Defining the Role of Tyrosine and Rational Tuning of Oxidase Activity by Genetic Incorporation of Unnatural Tyrosine Analogs

Article abstract of DOI:10.1021/ja5109936

While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pK_a and reduction potential in O₂ reduction have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O₂ reduction mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pK_a of the phenol ring and increasing reduction potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O₂ reduction activity and the fraction of H₂O formation with the pK_a of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H₂O₂, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivatives incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymatic activity beyond that of natural Tyr.

Full text of DOI:10.1021/ja5109936

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Communication
Defining the Role of Tyrosine and Rational Tuning of Oxidase
Activity by Genetic Incorporation of Unnatural Tyrosine Analogs
Yang Yu, Xiaoxuan Lv, Jiasong Li, Qing Zhou, Chang Cui, Parisa
Hosseinzadeh, Arnab Mukherjee, Mark J. Nilges, Jiangyun Wang, and Yi Lu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja5109936 • Publication Date (Web): 11 Feb 2015
Downloaded from http://pubs.acs.org on February 18, 2015
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Defining the Role of Tyrosine and Rational Tuning of Oxidase
Activity by Genetic Incorporation of Unnatural Tyrosine Ana-
logs
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†,‡
₸,‡
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¶
Yang Yu , Xiaoxuan Lv , Jiasong Li , Qing Zhou , Chang Cui , Parisa Hosseinzadeh , Arnab Mukherjee , Mark J.
₸
,*
†, ¶,§,*
£
Nilges , Jiangyun Wang , Yi Lu
₸
Laboratory of Nonꢀcoding RNA, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District,
Beijing, 100101, P. R. China
†
¶
§
£
Center of Biophysics and Computational Biology, Department of Chemistry, Department of Biochemistry, Illinois EPR
Research Center, University of Illinois at UrbanaꢀChampaign, Urbana, Illinois, 61801, USA
Supporting Information Placeholder
highly conserved Tyr, found by Xꢀray crystallography to be adjaꢀ
ABSTRACT: While a conserved tyrosine (Tyr) is found in oxiꢀ
17,18
cent to one of the histidine ligands to the Cu center.
It is sugꢀ
dases, the roles of phenol ring pK and reduction potential in oxyꢀ
B
a
gested that this Tyr donates one electron and one proton during
gen reduction have not been defined despite many years of reꢀ
search on numerous oxidases and their models. These issues repꢀ
resent major challenges in our understanding of O2 reduction
mechanism in bioenergetics. Through genetic incorporation of
unnatural amino acid analogs of Tyr, with progressively decreasꢀ
15,19
the reaction.
More importantly, it is proposed that the pK of
a
the phenol ring may play an important role in modulating oxidase
8,20
activity,
but experimental support for such a proposal remains
elusive despite decades of investigations on many oxidases and
their variants. CcO is a large (~200 kDa for bovine CcO) memꢀ
brane protein consisting of many other metalꢀbinding sites (e.g.,
CuA and heme a as electron donors) and tyrosines at other locaꢀ
ing pK of the phenol ring and increasing reduction potential, in
a
the active site of a functional model of oxidase in myoglobin, a
linear dependence of both the O reduction activity and the fracꢀ
2
tions, making it difficult to focus on the O reduction site and the
tion of H O formation with the pK of the phenol ring has been
2
2
a
conserved Tyr. More importantly, because mutations of this conꢀ
established. By using these unnatural amino acids as spectroscopꢀ
ic probe, we have provided conclusive evidence for the location of
a Tyr radical generated during reaction with H O , by the distincꢀ
21,22
served Tyr to other amino acids abolished the oxidase activity,
it has not been possible to address the role of the phenol pK in
a
2
2
oxidase activity at molecular details. These issues represent the
remaining major challenges in our understanding of the oxidase
mechanism in bioenergetics, and addressing them could lead to
the design of more efficient biocatalysts for applications such as
catalysts for fuel cells that use earth abundant metal ions (instead
of platinum) with low overꢀpotentials.
tive hyperfine splitting patterns of the halogenated tyrosines and
one of its deuterated derivatives incorporated at the 33 position of
the protein. These results demonstrate for the first time that enꢀ
hancing the proton donation ability of the Tyr enhances the oxiꢀ
dase activity, allowing the Tyr analogs to augment enzymatic
activity beyond that of natural Tyr.
