Structure and function of quinones in biological solar energy transduction: A high-frequency d-band EPR spectroscopy study of model benzoquinones

Article abstract of DOI:10.1021/jp210156a

Quinones are utilized as charge-transfer cofactors in a wide variety of reactions that are crucial for photosynthesis and respiration. In photosynthetic protein complexes, both Type I and Type II, including oxygenic and anoxygenic reaction centers contain quinone cofactors that are known to participate in electron- and proton-transfer processes. Type II reaction centers, purple bacterial reaction centers, and photosystem II utilize benzoquinone molecules, ubiquinone, and plastoquinone, respectively, to facilitate proton-coupled electron transfer reactions. Here, we report a systematic study of the principal components of the g-tensor of an extensive library of model benzosemiquinone anion radicals in both protic (2-isopropanol) and aprotic (dimethyl sulfoxide) solvents using high-frequency EPR spectroscopy. A detailed comparison of the experimental g-values of the benzosemiquinone models at D-band EPR frequency allows for the discrimination of substituent effects and solvent hydrogen bonds on the principal components of the g-tensor. Further, we compare the primary plastosemiquinone, Q_A^-, of photosystem II with the substituent and solvent hydrogen bond effects of benzosemiquinone models in vitro. This study significantly extends the experimental basis for elucidating the role of both molecular structure and interactions with environment on the functional tuning of quinone cofactors in biological solar energy transduction.

Full text of DOI:10.1021/jp210156a

ARTICLE
pubs.acs.org/JPCB
Structure and Function of Quinones in Biological Solar Energy
Transduction: A High-Frequency D-Band EPR Spectroscopy Study of
Model Benzoquinones
Ruchira Chatterjee,† Christopher S. Coates,† Sergey Milikisiyants,† Oleg G. Poluektov,‡ and K. V. Lakshmi*,†
†Department of Chemistry and Chemical Biology and The Baruch ’60 Center for Biochemical Solar Energy Research,
Rensselaer Polytechnic Institute, Troy, New York 12180, United States
‡Chemical Sciences and Engineering Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois 60439, United States
ABSTRACT: Quinones are utilized as charge-transfer cofactors
in a wide variety of reactions that are crucial for photosynthesis
and respiration. In photosynthetic protein complexes, both
Type I and Type II, including oxygenic and anoxygenic reaction
centers contain quinone cofactors that are known to participate
in electron- and proton-transfer processes. Type II reaction
centers, purple bacterial reaction centers, and photosystem II
utilize benzoquinone molecules, ubiquinone, and plastoqui-
none, respectively, to facilitate proton-coupled electron transfer reactions. Here, we report a systematic study of the principal
components of the g-tensor of an extensive library of model benzosemiquinone anion radicals in both protic (2-isopropanol) and
aprotic (dimethyl sulfoxide) solvents using high-frequency EPR spectroscopy. A detailed comparison of the experimental g-values of
the benzosemiquinone models at D-band EPR frequency allows for the discrimination of substituent effects and solvent hydrogen
bonds on the principal components of the g-tensor. Further, we compare the primary plastosemiquinone, QAꢀ, of photosystem II
with the substituent and solvent hydrogen bond effects of benzosemiquinone models in vitro. This study significantly extends the
experimental basis for elucidating the role of both molecular structure and interactions with environment on the functional tuning of
quinone cofactors in biological solar energy transduction.
