Ultrafast Photoinduced Electron Transfer in Green Fluorescent Protein Bearing a Genetically Encoded Electron Acceptor

Article abstract of DOI:10.1021/jacs.5b03652

Electron transfer (ET) is widely used for driving the processes that underlie the chemistry of life. However, our abilities to probe electron transfer mechanisms in proteins and design redox enzymes are limited, due to the lack of methods to site-specific

Full text of DOI:10.1021/jacs.5b03652

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Communication
Ultrafast Photo-induced Electron Transfer in Green Fluorescent
Protein Bearing a Genetically Encoded Electron Acceptor
Xiaoxuan Lv, Yang Yu, Meng Zhou, Cheng Hu, Feng Gao, Jiasong Li, Xiaohong
Liu, Kai Deng, Peng Zheng, Weimin Gong, Andong Xia, and Jiangyun Wang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b03652 • Publication Date (Web): 28 May 2015
Downloaded from http://pubs.acs.org on June 1, 2015
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Ultrafast Photo-induced Electron Transfer in Green Fluores-
cent Protein Bearing a Genetically Encoded Electron Accep-
tor
Xiaoxuan Lv,1,4 Yang Yu,3,4 Meng Zhou, 2 Cheng Hu, 1 Feng Gao, 1 Jiasong Li, 1 Xiaohong Liu, 1 Kai
Deng, 1 Peng Zheng, 1 Weimin Gong,1 Andong Xia,* ,2 and Jiangyun Wang*,1
1Laboratory of RNA Biology and Laboratory of Quantum Biophysics, Institute of Biophysics, Chinese Academy of
Sciences 15 Datun Road, Chaoyang District, Beijing 100101, China
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2Institute of Chemistry, Chinese Academy of Sciences Chinese Academy of Sciences, Beijing 100080, China.
3Department of Chemistry, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States
ABSTRACT: Electron transfer (ET) is widely used for
driving the processes that underlie the chemistry of life.
However, our abilities to probe electron transfer mechanisms
in proteins and design redox enzymes are limited, due to the
lack of methods to site-specifically insert electron acceptors
into proteins in vivo. Here we describe the synthesis and
genetic incorporation of 4-fluoro-3-nitrophenylalanine
(FNO2Phe), which has similar reduction potentials to
NAD(P)H and ferredoxin, the most important biological
reductants. Through the genetic incorporation of FNO2Phe
into green fluorescent protein (GFP) and femtosecond
transient absorption measurement, we show that photo-
induced electron transfer (PET) from the GFP chromophore
to FNO2Phe occurs very fast (within 11 picoseconds), which is
comparable to that of the first electron transfer step in
photosystem I, from P700* to A0. This genetically encoded,
low-reduction potential unnatural amino acid (UAA) can
significantly improve our ability to investigate electron
Scheme 1. The redox potential range of various metal
cofactors (in millivolts, versus the standard hydro-
gen electrode) in proteins and genetically encoded
unnatural amino acids. The structures of 4-fluoro-3-
transfer mechanisms in complex reductases, and facilitate
nitrophenylalanine, which is reported in this work,
the design of miniature proteins which mimic their
is highlighted in red.
functions.