Tyrosine (Tyr) is unique among all natural amino acids, as it is
capable of donating both an electron and a proton in enzymatic
reactions. As a result, a Tyr is found in the active sites of a numꢀ
ber of enzymes such as galactose oxidases, ribonucleotide reducꢀ
1
ꢀ8
tase, and cytochrome c oxidase (CcO). For instance, Stubbe,
Nocera and coworkers utilized a series of tyrosine analogs as
probes to study the protonꢀcoupled electron transfer in enzymes
9
ꢀ12
using Tyr radicals.
However, the exact role of Tyr in many
enzymatic activities remains to be understood. A primary example
is the Tyr found in the active site of CcO.
As the terminal oxidase in the respiration chain, CcO catalyzes
efficient reduction of O to H O, harvests the released chemical
2
2
Figure 1. Crystal structure of the Phe33Tyr/Leu29His/Phe43His
energy, and converts it into the proton gradient that drives the
synthesis of adenosine triphosphate, the energy form for most
sperm whale myoglobin (Phe33TyrꢀCu Mb, PDB ID: 4FWX) and
B
pK of tyrosine and tyrosine analogs. From top to bottom: tyroꢀ
1
3ꢀ16
a
cellular processes.
This reaction requires rapid transfer of four
sine, 3ꢀchlorotyrosine (ClTyr), 3,5ꢀdifluorotyrosine (F Tyr), 2,3,5ꢀ
2
electrons and four protons to molecular oxygen in a highly conꢀ
trolled manner in order to prevent the release of toxic reactive
oxygen species (ROS), such as superoxide and peroxide, which
difluorotyrosine (F Tyr).
3
In previous studies, we have prepared functional models of
oxidases by introducing two histidines (His29 and His43) and one
not only damage proteins through radical reactions, but also lower
8
the energy conversion efficiency due to incomplete O reduction.
Tyr (Tyr33) into the distal pocket of sperm whale myoglobin
2
23ꢀ31
Critical to controlling this important reaction is the presence of a
(Mb) (Figure 1),
Together with the His64 present in Mb, this
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protein, called Phe33TyrꢀCu Mb, mimics CcO not only structurꢀ
by Stubbe and coworkers, fluorine substitution on the Tyr’s
phenol ring can introduce a unique splitting pattern for the tyrosyl
B
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ally but also functionally, reducing O to H O with more than 500
2
2
28
turnovers. In contrast to CcO, the Phe33TyrꢀCu Mb model is
radical signal. Indeed, simulation of the radical in Phe33F Tyrꢀ
B
2
much smaller (18 kDa) and is free of other metalꢀbinding sites,
making it much easier to study the roles of the active site Tyr
using common spectroscopic methods. The Crystal structure of
Cu Mb gave three species with slightly different g values, with
B
the major species (59%) having a g tensor of 2.013, 2.007, 2.004
and splitting from the fluorine nucleus of 44, 27, 161 MHz, and
35, 17, 144MHz, respectively (Table S2). In comparison, simulaꢀ
Phe33TyrꢀCu Mb showed a water molecule (Wat1) in the heme
B
pocket within 2.5 Å of the hydroxyl group of Tyr33 (Figure 1),
which is potentially involved in the putative proton delivery from
Tyr33 to the hemeꢀbound oxygen. The other atoms in Tyr33,
however, do not appear proximal enough to directly interact with
the substrate in the active site. More importantly, unnatural amino
acids such as imiTyr that mimics the crossꢀlinked HisꢀTyr can be
tion of the radical from Phe33TyrꢀCu Mb gave a g tensor of
B
2.009, 2.004, 2.002 and splitting from ring protons of 26, 11, 19
MHz. This difference observed in Phe33F TyrꢀCu Mb arises
2
B
from replacement of the Tyr33 with F Tyr and strongly suggests
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that the radical in this reaction originates at position 33.