’ INTRODUCTION
solar energy transduction. This is also significant for the potential
application of quinones in the development of artificial photo-
synthetic systems for solar energy conversion.17ꢀ20
Quinones are utilized as charge-transfer cofactors in a wide
variety of reactions that are crucial for photosynthesis and
respiration.1ꢀ10 In photosynthetic protein complexes, both Type
I and Type II, including oxygenic and anoxygenic reaction
centers (RC), contain quinone cofactors that are known to
participate in electron- and proton-transfer processes. While
photosystem I (PSI) utilizes phylloquinone molecules for the
transfer of reducing equivalents through the electron transport
chain,9ꢀ11 the bacterial RC (BRC) and photosystem II (PSII)
utilize benzoquinone molecules to facilitate proton-coupled
electron transfer reactions.1ꢀ8 There are significant differences
in the function of the quinones in different RCs. Thus, similar
quinones can operate at up to 800 mV lower reduction potential
when present in type I RCs.12ꢀ16
The versatility of quinones and the diversity of their biological
function are controlled by both intra- and intermolecular inter-
actions. The differences in the functional specificity of quinones
is suggested to arise from the structure and location of the
quinone cofactor, the geometry of its binding site and the smart
matrix effects from the surrounding protein environment that
greatly influence the charge-transfer properties of quinones.14
Therefore, knowledge of electronic structure and its correlation
with the chemical structure of the quinones as well as the
interaction with the local protein environment is of importance
in understanding the versatility of quinone function in biological
Electron paramagnetic resonance (EPR) spectroscopy is a
powerful tool to study the electronic structure of paramagnetic
species.21ꢀ29 In particular, it is possible to resolve the electronic
g-tensor of organic radical species at higher EPR frequency.24,30ꢀ32
The EPR observables that are obtained from the analysis of high-
frequency EPR (HF EPR) spectra, such as the g-tensor, are highly
sensitive to subtle changes in the distribution of the electron spin
density. In the case of the semiquinone anion radical, the g-tensor
has been shown to depend not only on the chemical structure of the
semiquinone but also on the interactions of the semiquinone with
the surrounding matrix.33,34 Thus, the electronic g-tensor is an
important spectroscopic probe of the intra- and intermolecular
interactions of semiquinone radicals.
In recent years, the g-tensor of semiquinone anion radicals
both in vivo35,36 and in vitro37ꢀ39 has been an attractive subject
of experimental and theoretical investigations.40ꢀ42 Although the
results obtained in these studies have demonstrated the applica-
tion of HF EPR spectroscopy to investigate the interactions of
semiquinone anion radicals,43 such as hydrogen (H-) bonds to
Received: October 22, 2011
Revised:
November 27, 2011
Published: December 14, 2011
r
2011 American Chemical Society
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ARTICLE
of potassium tertiary-buteroxide solution (to IPA), was added
dropwise under an inert nitrogen gas atmosphere.49 The benzo-
semiquinone in solution was loaded into 0.3 mm (inner diam-
eter) quartz capillary tubes (VitroCon, Mountain Lakes, NJ),
and the sample was rapidly frozen at liquid helium temperature
(32 K) within the EPR resonator for the D-band HF EPR
spectroscopy measurements.
Growth and Isolation of Histidine-Tagged Photosystem II
Complexes from the PsbB Variant of Synechocystis PCC
6803. The hexa-histidine-tagged (HT) PsbB variant of Synecho-
cystis PCC 6803 was prepared by minor modification of pre-
viously published procedures.50 The PsbB variant of Synechocystis
PCC 6803 was grown on agar plates containing BG-11 medium
(1.76 M sodium nitrate (NaNO3), 30.3 mM magnesium sulfate
(MgSO4), 24.5 mM calcium chloride (CaCl2), 3.14 mM citric
acid and a mixture containing boric acid (H3BO3), manganese
chloride (MnCl2), zinc sulfate (ZnSO4), molybdic acid (H2MoO4),
cupric sulfate (CuSO4), and cobalt nitrate (Co(NO3)2)).51 The
single colonies of the PsbB variant of Synechocystis PCC 6803 were
harvested from the BG-11 agar plates, subcultured to 100 mL
growths and propagated to 18 L liquid cultures under constant
illumination at ∼30 °C using liquid BG-11 medium with 5 mM
glucose and 5 mM 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-
yl]amino]ethanesulfonic acid/potassium hydroxide ((TES)-KOH)
at pH 8.2.51 The cells were harvested by continuous-flow filtration
(Millipore, Billerica, MA) and resuspended in buffer containing
50 mM 2-(N-morpholino)ethanesulfonic acid/sodium hydroxide
(MES-NaOH) at pH 6.0, 5 mM CaCl2, 5 mM magnesium chloride
(MgCl2), and 25% w/v glycerol. The cells were broken using a
bead-beater (BioSpec, Bartlesville, OK), and the thylakoids were
separated by ultracentrifugation. The thylakoids were used to pre-
pare a detergent (dodecyl-β-D-maltoside) (β-DM)-solubilized ex-
tract of proteins in buffer containing 50 mM MES-NaOH, pH 6.0,
20 mM CaCl2, 5 mM MgCl2, 25% (w/v) glycerol, and 0.03% (w/v)
β-DM. The protein extract was subjected to purification on a Ni2+-
metal affinity chromatography column (Qiagen, Valencia, CA) to
isolate pure HT-PSII complexes.50 The isolation and purification
procedures were conducted in the dark at 4 °C. The resulting PSII
complexes were monitored by SDS-PAGE analysis.