fluorescent sensors,40 in vivo and in vitro. In previous works,
we and others have reported the genetic incorporation of the
unnatural amino acids (UAA) 3,5-dichlorotyrosine (Cl2Y),
The chemistry of life processes, most notably photosynthe-
39
3,5-difluorotyrosine(F2Y),38,40 and Dopa, which have redox
sis, are driven largely through the directional movement of
potentials spanning from 400 mV to 800 mV. While we have
electrons in proteins.1-18 While natural amino acids (such as
demonstrated that upon Cu(II) binding, the genetically en-
cysteine, tyrosine, and tryptophan) can serve as electron do-
coded UAA 3-pyrazole-tyrosine(pyTyr)41 can be used as an
nors, they are not known to be used as electron acceptors.19-24
efficient electron acceptor, the reduction potential of the
To facilitate efficient electron transfer in living systems, na-
pyTyr/Cu(II) complex (168 mV) is much higher than
ture has employed numerous metal-containing electron-
NAD(P)H, and therefore precludes the design of
accepting cofactors such as iron-sulfur clusters, copper and
reductases.42
haem, which have broadly tunable redox potentials (Scheme
In this work, we describe the facile synthesis and genetic
incorporation of 4-fluoro-3-nitrophenylalanine (FNO2Phe, or
FN) 1. The crystal structure of the Aequorea victoria green
fluorescent protein (GFP) bearing FNO2Phe at a specific site
(Figure 2) reveals that the small size of the FNO2Phe does
not cause significant structural changes to GFP.
1 and Figure S1).1,25-33 For example, three iron-sulfur clusters
are present in photosystem I, which together form a facile
electron accepting route to the photochemical reduction of
NAD(P)+ to produce NAD(P)H.25 However, the factors con-
trolling the redox potentials of metal cofactors are still poorly
understood, and it is difficult to introduce metal binding
sites with desirable redox potentials into proteins.34-36 Fur-
thermore, the mechanism of metal-cofactor insertion into
proteins in vivo is extremely complicated and remains an
important frontier of bioinorganic chemistry.37
Through femtosecond transient absorption measurement,
we establish that electron transfer from the GFP chromo-
phore to FNO2Phe occurs very fast, within 11 picoseconds, in
a distance-dependent manner. This rate is comparable to
that of the first electron transfer step in photosystem I, from
P700* to A0, which occurs within 10-30 picoseconds after
P700 photo-excitation.43 (Figure S2) The GFP mutants bear-
ing FNO2Phe reported in this work recapitulate two im-
portant properties of photosystem I: very fast forward elec-
tron transfer rate, and photo-induced production of strongly
New methods for the genetic incorporation of metal-
independent redox probes with various reduction potentials
greatly expand our ability to investigate electron transfer
mechanism in proteins,38 design oxidases and reductases,39
and construct photo-induced electron transfer (PET)
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reducing species. Further development along this route may
for NO2Phe. We therefore chose FNO2Phe for all our subse-
quent investigations.
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result in miniature protein systems which recapitulate the
most important property of PS I, the photocatalytic produc-
tion of NAD(P)H.
The UAAs FNO2Phe and NO2Phe (Scheme S1)45-47 have
peak potentials comparable to that of the iron-sulfur clusters
and NAD(P)H at -310 mV and -470 mV, respectively, as
shown by cyclic voltammetry measurements (Figure S8).44
FNO2Phe was synthesized in two steps using a previously
reported method with minor modifications (Scheme S1).48 To
selectively incorporate FNO2Phe at specific sites in proteins
produced in E. coli, a mutant Methanocaldococcus jannaschii
tyrosyl amber suppressor tRNA (MjtRNATyrCUA)/tyrosyl-tRNA
synthetase (MjTyrRS) pair was evolved to incorporate
FNO2Phe in response to the TAG codon, as previously re-
ported.49 One MjTyrRS clone emerged after three rounds of
positive selection and two rounds of negative selection. This
clone was named FNO2PheRS (Table S1).