2
9
readily introduced into the Phe33TyrꢀCu Mb. This approach
B
complements synthetic modeling, by which a Tyr has been introꢀ
32ꢀ34
duced into models of hemeꢀcopper centers.
Herein we report
introduction of a series of unnatural amino acid analogs of Tyr
with different pK ’s and reduction potentials of the phenol ring
a
into the Cu Mb at position 33 to provide conclusive evidence for
B
the roles the pK and reduction potential in regulating oxidase
a
activity.
We chose three unnatural amino acid analogs of Tyr with proꢀ
gressively lower pK of the phenol group than that of the native
a
Tyr (pKa =10.0):
3ꢀchlorotyrosine (ClTyr, pK =8.3), 3,5ꢀ
a
difluorotyrosine (F Tyr, pK = 7.2), and 2,3,5ꢀtrifluorotyrosine
2
a
(
F Tyr, pK =6.4) (Figure S1). The ClTyr was synthesized through
3 a
35
a oneꢀstep chemical transformation as described previously
while F2Tyr and F3Tyr were synthesized through an enzymatic
reaction catalyzed by tyrosine phenol lyase.
10
These tyrosine
analogs were incorporated into the protein in response to a TAG
amber stop codon by evolved tyrosyl amber suppressor tRNA
Tyr
(MjtRNA
)/tyrosylꢀtRNA synthetase (MjTyrRS) pairs from
CUA
29,36,37
Methanocaldococcus jannaschii.
The successful preparation
of the proteins was confirmed by electrospray ionization mass
spectrometry (Table S1). The asꢀpurified proteins do not contain
any metal in the Cu site. No copper ion was added in this work,
B
as previous studies have shown that the presence of copper does
not have any influence on the oxidase activity of Phe33Tyrꢀ
28
Cu Mb. The UVꢀvis absorption spectrum of the Phe33ClTyrꢀ
Figure 2. From top to bottom: Xꢀband EPR spectra of
B
Cu Mb as purified showed a Soret band at 407 nm and visible
Phe33TyrꢀCu Mb and the radicals formed in the reactions of
B
B
absorption bands at 501, 538 and 629 nm (Figure S2). Upon reꢀ
duction by adding excess dithionite, the deoxy form of the protein
exhibited a Soret band at 431 nm and a visible absorption band at
Phe33TyrꢀCu Mb, Phe33F TyrꢀCu Mb, Phe33ClTyrꢀCu Mb, and
B
2
B
B
Phe33D ClTyrꢀCu Mb with H O . S9pectra simulated by
2
B
2
2
3
SIMPOW6 are drawn in red dotted lines.
5
58 nm. Both UVꢀvis spectra of the met and deoxy forms of the
To provide further support for this assignment, we replaced the
Phe33ClTyrꢀCu Mb are almost identical to the corresponding
B
Phe33 with 3ꢀchlorotyrosine (Phe33ClTyrꢀCu Mb) and obtained
B
spectra of WTMb or Phe33TyrꢀCu Mb, as do the UVꢀvis spectra
B
its EPR spectrum under the same conditions. Since the chloroꢀ
substitution can introduce quadruple splitting with a weak couꢀ
pling constant that is then further broadened by the presence of
of the proteins containing the F Tyr and F Tyr (Figure S2). Furꢀ
2
3
thermore, the EPR spectrum of metꢀPhe33TyrꢀCu Mb is very
B
similar to that of WTMb, while the spectra of metꢀPhe33ClTyrꢀ
35
37
both Cl and Cl isotopes, its contribution to the radical hyperꢀ
fine splitting should be minimal. Indeed, much less hyperfine
splitting was observed in the EPR spectrum of Phe33ClTyrꢀ
Cu Mb than those of either Phe33TyrꢀCu Mb or F Phe33F Tyrꢀ
Cu Mb and metꢀPhe33F TyrꢀCu Mb display an additional rhomꢀ
B
2
B
bic signal at g ≈ 6 (Figure S3). These results suggest that introducꢀ
ing the Tyr analogs has minimal perturbation on the structures of
the protein models.