Figure 1. Chemical structure and substituent groups of (A) BQ: R1 =
R2 = R3 = R4 = H. pPBQ: R1 = phenyl, R2 = R3 = R4 = H. DPBQ: R1 =
R3 = phenyl, R2 = R4 = H. DMBQ: R1 = R3 = methyl, R2 = R4 = H.
TMBQ: R1 = R2 = R3 = R4 = methyl. DCBQ: R1 = R3 = chloro, R2 =
R4 = H. TCBQ: R1 = R2 = R3 = R4 = chloro and (B) plastoquinone, PQ.
the carbonyl keto oxygen atom(s) of the semiquinone and π-
stacking interactions with the semiquinone ring, the specific role
of these interactions in the functional tuning of quinones remains
unknown. Further, the effect of H-bonds of model semiquinones
in protic solvents has been previously investigated by HF EPR
spectroscopy;44ꢀ46 however, there exists a lack of comparative
experimental data on the g-tensor of semiquinone radicals in an
aprotic environment,47,48 which hinders the development of
theoretical models.
We report a systematic HF EPR spectroscopy study of the
principal components of the g-tensor of an extensive library of
model benzosemiquinone anion radicals in an aprotic (dimethyl
sulfoxide; DMSO) and protic (2-isopropanol; IPA) solvent. In
this study, the increased electron Zeeman interaction at D-band
(130 GHz) microwave frequency leads to the complete resolu-
tion of spectral features arising from the three canonical orienta-
tions of the g-tensor of the benzosemiquinone models. A detailed
comparison of the experimental g-values of the benzosemiqui-
none models allows for the isolation of substituent effects and
solvent H-bonds on the principal components of the g-tensor.
Further, comparison of the EPR properties of the primary
plastosemiquinone, QAꢀ, of PSII with those of model benzose-
miquinones in vitro provides a better understanding of the tuning
and control of quinone cofactors in photosynthetic reaction
centers.
The HT-PSII complexes were manganese (Mn)-depleted in
buffer containing 50 mM MES-NaOH, pH 6.0, 20 mM CaCl2,
5 mM MgCl2, 25% w/v glycerol, 0.03% w/v β-DM, 10 mM
hydroxylamine hydrochloride (NH2OH HCl), and 10 mM so-
3
dium ethylenediamine tetraacetic acid (Na-EDTA).52 The Mn-
depleted HT-PSII was washed in a buffer containing 50 mM
MES-NaOH, pH 6.0, 20 mM CaCl2, 5 mM MgCl2, 25% w/v
glycerol, 0.03% w/v β-DM, and 5 mM Na-EDTA. The high-spin
(electron spin S = 2) nonheme iron center, Fe(II), in Mn-
depleted HT-PSII was converted to its low-spin (electron spin
S = 0) state by incubating with 350 mM potassium cyanide (KCN)
at pH 6.5 for 3.5 h in the dark at 4 °C.53 The Mn-depleted, CN-
treated HT-PSII was suspended in a deuterated glycine buffer at
pH 10.02, where deuterated water (D2O) was used in place of
H2O in the buffer. After repeated freezeꢀpumpꢀthaw cycles, the
Mn-depleted, CN-treated HT-PSII in deuterated buffer was
treated with sodium dithionite to chemically reduce the neutral
primary plastoquinone, QA, to the reduced plastosemiquinone
state, QAꢀ. The Mn-depleted, CN-treated HT-PSII with the
’ MATERIALS AND METHODS
Preparation of Benzosemiquinone Anion Radicals. The
library of benzoquinone model compounds used in this study
was obtained from commercial sources. 1,4-Benzoquinone (BQ )
(98%), 2,5-dimethyl-1,4-benzoquinone (DMBQ) (99%), 2,3,5,
6-tetramethyl-1,4-benzoquinone (TMBQ) (99%), and 2-phen-
yl-1,4-benzoquinone (pPBQ) (99%) were purchased from Acros
Organics (Morris Plains, NJ). 2,3,5,6-Tetrachloro-1,4-benzoqui-
none (TCBQ) (99%) was purchased from Sigma-Aldrich (St.
