in 52− and in homoleptic 32−, while the FeS−S bonds in
52− change much less. This might give rise to a smaller
reorganization energy, λ, during PCET, which in turn leads to
faster PCET between the cluster and TEMPO. It has been
shown before that Marcus’s theory, which had initially been
established for ET reactions, can also be applied to PCET
reactions.34,35 Thus, a small reorganization energy λ should be
advantageous not only for fast electron transfer, but for fast
PCET as well. However, at present it cannot be excluded that
steric effects, viz., a different TEMPO accessibility of the
backbone N−H groups caused by different substituents at the
nearby bridging C atom (Ph/H in 5H2− versus n-Pr/n-Pr in
3H2−), may also play a role; see Figure S19 in the Supporting
Information for illustrative space-filling models generated from
the crystal structures of mixed-valent 33− and 53−.
To examine its PCET reactivity, mixed-valent 5H2−
(generated in situ by addition of 1 equiv of 4-DMAPH(BF4)
to 53−) was treated with the nitroxyl radical TEMPO. Full
conversion to deprotonated diferric 52− and 1 equiv of
TEMPO-H was ascertained by 1H NMR spectroscopy. Because
of the moderate N−H BDFE of 5H2−, calculation of the free
energy of the reaction, ΔG°CPET (CPET = concerted proton
and electron transfer), gives a sizable value, −26.4 kJ mol−1 (see
the Supporting Information). To obtain mechanistic insight,
double-mixing stopped-flow measurements were undertaken at
varying temperatures under pseudo-first-order conditions using
an excess of TEMPO, yielding kinetic parameters of the
reaction (see the Supporting Information). At 20 °C, a second-
order rate constant k = (9.5
1.2) × 104 M−1 s−1 was
determined, and the transition-state parameters ΔH‡ = 8.7
1.0 kJ mol−1 and ΔS‡ = −120 40 J mol−1 K−1 were derived
from an Eyring plot (Figure S15, Supporting Information). For
the free energy of the transition state, ΔG‡, at 293 K a value of
43.8 0.3 kJ mol−1 was thus calculated (see the Supporting
Information).
To verify that the reaction follows a concerted and not a
stepwise pathway, the initial steps of the alternative pathways
were examined. These are, starting from 5H2− and TEMPO,
either proton transfer or electron transfer, leading to 53− (and
TEMPO-H•+) or 5H− (and TEMPO−), respectively. To
ascertain the favored pathway, the respective activation energies
have been compared. Since the activation energies ΔG‡ must be
at least as high as the free energies ΔG°CPET, these values are a
conservative lower limit to ΔG‡ (for calculation of the free
energies ΔG°PT and ΔG°ET, see the Supporting Information).
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Present Address
CONCLUSIONS
■
§C.T.S.: Department of Chemistry, University of Utah, 315 S.
1400 East, Salt Lake City, UT 84112.
In summary, we report a synthetic analogue for the Rieske
cofactor that not only emulates the heteroleptic {His2Cys2}
ligation of the biological antetype, but also represents a
functional model undergoing fast concerted electron and
proton transfer. All four species in the PCET square scheme
have been thoroughly characterized, and three of them, namely,
diferric 52−, protonated diferric 5H2−, and mixed-valent 53−,
could be studied by single-crystal X-ray diffraction. This
provides unprecedented structural information and reveals
that the [2Fe−2S] core undergoes only minor structural
changes upon protonation or reduction, which is in line with
low reorganization energies upon PCET. However, subtle
variations of the [2Fe−2S] core in the diferric 52− and mixed-
valent 53− states reflect that the additional electron is largely
localized at the N-coordinated Fe site, in accordance with EPR
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Eberhard Bothe and Dr. Eckhard Bill (Max
Planck Institute for Chemical Energy Conversion, Mulheim,
̈
Germany) for CPC and EPR measurements. Financial support
by the DFG (International Research Training Group GRK
1422 “Metal Sites in Biomolecules: Structures, Regulation and
Mechanisms”; see www.biometals.eu) and the Cusanuswerk
(Ph.D. fellowship for A.A.) is gratefully acknowledged. C.T.S.
(Grant GM099316) and J.M.M. (Grant GM50422) gratefully
acknowledge the U.S. National Institutes of Health.
and Mossbauer evidence. It is somewhat surprising and
̈
counterintuitive though that the more pronounced valence
localization caused by the heteroleptic {N2/S2} ligation does
not lead to larger core structural changes compared to
homoleptically coordinated [2Fe−2S] complexes.
REFERENCES
■
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fully elucidated. Both the difference in pKa for diferric 5H− and
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ASSOCIATED CONTENT
■
(15) Albers, A.; Bayer, T.; Demeshko, S.; Dechert, S.; Meyer, F.
Chem.Eur. J. 2013, 19, 10101.
S
* Supporting Information
Synthetic procedures, complete experimental details, H NMR
and UV−vis titration, additional EPR and Mossbauer spectra,
details of the kinetic investigations, and crystallographic details
(CIF) for (CoCp*2)25, (NEt4)35·DMF, and (NEt4)5H·2DMF·
Et2O. This material is available free of charge via the Internet at
http://pubs.acs.org.
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̈
̈
AUTHOR INFORMATION
■
Corresponding Author
franc.meyer@chemie.uni-goettingen.de
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Journal of the American Chemical Society
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