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To determine whether FNO2PheRS can facilitate the in-
corporation of FNO2Phe into proteins with high efficiency
and fidelity, an amber stop codon was substituted for residue
Ser4 in sperm whale myoglobin or Tyr151 in GFP. Protein
synthesis was carried out in E. coli in the presence of UAA
specific TyrRS mutants, MjtRNATyrCUA, and 1 mM UAA, or in
the absence of UAA as a negative control. Analysis of the
purified protein by SDS-PAGE showed that full-length myo-
globin and GFP expressed only in the presence of FNO2Phe
(Figure 1), indicating that FNO2PheRS was specifically active
for FNO2Phe but inactive for any natural amino acids. The
yields of mutant myoglobin and GFP were 10 mg/L and 20
mg/L, respectively. For comparison, the yields of wild-type
sperm whale myoglobin (wtMb) and GFP were 50 mg/L and
100 mg/L, respectively. ESI-MS analysis FNO2Phe encoded
mutant GFP (GFP151FNO2Phe) gave average masses of 27756
Da, in agreement with the calculated mass (Figure 1C). The
N-terminus methionine is cleaved, one water molecule and
two hydrogen atoms are lost during chromophore matura-
tion. Subsequent tryptic digestion and tandem mass spec-
trometric analysis confirmed the presence of FNO2Phe at
position 151 of the GFP (Figure S4).The high-resolution crys-
tal structure of GFP151FNO2Phe shows that the replacement
of Tyr151 by FNO2Phe causes little perturbation to the overall
structure of GFP (Figure 2). The fluorine atom and nitro
group are co-planar with the benzene ring. The planar struc-
ture of FNO2Phe should be favorable for minimizing reorgan-
ization energy upon 1 electron reduction, since the nitroben-
zene anion radical also has a planar structure.46
Figure 1. Coomassie-stained SDS-PAGE of TAG4 mu-
tants myoglobin (A) or TAG151 mutants GFP (B) ex-
pressed in the presence or absence of 1 mM UAA. C)
ESI-MS spectra of the GFP151FNO2Phe mutant. The
insert shows the deconvoluted spectrum. Expected
mass: 27756 Da; observed mass: 27756 Da.
Figure 2. Crystal structure of the GFP151FNO2Phe
complex (pdb code: 4ON8). The GFP chromophore,
and residues 151, 149, 95 and 17 are shown as sticks.
To analyze the rate and distance-dependence of PET be-
tween the GFP chromophore and the electron accepting
We then measured the relative fluorescence intensity of
5uM wild-type GFP, GFP151FNO2Phe and GFP151 NO2Phe. As
shown in Figure S6, GFP151FNO2Phe exhibited 66% quench-
ing compared to wild type GFP. By contrast, GFP151NO2Phe
showed less than 5% quenching (see Figure S6). Since the
absorption spectrum of FNO2Phe does not overlap with the
emission spectrum of GFP (see Figure S7), the fluorescence
quenching is not caused by fluorescence resonance energy
transfer (FRET). The difference in quenching can be ex-
plained by the difference in reduction potentials between
FNO2Phe and NO2Phe. The reduction potential of FNO2Phe
is 160 mV lower than that of NO2Phe, so there is a stronger
driving force for PET from the GFP chromophore to
FNO2Phe, resulting in stronger quenching than is observed
FNO2Phe, femtosecond transient absorption measurements
of wtGFP and GFP mutants were performed using a 400 nm
pump laser. FNO2Phe alone has no absorption at 400 nm
(Figure S7). The transient absorption spectrum of wtGFP fits
well to a sequential model (Scheme S2), with two compo-
nents having decay times of ~ 1.3 ps and ~3.0 ns (Figure 3A
and Table S2), consistent with previous investigations.50-53
These results indicate that after photoexitation of the proto-
nated neutral GFP chromophore, deprotonation occurs with-
in 1.3 ps, resulting in the formation of anionic I* with an ex-
cited-state absorption (ESA) around 440 nm and a stimulat-
ed emission (SE) around 509 nm.
The ESA and SE of GFP151FNO2Phe show a fast decay com-
pared to the wtGFP (Figure 3), indicating efficient electron
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transfer from the GFP chromophore to FNO2Phe. To extract
Table 1. Distance-dependent electron transfer rate
of wtGFP and mutants.
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the electron transfer rate, a kinetic scheme shown in Scheme
S2 is proposed, which indicates electron transfer from I* to
FNO2Phe to yield the FNO2Phe anion radical (FN-).