Louis, MO). 2,5-Dichloro-1,4-benzoquinone (DCBQ) (98%)
and 2,5-diphenyl-1,4-benzoquinone (DPBQ) (99%) were pur-
chased from Pfaltz & Bauer (Waterbury, CT) (Figure 1). The
benzosemiquinone anion radicals were generated by dissolving
the quinone (1 mM to 10 mM) in dimethyl sulfoxide (DMSO)
or isopropyl alcohol (IPA), and the solution was purged with dry
nitrogen gas. To this mixture, a reducing agent, either 10 M
aqueous sodium hydroxide (to DMSO) or a minimum amount
ꢀ
reduced QA semiquinone state was used for the D-band
(130 GHz) HF EPR spectroscopy measurements.
High-Frequency D-Band EPR Spectroscopy. The HF EPR
spectra were recorded on a home-built D-band (130 GHz)
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The Journal of Physical Chemistry B
ARTICLE
Figure 3. First derivative of the spinꢀecho-detected field-sweep
D-band (130 GHz) EPR spectrum of model benzosemiquinone anion
radicals with IPA as the solvent. (A) 1,4-benzoquinone (BQ), (B)
plastoquinone-9 (PQ), (C) 2-phenyl-1,4-benzoquinone (pPBQ), (D)
2,5-diphenyl-1,4-benzoquinone (DPBQ), (E) 2,5-dimethyl-1,4-benzo-
quinone (DMBQ), (F) 2,3,5,6-tetramethyl-1,4-benzoquinone (TMBQ),
(G) 2,5-dichloro-1,4-benzoquinone (DCBQ), and (H) 2,3,5,6-tetra-
chloro-1,4-benzoquinone (TCBQ). The experimental and simulated
spectra are shown as black and red traces, respectively.
Figure 2. First derivative of the spinꢀecho-detected field-sweep
D-band (130 GHz) EPR spectrum of model benzosemiquinone anion
radicals with DMSO as the solvent. (A) 1,4-benzoquinone (BQ), (B)
plastoquinone-9 (PQ), (C) 2-phenyl-1,4-benzoquinone (pPBQ), (D)
2,5-diphenyl-1,4-benzoquinone (DPBQ), (E) 2,5-dimethyl-1,4-benzo-
quinone (DMBQ), (F) 2,3,5,6-tetramethyl-1,4-benzoquinone (TMBQ),
(G) 2,5-dichloro-1,4-benzoquinone (DCBQ), and (H) 2,3,5,6-tetra-
chloro-1,4-benzoquinone (TCBQ). The experimental and simulated
spectra are shown as black and red traces, respectively.