B
B
2
2
Cu Mb (Figure 2). The EPR spectrum from Phe33ClTyrꢀCu Mb
B
B
In a previous study, a tyrosineꢀbased radical was observed in
the reaction of the Mbꢀbased oxidase with O and H O , stressing
was simulated by removing ring protons. Finally, in addition to
the halogen on the phenol ring, substitution of the protons with
2
2
2
38
the central role of Tyr in oxidase reaction. Removing the three
deuterons could also induce a change in the hyperfine splitting
40,41
other tyrosines in Phe33TyrꢀCu Mb (Tyr 103, 146 and 151) reꢀ
pattern.
To eliminate the splitting from the protons, we synꢀ
B
sulted in an almost identical EPR signal, providing support that
thesized β,β ꢀD ꢀ3ꢀClTyr (D ClTyr) from β,βꢀD Tyr in one step
2 2 2
3
8
Tyr33 may be the location of the tyrosyl radical. Inspired by
with 45% yield. This unnatural amino acid was then incorporated
into myoglobin using the same tRNA/aaRS system for the
Phe33ClTyrꢀCu Mb to give Phe33D ClTyrꢀCu Mb. Compared to
1
0ꢀ12
Stubbe and coworkers’ work,
we used EPR to characterize the
reaction between H O and ferric Phe33TyrꢀCu Mb and its variꢀ
2
2
B
B
2
B
ants, to provide a direct evidence of the location of the tyrosyl
radical. As shown in Figure 2, upon reaction with stoichiometric
amount of H O , the Phe33TyrꢀCu Mb exhibited a free radical
the spectra from Phe33ClTyrꢀCu Mb, Phe33D ClTyrꢀCu Mb
B 2 B
gives a different splitting pattern that can be simulated by changꢀ
ing β protons to deuterons. Together, these results provide concluꢀ
sive evidences that the tyrosyl radical is located at residue Tyr33.
2
2
B
signal typical of a tyrosyl radical, distinctive from those from
10
WTMb or Cu Mb (without the Phe33Tyr mutation). As shown
B
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Having identified the location of Tyr radical, we then investiꢀ
gated the relationship between the oxidase activity and the properꢀ
pK and reduction potential into specific sites of designed proteins
should greatly improve our ability to elucidate the precise roles of
Tyr in enzymatic reactions and to design redoxꢀactive enzymes.
a
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ties of the Tyr, such as pK of the phenol and the reduction potenꢀ
a
tial of the amino acid. The rates of oxygen reduction catalyzed by
6
ꢁM myoglobin mutants containing Tyr33 or its unnatural amino
acid analogs were measured by a Clarkꢀtype O electrode in 20
2
mM tris(hydroxymethyl)aminomethane (Tris) buffer at pH 7.4,
using a protocol reported previously for both HCOs and their
28,29,42
ꢀ
2ꢀ
models.