Mutant
GFP149FNO2Phe 11 ± 0.55
distance kET (s-1)

ET (ps)

(Å )
8.8
10.3
11.7
12.9
(9.09±0.45)×1010
GFP151FNO2Phe
GFP95FNO2Phe
GFP17FNO2Phe
73 ± 3.5
183 ± 11
253 ± 13
(1.37±0.07)×1010
(5.46±0.33)×109
(3.95±0.20)×109
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Based on the electron transfer time constants (ET) of GFP-
FNO2Phe mutants obtained from the fitting procedure (see
Figure S10 and Table 1), we are able to calculate the electron
transfer rate from the GFP chromophore to FNO2Phe
(kET=1/ET).56 GFP149FNO2Phe gives the shortest electron
transfer time constant of 11 ps, which corresponds to an elec-
tron transfer rate constant of 9.09×1010 s-1. The ET rate con-
stants
of
GFP151FNO2Phe,
GFP17FNO2Phe
and
GFP95FNO2Phe were slower, at 1.37×1010 s-1, 5.46×109 s-1 and
3.95×109 s-1, respectively. The edge-to-edge distance from the
GFP chromophore to residue FNO2Phe 151 was 10.3 Å , accord-
ing to the GFP151FNO2Phe structure (Figure 2). Edge-to-edge
distances from the GFP chromophore to residues
FNO2Phe149, FNO2Phe17 and FNO2Phe95 were estimated by
measuring the shortest distance between the GFP chromo-
phore and the carbons of residues Asn149, Glu17 and
Glu95, respectively (Figure 2).57-60 Taken together these re-
sults demonstrate that PET from the GFP chromophore to
FNO2Phe can occur, and that the rate of PET decreases ex-
ponentially as distance increases (Table 1).57 The distance
decay factor (β=0.9 Å−1) is close to that of a single-step tun-
neling reaction (Figure S11).58,59
Figure 3. A) Evolution of the transient absorption
spectra of wtGFP and GFP151FNO2Phe. The spectra
are obtained at different delay times (pump at 400
nm). The inset shows the kinetic traces at specific
wavelengths. B) Species associated difference spec-
tra (SADS) and C) Time-dependent concentrations of
transient species obtained from target global fitting
combined with singular value decomposition (SVD).
In conclusion, we have demonstrated the genetic incorpo-
ration of FNO2Phe as an efficient electron accepting probe
which has reduction potential similar to that of iron-sulfur
clusters and NAD(P)H. This new probe is well-suited for
investigating electron transfer mechanism for reductases,
such as photosystem I, hydrogenase, sulfite (nitrite) reduc-
tase and nitrogenase, thus facilitating the design of miniature
proteins mimicking their functions. In these complex en-
zymes, multi-step electron hopping through iron-sulfur clus-
ters is employed to optimize electron transfer rates. Since it
is difficult to incorporate iron-sulfur cluster in designed
metalloenzymes,61 the genetically coded FNO2Phe may pro-
vide a suitable alternative for designing complex metalloez-
nymes. The fluorine atom on FNO2Phe should also be helpful
for EPR and NMR assignments for electron transfer studies.
A target global fitting procedure based on the proposed
kinetic model combined with singular value decomposition
(SVD)50-54 was then employed to extract the time-dependent
concentrations of transient species and species associated
50-54
difference spectra (SADS)
from the transient data as
shown in Figure 3. The fitted time constants of wtGFP and
GFP mutants are listed in Table S2. Besides the 1.8 ps excited
state deprotonation reaction from A* to I*, and a 3.2 ns decay
from I* to ground state, a new decay process with a time-
constant of 73 ps was obtained, resulting from the electron
transfer from the GFP chromophore to FNO2Phe. Our SADS
data (Figure 3B) suggests the formation of the FNO2Phe ani-
on radical, since a new peak around 470 nm appeared in
GFP151FNO2Phe, but not in wtGFP. This is consistent with
previous electrochemical studies, which have reported that
the nitrobenzene anion radical has absorbance maxima
around 470 nm.55 Furthermore, the similar transient absorp-
tion spectra of GFP149FNO2Phe, GFP95FNO2Phe and
GFP17FNO2Phe are given in Figure S9, and the time-
dependent concentrations of transient species and species
associated difference spectra (SADS) are also extracted based
on the same kinetic model as shown in Scheme S2. The fitted
time constants of these GFP mutants are also listed in Table
S2. In order to show the quality of fitting, kinetics at several
selected wavelengths are plotted together with a global fit of
all the collected time traces (Figure S10).