pulsed spectrometer described previously.31,54 The HF EPR
spectroscopy measurements were performed at 32 K. The
temperature of the EPR sample was regulated by an Oxford
ITC 503 temperature controller with an Oxford CF 1200
continuous-flow helium cryostat (Oxford Instruments, Oxford-
shire, UK). The spinꢀecho-detected field-sweep HF EPR spec-
tra were obtained using microwave pulses of 90 and 120 ns
duration with a separation of 250 ns and a repetition rate of
40 Hz. The HF EPR spectra were processed using the software
program SpecLab, and the numerical first derivative of the
spinꢀecho-detected HF EPR spectra were simulated using the
software program, SimBud (kindly provided by Dr. Astashkin at
the Electron Paramagnetic Resonance Facility, Department of
Chemistry, University of Arizona, Tucson, AZ). For the spectral
simulations, the anisotropic g-tensor was used to reproduce the
spectral features that are observed in the experimental EPR
spectra. Other magnetic interactions that are much smaller in
magnitude and hence unresolved in the HF EPR spectra (e.g., the
electronꢀnuclear hyperfine interactions) were approximated by
the intrinsic spectral line width that was assumed as a Gaussian
function. In the present study, the relative g-values are measured
with high precision (∼ 10ꢀ4). However, the absolute g-values
could include systematic errors due to difficulty in the determi-
nation of the precise static magnetic field experienced by the EPR
sample. As a reference, the value of the z-component of the
g-tensor, gZ, was set to the g-value of a free electron, g = 2.00232.
This is a valid approximation as the deviations of the gZ com-
ponent are usually on the order of 10ꢀ5, which is much smaller
than the variation of the values of the gX and gY components that
are observed in this study (∼10ꢀ3).39 The experimental and
simulated HF EPR spectra that are presented here were prepared
in Matlab R2008a.
Figure 4. First derivative of the spinꢀecho-detected field-sweep
D-band (130 GHz) EPR spectrum of (A) the plastosemiquinone anion
radical in DMSO, (B) the plastosemiquinone anion radical in IPA, and
(C) the plastosemiquinone anion radical of PSII, QAꢀ, in aqueous
buffer. The experimental and simulated spectra are shown as black and
red traces, respectively.
When recorded at conventional X-band microwave frequency
(9ꢀ10 GHz), the spinꢀecho-detected EPR spectra are often
distorted due to the presence of pronounced nuclear modula-
tions when the anisotropy of the electronꢀnuclear hyperfine
interaction is on the same order of magnitude as the nuclear
Zeeman interaction. In the present study, the hyperfine interaction
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Table 1. Principal Components of the g-Tensor of the Model Benzosemiquinones Obtained from Spectral Line Shape
Simulations of the Experimental HF D-band (130 GHz) EPR Spectra in DMSO and IPA, Respectively, and the Plastosemiquinone
Anion, Q Aꢀ, of PSII
simulation parameters in
simulation parameters in DMSOa
IPAa
semiquinone (abbreviation)
1,4-benzoquinone (BQ )
gX
gY
gZ
gX
gY
gZ
2.00659
2.00535
2.00232
2.00232
2.00232
2.00232
2.00232
2.00232
2.00232
2.00232
2.00232
2.00647 2.005315 2.00232
2.00635 2.00525 2.00232
2.00656 2.00526 2.00232
2.00675 2.00523 2.00232
2.00623 2.00529 2.00232
2.00624 2.00526 2.00232
2.007295 2.00597 2.00232
2.00687 2.00602 2.00232
plastoquinone-9 (PQ )
2.00665
2.00530
2-phenyl-1,4-benzoquinone (pPBQ)
2,5-diphenyl-1,4-benzoquinone (DPBQ )
2,5-dimethyl-1,4-benzoquinone (DMBQ )
2,3,5,6-tetramethyl-1,4-benzoquinone (TMBQ )
2,5-dichloro-1,4-benzoquinone (DCBQ )
2,3,5,6-tetrachloro-1,4-benzoquinone (TCBQ )
QAꢀ in PSII (aqueous buffer)
2.00695
2.005295
2.005286
2.00528
2.007265
2.00641
2.006495
2.00757
2.00533
2.00697
2.00882
2.00695
2.00675
2.00527
(aqueous buffer)
(aqueous buffer)
(aqueous buffer)
a The principal values of the g-tensor are assigned in the following order: gX > gY > gZ.39
