A Designed Metalloenzyme Achieving the Catalytic Rate of a Native Enzyme

Article abstract of DOI:10.1021/jacs.5b07119

Terminal oxidases catalyze four-electron reduction of oxygen to water, and the energy harvested is utilized to drive the synthesis of adenosine triphosphate. While much effort has been made to design a catalyst mimicking the function of terminal oxidases, most biomimetic catalysts have much lower activity than native oxidases. Herein we report a designed oxidase in myoglobin with an O₂ reduction rate (52 s^-1) comparable to that of a native cytochrome (cyt) cbb₃ oxidase (50 s^-1) under identical conditions. We achieved this goal by engineering more favorable electrostatic interactions between a functional oxidase model designed in sperm whale myoglobin and its native redox partner, cyt b₅, resulting in a 400-fold electron transfer (ET) rate enhancement. Achieving high activity equivalent to that of native enzymes in a designed metalloenzyme offers deeper insight into the roles of tunable processes such as ET in oxidase activity and enzymatic function and may extend into applications such as more efficient oxygen reduction reaction catalysts for biofuel cells.

Full text of DOI:10.1021/jacs.5b07119

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Communication
A Designed Metalloenzyme Achieving the Catalytic Rate of a Native Enzyme
Yang Yu, Chang Cui, Xiaohong Liu, Igor D. Petrik, Jiangyun Wang, and Yi Lu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b07119 • Publication Date (Web): 28 Aug 2015
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A Designed Metalloenzyme Achieving the Catalytic Rate of a
Native Enzyme
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Yang Yu†,‡, Chang Cui¶,‡, Xiaohong Liu₸,‡, Igor D. Petrik¶, Jiangyun Wang₸,*, Yi Lu†, ¶,*
₸Laboratory of Non-coding 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, University of Illinois at Urbana-
Champaign, Urbana, Illinois, 61801, USA
Supporting
Information
Placeholder
gradient that drives the synthesis of adenosine triphosphate,
the universal source of energy for most biological processes.2
In addition to their important role in bioenergetics, terminal
oxidases are also of interest to biofuel cell research because
the oxidases are among the most efficient oxygen reduction
reaction (ORR) catalysts, having the lowest over-potentials
and using
ABSTRACT: Terminal oxidases catalyze four-electron reduc-
tion of oxygen to water, and the energy harvested is utilized
to drive ATP synthesis. While much effort has been made to
design a catalyst mimicking the function of terminal oxidas-
es, most biomimetic catalysts have much lower activity than
a native oxidase. We herein report a designed oxidase in my-
oglobin with an O2 reduction rate (52 s-1) comparable to that
of a native cyt cbb3 oxidase (50 s-1) under identical conditions.
We achieved this goal by engineering more favorable electro-
static interactions between a functional oxidase model de-
signed in sperm whale myoglobin and the native redox part-
ner of terminal oxidases, cyt b5, resulting in a 400 fold ET
rate enhancement. Achieving high activity equivalent to na-
tive enzymes in a designed metalloenzyme offers deeper in-
sight into the roles of tunable processes like ET in oxidase
activity and enzymatic function and may extend into applica-
tions such as more efficient oxygen reduction reaction cata-
lysts for biofuel cells.
Metalloenzymes normally exhibit much higher activities
under milder conditions than small inorganic catalysts, even
though the metalloenzymes use only a limited number of
physiologically available metal ions or metal-containing co-
factors. Rational design of metalloenzymes that mimic native
enzymes allows elucidation of structural features and mech-
anisms responsible for high activity and efficient use of
earth-abundant metal cofactors, and facilitates the design of
more robust and cost-effective catalysts for many applica-
tions.1 A key measure of success in this endeavor is the activi-
ty of the designed enzymes compared with the native en-
zymes they model. While tremendous progress has been
made in the field, most reported metalloenzymes display
activities that are far below those of the native enzymes that
they mimic.
Figure 1. (a) Structures of G65Y-CuBMb, showing the engi-
3
neered lysines in blue and cyt b5 (PDB ID: cyt b5 1CYO ;
F33Y-CuBMb 4FWY4, rendered through VMD5) (b) Oxidase
activity of G65Y-CuBMb in comparison with that of native
cyt cbb3 oxidase and G65YCuBMb with the same concentra-
tion, under the typical condition. Typical condition: NADH 2
mM, cyt b5 reductase 80 nM, cyt b5 5 μM, G65Y-CuBMb(+6)
50 nM. The black arrows indicate the addition of reductant
and the double arrow shows the injection of native cyt cbb3
oxidase.
An excellent example of metalloenzymes of interest are