Using this new method and femtosecond transient absorp-
tion measurement, we demonstrated that photo-induced
electron transfer (PET) between green fluorescent protein
(GFP) chromophore and protein-bound electron acceptor
can occur rapidly, within 11 picoseconds, suggesting that GFP
may be optimized for PET reaction.
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(27) Park, J. S.; Karnas, E.; Ohkubo, K.; Chen, P.; Kadish, K. M.;
Fukuzumi, S.; Bielawski, C. W.; Hudnall, T. W.; Lynch, V. M.; Sessler,
J. L. Science 2010, 329, 1324.
(28) Beinert, H.; Holm, R. H.; Munck, E. Science 1997, 277, 653.
(29) Liu, J.; Chakraborty, S.; Hosseinzadeh, P.; Yu, Y.; Tian, S. L.;
Petrik, I.; Bhagi, A.; Lu, Y. Chem. Rev. 2014, 114, 4366.
(30) Rivalta, I.; Brudvig, G. W.; Batista, V. S. Curr. Opin. Chem. Biol.
2012, 16, 11.
Corresponding Author
1
2
3
4
*jwang@ibp.ac.cn
*andong@iccas.ac.cn
Author Contributions
4These authors contributed equally to this work.
5
6
7
8
9
(31) Tobin, P. H.; Wilson, C. J. J. Am. Chem. Soc. 2014, 136, 1793.
(32) Yu, F. T.; Penner-Hahn, J. E.; Pecoraro, V. L. J. Am. Chem. Soc.
2013, 135, 18096.
(33) J. J. Fraustó da Silva, R. J. P. Williams, The Biological Chemistry
of the Elements, Oxford University Press: Oxford, 2001.
(34) Liu, J.; Meier, K. K.; Tian, S. L.; Zhang, J. L.; Guo, H. C.; Schulz,
C. E.; Robinson, H.; Nilges, M. J.; Munck, E.; Lu, Y. J. Am. Chem. Soc.
2014, 136, 12337.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
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We gratefully acknowledge the Major State Basic Research
Program of China (2015CB856203), the National Science
Foundation of China (21325211, 91313301, 21333012), CAS grant
(KJZD-EW-L01), Tianjin Municipal Grant (Y1M6101241). We
thank Xiang Ding at the Core Facility for Protein Research,
CAS for MS spectrum.
(35) Bhagi-Damodaran, A.; Petrik, I. D.; Marshall, N. M.; Robinson,
H.; Lu, Y. J. Am. Chem. Soc. 2014, 136, 11882.
(36) Liu, J.; Chakraborty, S.; Hosseinzadeh, P.; Yu, Y.; Tian, S.;
Petrik, I.; Bhagi, A.; Lu, Y. Chem. Rev. 2014, 114, 4366.
(37) Gaetke, L. M.; Chow, C. K. Toxicology 2003, 189, 147.
(38) Minnihan, E. C.; Young, D. D.; Schultz, P. G.; Stubbe, J. J. Am.
Chem. Soc. 2011, 133, 15942.
(39) Alfonta, L.; Zhang, Z. W.; Uryu, S.; Loo, J. A.; Schultz, P. G. J.
Am. Chem. Soc. 2003, 125, 14662.