Table 2. Effective g-Tensor, ΔgX, with the Corresponding Redox Potentials of Model Benzosemiquinonesa
semiquinone (abbreviation)
ΔgX in DMSO
ΔgX in IPA
redox potential (V)
1,4-benzoquinone (BQ )
0.00427
0.00415
0.00403
0.00424
0.00443
0.00391
0.00392
0.00498
0.00455
N/A
ꢀ0.043b
ꢀ0.351b
ꢀ0.034b
ꢀ0.025b
ꢀ0.210b
ꢀ0.385b
0.227b
plastoquinone-9 (PQ )
0.00433
2-phenyl-1,4-benzoquinone (pPBQ )
2,5-diphenyl-1,4-benzoquinone (DPBQ )
2,5-dimethyl-1,4-benzoquinone (DMBQ )
2,3,5,6-tetramethyl-1,4-benzoquinone (TMBQ )
2,5-dichloro-1,4-benzoquinone (DCBQ )
2,3,5,6-tetrachloro-1,4-benzoquinone (TCBQ )
QAꢀ in PSII (aqueous buffer)
0.00463
0.00494
0.00409
0.00418
0.00525
0.00650
0.476b
ꢀ0.148c
0.00443 (aqueous buffer)
a The redox potentials were measured in acetonitrile, and the values were referenced to ferrocene.50 b From Weyers et al.50 c From Ishikita and Knapp.61
of the benzosemiquinone anion radicals is typically in the range of
∼10ꢀ20 MHz,39,49 which is much smaller than the nuclear
Zeeman interaction which is approximately 200 MHz at 130 GHz
EPR frequency. Thisresultsin thecomplete suppressionofnuclear
modulations as is indeed confirmed in this study.
anion radicals in DMSO are well-resolved at D-band EPR
frequency. Qualitative examination of the experimental HF
EPR spectra (black traces) that are displayed in Figure 2 reveals
that the gX and gY components of the g-tensor of the benzo-
semiquinone models depend on the substituent group(s) on the
benzosemiquinone ring. Additional effects due to H-bonding
interactions with the surrounding matrix are absent in these
spectra as DMSO is an aprotic solvent.
’ RESULTS
Shown in Figures 3AꢀH are the D-band experimental (black
trace) and simulated (red trace) first-derivative spinꢀecho-detected
magnetic field-sweep EPR spectra of a frozen solution of model
benzosemiquinone anion radicals (described above in Figure 2) in
the protic solvent, IPA. As can be seen from the spectra shown in
Figure 3AꢀH, the principal components of the g-tensor of the
benzosemiquinone anion radicals in IPA are also well-resolved at
D-band EPR frequency. Further, comparison of the g-tensors of the
model benzosemiquinone anion radicals in DMSO and IPA indicates
that in addition to substituent effects, the g-tensor of the model
benzosemiquinone anion radicals in IPA is affected by interactions
with the solvent. In Figure 3, the gX and gY components of the
g-tensor display pronounced influence of the presence of H-bonds of
the benzosemiquinone radicals with IPA.
Shown in Figures 2AꢀH are the D-band experimental (black
trace) and simulated (red trace) first-derivative spinꢀecho-detected
magnetic field-sweep EPR spectra of frozen solutions of the
model benzosemiquinone anion radicals, 1,4-benzoquinone
(BQ), plastoquinone-9 (PQ), 2-phenyl-1,4-benzoquinone (pPBQ),
2,5-diphenyl-1,4-benzoquinone (DPBQ), 2,5-dimethyl-1,4-ben-
zoquinone (DMBQ), 2,3,5,6-tetramethyl-1,4-benzoquinone
(TMBQ), 2,5-dichloro-1,4-benzoquinone (DCBQ), and 2,3,5,6-
tetrachloro-1,4-benzoquinone (TCBQ), in the aprotic solvent,
DMSO. The investigation of the semiquinone anion radicals at
D-band EPR frequency, which is more than an order of magni-
tude higher than the conventional X-band EPR frequency, leads
to enhanced resolution of the spectral features from the three
principal components of the g-tensor. This is due to the increase
in the resolution of the g-factor at higher magnetic field. As can be
seen from the spectra shown in Figure 2AꢀH, the principal
components of the g-tensor of the model benzosemiquinone
Figure 4AꢀC displays the experimental (black trace) and
simulated (red trace) D-band EPR spinꢀecho-detected magnetic
field-sweep EPR spectra of the primary plastosemiquinone anion,
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The Journal of Physical Chemistry B
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Figure 6. Correlation between the effective gX component of the
g-tensor, ΔgX, in DMSO and the redox potential of the corresponding
model benzosemiquinones.