The rates of O2 and O2 generation were deterꢀ
mined by comparing the rates of oxygen consumption in the presꢀ
ence and absence of superoxide dismutase (SOD) and catalase,
2
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respectively, as described previously. Since the SOD and cataꢀ
ꢀ
2ꢀ
lase will convert O2 or O2 to O and H O, lowering apparent O
2
2
2
ꢀ
2ꢀ
consumption rates, the fraction of O , O and H O generation
2
2
2
can be calculated based on the known stoichiometry of the above
reactions. Remarkably, the oxidase activities of these models are
inversely proportional to the pK of the phenol ring of Tyr33 and
a
its analogs (Figure 3A), supporting the proposed role of Tyr in
oxidase activity by providing the proton of the Tyr to facilitate Oꢀ
O bond cleavage and formation of H O. Furthermore, the percent
2
water formation is also inversely proportional to the pK of the
a
phenol rings (Figure 3B). In addition, to investigate the role of
the reduction potential of these tyrosines in the oxidase activity,
we measured the reduction potential of the Tyr and its analogs at
pH 13, the pH at which the phenol is fully deprotonated so that
10
the potential is independent of the pK (Figure S4). Since oxidaꢀ
a
tion of Tyr is an irreversible oneꢀelectron process on the elecꢀ
43
trode, we only obtained the peak potential (E ). As the halogenꢀ
p
ation decreases phenol pK ’s from 9.8 to 6.4, the E increases
a
p
from 672 mV to 850 mV in the same order, making the two facꢀ
tors difficult to separate. Interestingly, the oxidase activity of
these proteins increases as the E (at pH 13) of the Tyr and its
p
analogs increases (Figure 3C). However, we found poor correlaꢀ
tion between the reduction potentials of the Tyr and its analogs at
2
pH 7 and their oxidase activity (R =0.46, Figure S5).
To further demonstrate the robustness of our oxidase models,
we carried out multiple turnover experiments with a previously
28
established protocol. As shown in Figure 3D, the Phe33ClTyrꢀ
Cu Mb mutant was able to catalyze O reduction for more than
B
2
1200 turnovers without significant reduction of catalytic rate.
Under similar conditions, the Phe33TyrꢀCu Mb could only cataꢀ
B
2
8
lyze the reaction for less than 500 turnovers. Therefore, introꢀ
ducing the unnatural amino acids has allowed us not only to clariꢀ
fy the role of Tyr, but also to increase the activity and turnovers of
the enzyme.
Figure 3. The pK of phenols on the Tyr and its analogs affects
a
In summary, through the genetic incorporation of a series of
unnatural amino acid analogs of Tyr, with progressively decreasꢀ
oxidase activity. A) Correlation of oxidase activity of Phe33Tyrꢀ
Cu Mb, Phe33ClYꢀCu Mb, Phe33F YꢀCu Mb, and Phe33F Yꢀ
B
B
2
B
3
2
ing pK ’s of the phenol ring and increasing reduction potentials
a
Cu Mb vs. pK of phenols on the Tyr and its analogs, R =ꢀ0.98;
B a
into the active site of a functional model of an oxidase in myogloꢀ
bin, an inversely proportional correlation was found between pheꢀ
B) Correlation of water produced in oxygen reduction reaction
performed by these proteins vs. the pK of phenols on the Tyr and
a
2
nol pK in the Tyr and Tyr analogs and oxidase activity and selecꢀ
a
its analogs, R =0.94; C) Correlation of oxidase activity of
tivity of the reaction towards water production in these models.
Using unnatural amino acids as spectroscopic probes, the radical
generated in the reaction of myoglobinꢀbased functional oxidase
Phe33TyrꢀCu Mb, Phe33ClYꢀCu Mb, Phe33F YꢀCu Mb, and
B
B
2
B
Phe33F YꢀCu Mb vs. peak potential at pH 13 (E ) of the correꢀ
3
B
p
2
sponding Tyr and Tyr analogs, R =0.95; D) O ꢀreduction turnover
2
models with H O is unambiguously assigned to the active site
2
2
number by Phe33ClTyrꢀCu Mb and Phe33TyrꢀCu Mb measured
B
B
Tyr evidenced by hyperfine splitting patterns of the halogenated
tyrosines and one of its deuterated derivatives.
during the stepwise addition of O2.