(40) Liu, X. H.; Jiang, L.; Li, J. S., Wang, L.; Yu, Y.; Zhou, Q.; Lv, X.;
Gong, W.; Lu Y.; Wang, J. Y. J. Am. Chem. Soc. 2014, 136, 13094.
(41) Liu, X. H; Li, J.; Dong, J.; Hu, C.; Gong, W.; Wang, J. Angew.
Chem. Intl. Ed. 2012, 51, 10261.
(42) Rae, T. D.; Schmidt, P. J.; Pufahl, R. A.; Culotta, V. C.;
O'Halloran, T. V. Science 1999, 284, 805.
(43) Hastings, G.; Kleinherenbrink, F. A. M.; Lin, S.; Mchugh, T. J.;
Blankenship, R. E. Biochemistry 1994, 33, 3193.
(44) Dunnivant, F. M.; Schwarzenbach, R. P.; Macalady, D. L.
Environ. Sci. Technol. 1992, 26, 2133.
(45) Tsao, M. L.; Summerer, D.; Ryu, Y.; Schultz, P. G. J. Am. Chem.
Soc. 2006, 128, 4572.
(46) Peters, F. B.; Brock, A.; Wang, J.; Schultz, P. G. Chem. Biol.
2009, 16, 148.
(47) Wang, Y. S.; Fang, X.; Chen, H. Y.; Wu, B.; Wang, Z. U.; Hilty,
References
(1) Wasielewski, M. R. Chem. Rev. 1992, 92, 435.
(2) Moser, C. C.; Keske, J. M.; Warncke, K.; Farid, R. S.; Dutton, P. L.
Nature 1992, 355, 796.
(3) Migliore, A.; Polizzi, N. F.; Therien, M. J.; Beratan, D. N. Chem.
Rev. 2014, 114, 3381.
(4) Cordes, M.; Giese, B. Chem. Soc. Rev. 2009, 38, 892.
(5) Winkler, J. R.; Gray, H. B. Chem. Rev. 2014, 114, 3369.
(6) Barber, J.; Andersson, B. Nature 1994, 370, 31.
(7) Schultz, D. M.; Yoon, T. P. Science 2014, 343, 985.
(8) Skourtis, S. S.; Beratan, D. N. Science 2007, 316, 703.
(9) Wang, H. Y.; Lin, S.; Allen, J. P.; Williams, J. C.; Blankert, S.;
Laser, C.; Woodbury, N. W. Science 2007, 316, 747.
(10) Genereux, J. C.; Barton, J. K. Chem. Rev. 2010, 110, 1642.
(11) Hammes-Schiffer, S.; Stuchebrukhov, A. A. Chem. Rev. 2010, 110,
6939.
(12) Giese, B.; Graber, M.; Cordes, M. Curr. Opin. Chem. Biol. 2008,
12, 755.
(13) Merino, E. J.; Boal, A. K.; Barton, J. K. Curr. Opin. Chem. Biol.
2008, 12, 229.
(14) Seefeldt, L. C.; Hoffman, B. M.; Dean, D. R. Curr. Opin. Chem.
Biol. 2012, 16, 19.
(15) Balzani, V.; Credi, A.; Venturi, M. Curr. Opin. Chem. Biol. 1997,
1, 506.
(16) Boekema, E. J.; van Breemen, J. F.; van Roon, H.; Dekker, J. P.
Biochemistry 2000, 39, 12907.
(17) Stewart, D. H.; Nixon, P. J.; Diner, B. A.; Brudvig, G. W.
Biochemistry 2000, 39, 14583.
(18) Vasil'ev, S.; Bruce, D. Biochemistry 2000, 39, 14211.
(19) Liu, Z.; Tan, C.; Guo, X.; Li, J.; Wang, L.; Sancar, A.; Zhong, D.
Proc. Natl. Acad. Sci. 2013, 110, 12966.