Figure 5. Correlation of the effective g-component of the g-tensor, ΔgX
and ΔgY, of the model benzosemiquinones in DMSO.
Q Aꢀ, of Mn-depleted, CN-treated PSII and the plastosemiqui-
noneanion radical in IPA and DMSO, respectively. Once again, as
can be seen in Figure 4AꢀC, the three principal components of
the g-tensor of the plastosemiquinone anion radical both in vivo
(Mn-depleted, CN-treated PSII) and in vitro (IPA and DMSO)
are completely resolved at D-band EPR frequency.
The experimental spinꢀecho-detected magnetic field-sweep
EPR spectra (black traces) in Figures 2ꢀ4 can be accurately
reproduced by numerical simulations (red traces). The corre-
sponding principal values of the g-tensor that are obtained from
spectral simulations of the EPR spectra of the benzosemiquinone
anion radicals are summarized in Table 1.
Figure 7. Orientation of the principal components of the g-tensor with
respect to the molecular plane of the benzquinone ring.
’ DISCUSSION
The increased electron Zeeman interaction at D-band (130 GHz)
EPR frequency leads to resolution of the g-tensor of the benzose-
miquinone anion radical. In this study, through spectral simulations
of the experimental HF EPR spectra, we compile the principal
components of the g-tensor of an extensive library of model
benzosemiquinones and the primary plastosemiquinone, Q Aꢀ, of
PSII (Table 1). Listed in Table 2 is the effective gX component of the
g-tensor, ΔgX, with the corresponding redox potential of the
benzosemiquinones. As can be seen in Table 2 and Figure 5, there
is a clear relationship between the value of ΔgX in the aprotic solvent,
DMSO, and the nature of the substituents that are present on the
benzosemiquinone. A similar trend is also observed in the protic
solvent, IPA (Table 2). The effective gX component of the g-tensor,
ΔgX, also displays a dependence on the redox potential of the
benzoquinone. As can be seen in Table 2 and Figure 6, there is an
increase in ΔgX with an increase (to a more positive value) of the
redox potential of the corresponding benzoquinone.
The dependence of Δg on the nature of the substituents can
be interpreted on the basis of the g-factor theory previously
published by Stone.55 Within a simplified Stone’s theory, the
deviation of the principal components of the g-tensor (Figure 7)
from the free electron g-value for the benzosemiquinone anion
radical, where the g-anisotropy originates from the excitation of
an electron in a nonbonding lone-pair orbital on the keto oxygen
atom to the half-occupied π orbital, can be expressed as39
2ξFπ0 Cx
2
ΔgY ¼
ð2Þ
ꢁ
ΔEnπ
where ξ is the spinꢀorbit coupling parameter, Fπ0 is the spin
density of the π orbital on the oxygen atom, and Cx and Cy
determine the admixtures of the px and py orbitals in the lone-pair
orbitals of oxygen atom, respectively. ΔEnπ* is the difference in
energy between the lone-pair orbitals and the half-occupied π
orbital of the benzosemiquinone. As described by Burghaus and
co-workers, a higher value of Cy explains the higher susceptibility
of the value of the gX component of the g-tensor to the presence
of substituent groups on the benzosemiquinone ring.39 Similarly,
gX would also have higher susceptibility to the polarity of the
solvent.