Multiꢀelectron processes occur in numerous essential chemical
and biological reactions such as respiration and solar energy
conversions. While chemists have often achieved these functions
using metal ions such as Pd, Ru, and Rh that can undergo multiꢀ
electron processes, nature uses redoxꢀactive tyrosine residues in
conjunction with earth abundant transition metals such as Mn, Fe,
and Cu. More importantly, protons are often involved in such
multiꢀelectron processes, a role that tyrosines can also fulfill. Our
ability to genetically encode unnatural amino acids with tunable
Indeed, by directly incorporating the unnatural amino acids into
a functional oxidase model in response to the amber codon TAG
in E. coli, we have successfully provided evidence, for the first
time in a metalloprotein, that the enhanced proton donating ability
of the phenol ring of the Tyr is responsible for the high oxidase
activity and selectivity for water production. Since the syntheses
of these unnatural amino acids are facile with high yields and the
mutant proteins can be easily obtained at milligram quantity,
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incorporation of these unnatural amino acids offers exciting new
(17) Tsukihara, T.; Aoyama, H.; Yamashita, E.; Tomizaki, T.;
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opportunities to design metalloenzymes with functional properties
even better than those of native enzymes and to address a variety
of unresolved questions regarding the activity in these enzymes.
Yamaguchi, H.; ShinzawaꢀItoh, K.; Nakashima, R.; Yaono, R.;
Yoshikawa, S. Science 1996, 272, 1136.
(18) Yoshikawa, S.; ShinzawaꢀItoh, K.; Nakashima, R.; Yaono,
R.; Yamashita, E.; Inoue, N.; Yao, M.; Fei, M. J.; Libeu, C. P.;
Mizushima, T.; Yamaguchi, H.; Tomizaki, T.; Tsukihara, T.
Science 1998, 280, 1723.
ASSOCIATED CONTENT
(19) Proshlyakov, D. A.; Pressler, M. A.; DeMaso, C.; Leykam,
Supporting Information
J. F.; DeWitt, D. L.; Babcock, G. T. Science 2000, 290, 1588.
(20) Pratt, D. A.; Pesavento, R. P.; van der Donk, W. A. Org.
Lett. 2005, 7, 2735.
(21) Das, T. K.; Pecoraro, C.; Tomson, F. L.; Gennis, R. B.;
Rousseau, D. L. Biochemistry 1998, 37, 14471.
Experimental details about F Tyr synthesis, pK measurement,
2
a
protein expression and purification, UVꢀvis spectra, and oxidase
activity assay. This material is available free of charge via the
Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
(
Corresponding Author
(
jwang@ibp.ac.cn, yiꢀlu@illinois.edu.
122, 8192.
(25) Wang, N.; Zhao, X.; Lu, Y. J. Am. Chem. Soc. 2005, 127,
16541.
(26) Zhao, X.; Nilges, M. J.; Lu, Y. Biochemistry 2005, 44,
Author Contributions
‡These authors contributed equally.
6
559.
27) Zhao, X.; Yeung, N.; Wang, Z.; Guo, Z.; Lu, Y.
Biochemistry 2005, 44, 1210.
28) Miner, K. D.; Mukherjee, A.; Gao, Y.ꢀG.; Null, E. L.;
(
Notes
The authors declare no competing financial interests.
(
Petrik, I. D.; Zhao, X.; Yeung, N.; Robinson, H.; Lu, Y. Angew.
Chem. Int. Ed. 2012, 51, 5589.
(29) Liu, X.; Yu, Y.; Hu, C.; Zhang, W.; Lu, Y.; Wang, J.
Angew. Chem. Int. Ed. 2012, 51, 4312.
ACKNOWLEDGMENT
We gratefully acknowledge the Major State Basic Research Proꢀ
gram of China (2015CB856203), National Science Foundation of
China (21325211 and 91313301) to J.W., and the US National
Institutes of Health (GM06221) to Y.L.
(
30) Lu, Y.; Yeung, N.; Sieracki, N.; Marshall, N. M. Nature
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32) Kim, E.; Chufán, E. E.; Kamaraj, K.; Karlin, K. D. Chem.
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