(20) Liu, Z.; Zhang, M.; Guo, X.; Tan, C.; Li, J.; Wang, L.; Sancar, A.;
Zhong, D. Proc. Natl. Acad. Sci. 2013, 110, 12972.
(21) Shih, C.; Museth, A. K.; Abrahamsson, M.; Blanco-Rodriguez, A.
M.; Di Bilio, A. J.; Sudhamsu, J.; Crane, B. R.; Ronayne, K. L.; Towrie,
M.; Vlcek, A.; Richards, J. H.; Winkler, J. R.; Gray, H. B. Science 2008,
320, 1760.
C.; Liu, W. R. ACS Chem. Biol. 2013, 8, 405.
(48) Vergne, C.; BoisChoussy, M.; Ouazzani, J.; Beugelmans, R.;
Zhu, J. P. Tetrahedron-Asymmetr 1997, 8, 391.
(49) Wang, J.; Zhang, W.; Song, W.; Wang, Y.; Yu, Z.; Li, J.; Wu, M.;
Wang, L.; Zang, J.; Lin, Q. J. Am. Chem. Soc. 2010, 132, 14812.
(50) Kennis, J. T.; Larsen, D. S.; van Stokkum, I. H.; Vengris, M.; van
Thor, J. J.; van Grondelle, R. Proc. Natl. Acad. Sci. 2004, 101, 17988.
(51) Leiderman, P.; Genosar, L.; Huppert, D.; Shu, X.; Remington, S.
J.; Solntsev, K. M.; Tolbert, L. M. Biochemistry 2007, 46, 12026.
(52) Shi, X.; Abbyad, P.; Shu, X.; Kallio, K.; Kanchanawong, P.;
Childs, W.; Remington, S. J.; Boxer, S. G. Biochemistry 2007, 46,
12014.
(53) Kennis, J. T. M.; van Stokkum, I. H. M.; Peterson, D. S.; Pandit,
A.; Wachter, R. M. J. Phys. Chem. B 2013, 117, 11134.
(54) van Wilderen, L. J. G. W.; Lincoln, C. N.; van Thor, J. J. Plos
One 2011, 6, e17373.
(55) Compton, R. G.; Dryfe, R. A. W.; Fisher, A. C. J. Chem. Soc.,
Perk. Trans. 1994, 1581.
(56) Hay, S.; Wallace, B. B.; Smith, T. A.; Ghiggino, K. P.;
Wydrzynski, T. Proc. Natl. Acad. Sci. 2004, 101, 17675.
(57) Gray, H. B.; Winkler, J. R. Biochim Biophys Acta. 2010, 1797,
1563.
(22) Doose, S.; Neuweiler, H.; Sauer, M. Chemphyschem 2009, 10,
1389.
(23) Dutta, P. K.; Lin, S.; Loskutov, A.; Levenberg, S.; Jun, D.; Saer,
R.; Beatty, J. T.; Liu, Y.; Yan, H.; Woodbury, N. W. J. Am. Chem. Soc.
2014, 136, 4599.
(24) Gritsch, P. J.; Leitner, C.; Pfaffenbach, M.; Gaich, T. Angew.
Chem. Int. Ed. 2014, 53, 1208.
(25) Osyczka, A.; Moser, C. C.; Daldal, F.; Dutton, P. L. Nature 2004,
427, 607.
(26) Mayo, S. L.; Ellis, W. R.; Crutchley, R. J.; Gray, H. B. Science
1986, 233, 948.
(58) Page, C. C.; Moser, C. C.; Chen, X. X.; Dutton, P. L. Nature
1999, 402, 47.
(59) Marcus, R. A. J. Chem. Phys. 1956, 24, 966.
(60) Gray, H. B.; Winkler, J. R. Q Rev Biophys 2003, 36, 341.
(61) Hu, C.; Chan, S. I.; Sawyer, E. B.; Yu, Y.; Wang, J. Y. Chem Soc
Rev 2014, 43, 6498.
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