Further examination of the g-tensors in Table 2 and Figure 5
reveals that the presence of a methyl group substituent reduces
the g-anisotropy, ΔgX, of the benzosemiquinone. This is because
a methyl group is electron donating in nature. This leads to a
reduction of the electron spin density on the oxygen atom(s)
through hyperconjugation effects.39,56,57 However, this effect is
clearly not additive with respect to the number of methyl groups
that are present on the benzosemiquinone. This could be due to
the presence of steric interaction between the multiple methyl
groups on the ring. Similarly, the presence of a phenyl group
leads to a larger ΔgX and this effect is enhanced with a larger
number of phenyl group substituents. This is possibly due to the
2ξFπ0 Cy
2
ΔgX
¼
ð1Þ
ꢁ
ΔEnπ
680
dx.doi.org/10.1021/jp210156a |J. Phys. Chem. B 2012, 116, 676–682

The Journal of Physical Chemistry B
ARTICLE
dual nature of the phenyl group substituent that is electron-
withdrawing based on resonance effects and is electron-donating
through inductive effects. This could lead to an overall increase in
the electron spin density on the oxygen atom(s) of the benzo-
semiquinone.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (518) 276 3271. Fax: (518) 276 4887. E-mail: lakshk@
rpi.edu.
There are large differences in ΔgX and ΔgY values that are
observed in the presence of halogen groups, such as chloro-
substituted benzosemiquinones (Table 2 and Figure 5).58,59 This
is in agreement with Stone theory as a chloro group has a larger
spinꢀorbit coupling constant (ξ = 587 cmꢀ1).59 It has been
suggested that the positive shift of the gX and gY components is
due to the participation of the chlorine pz orbitals in the
delocalized molecular π orbital of the benzosemiquinone ring.58
Further, the electron-withdrawing nature of the chloro group
also decreases the overall electron spin density distribution
across the benzosemiquinone. This results in an increase of the
electron spin density on the oxygen atom(s).
’ ACKNOWLEDGMENT
We thank Dr. Andrei Astashkin (Electron Paramagnetic Reso-
nance Facility, Department of Chemistry, University of Arizona,
Tucson, AZ) for providing the spectral processing software,
SpecLab, and the EPR spectral simulation program, SimBud. We
also thank Amanda Weyers for assistance with sample prepara-
tion. This work was supported by the Division of Chemical Sciences,
Geosciences, and Biosciences, Office of Basic Energy Sciences of
the U.S. Department of Energy through Grants DE-FG02-
07ER15903 (to K.V.L.) and DE-AC02-06CH11357 (to O.G.P.).
On the basis of the Stone theory, it is expected that H-bond
interactions at the carbonyl oxygen atom(s) of the benzosemi-
quinone would influence ΔgX and could serve as direct probe of
H-bond interactions. As can be seen in Table 2, the H-bond
interaction of the benzosemiquinone in the protic solvent, IPA,
reduces the value of ΔgX. It has been suggested by Burghaus and
co-workers that the reduction of ΔgX upon increase in the
electron spin density of the π orbital, Fπ0, can be attributed to
the increase of the polarity of the CꢀO bond in the presence of a
positively charged H atom and a lowering of the energy of the
lone-pair orbital, Enπ*, due to the formation of a bond between
the orbital of the oxygen lone pair and the 1s orbital of the
H-bonded atom leading to the subsequent increase of the energy
gap in eqs 1 and 2.39 These effects lead to the lowering of the
value of gX as the lone pair orbital is closely directed along the
y axis (as opposed to the x axis). The g-tensor values obtained
here are in good agreement with previous studies in literature.39
Finally, we compare the g-tensor of the plastosemiquinone
in vivo, QAꢀ of PSII, with the g-tensor of the plastosemiquinone
in vitro (Table 2). As can be seen, there is an increase in ΔgX
when the plastosemiquinone is closely interacting with the
surrounding protein environment of PSII. This is a direct
consequence of the difference in the nature of the H-bond
interactions in vivo and in vitro. In vitro, we observe highly
symmetric H-bonds formed between the semiquinone radical
and the protic solvent. However, it is known from previous
’ ABBREVIATIONS
EPR electron paramagnetic resonance spectroscopy
HF EPR high-frequency EPR spectroscopy
RC
BRC
PSI
photosynthetic reaction center
bacterial reaction center
photosystem I
PSII
BQ
photosystem II
benzoquinone
H-bond hydrogen bond
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