terminal oxidases in the respiratory chain, in which O2 is
reduced to H2O in a four electron (4e-) process, and the en-
ergy harvested from this reduction is converted into a proton
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earth abundant metal ions such as iron and copper, unlike
ferric G65Y-CuBMb(+6) is similar to that of G65Y-CuBMb
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the best industrial ORR fuel cell catalysts, which require pre-
cious platinum.6 To understand the efficiency of this im-
portant class of metalloenzymes, many synthetic models
have been made to mimic their structures and functions,
with particular focus on the heme-copper center at the active
site of most oxidases.7 Despite years of effort by many
groups, the activities of the biomimetic compounds in ho-
mogenous solution are still far below those of native oxidas-
es.8
(Figure S3), suggesting that the mutations cause minimal
perturbation to the overall structure and the structure of the
heme active site.
To determine the oxidase activity measured by the rate of
O2 reduction, we used an O2-selective electrode commonly
used in monitoring catalytic activity in native oxidases.10, 14 In
the presence of 80 nM NADH-cyt b5 reductase, the physio-
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logical redox partner of cyt b5 and 5 μM of cyt b5, the con-
9
centration of O2 in the presence of 50 nM G65Y-CuBMb(+6)
decreased rapidly (Figure 1b), with the rate of the O2 reduc-
tion being 52 s-1. The reaction was carried out in 5 mM potas-
sium phosphate buffer, pH 6, which has been shown to be
the optimal pH for O2 reduction in this and other CuBMb
variants.4 The activity decreased with higher ionic strength
(Figure S4), suggesting that the ionic strength affects the
electrostatic interaction between Mb and cyt b5, which is
consistent with those reported by Hoffman and coworkers in
the similar system.16 Therefore the low ionic strength (5 mM)
was used in this study. Similar to G65Y-CuBMb,4 which has
been shown to mimic copper-independent cyt bd oxidase,17
addition of copper to G65Y-CuBMb(+6) did not increase oxi-
dase activity.4 As a result, no copper was added in the reac-
tion. In contrast, the G65Y-CuBMb without the
D44K/D60K/E85K mutations exhibited a rate of 0.3 s-1 under
the same condition. Furthermore, the protein without the
G65Y mutation (CuBMb(+6)) also exhibited a low O2 reduc-
tion rate 8.1 s-1, which affirms the importance of the G65Y
mutation in oxidase activity.4 Remarkably, the O2 reduction
rate of G65Y-CuBMb(+6) (52 s-1) is so fast that it is similar to
that of native cbb3 oxidase (50 s-1) measured under the same
condition.
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As an alternative approach to biomimetic studies of native
enzymes using small organic molecules, we use small and
stable proteins such as myoglobin (Mb) as scaffolds to design
structural features that mimic native enzymes.4, 9 Mb func-
tions as an O2 storage protein in biology. To transform the
Mb into an oxidase, we have introduced conserved structural
elements of heme-copper oxidases, one Tyr and two His resi-
dues, into the distal pocket of Mb. One such variant,
L29H/F43H/G65Y Mb (called G65Y-CuBMb) was able to re-
duce O2 to water, with catalytic oxygen reduction rate of 0.30
s-1 (Figure S1) and >1,000 turnovers, using ascorbate as a re-
ductant and TMPD as a redox mediator.4
While the above results demonstrate that we can rationally
design a functional enzyme with a high number of turnovers,
the catalytic rate (0.30 s-1) is still far below that of native en-
zymes (e.g., cyt cbb3 oxidase from Rhodobacter sphaeroides
has O2 reduction rate of 52 s-1).10 Our preliminary investiga-
tions of the G65Y-CuBMb and similarly designed Mb model
enzymes suggest that electron transfer (ET) into the catalytic
center may be rate limiting. This finding is consistent with
the studies of native cyt c oxidase11 and some synthetic mod-
els that showed that the electron delivery into the active site
might be a rate-limiting step.7b, 12 In searching for new redox
partners of Mb to increase the ET rate and potentially cata-
lytic activity, we were excited to learn the work by Hoffman
and coworkers, who replaced three negatively charged amino
acids (Asp44, Asp60 and Glu85) in myoglobin with three
positively charged lysines to improve electrostatic interac-
tions between the Mb and its redox partner, cyt b5, which is
negatively charged.13 Such a D44K/D60K/E85K variant, called
Mb(+6), enhanced the rate of ET from the triplet state of Zn-
deuteroporphyrin-substituted Mb to ferric cyt b5 by >2 orders
of magnitude (from 5.5x103 s-1 to ~ 1.0x106 s-1).13a Inspired by
this work, we report herein that introducing the
D44K/D60K/E85K mutations into G65Y-CuBMb (named
G65Y-CuBMb(+6) hereafter, see Figure 1a) resulted in dra-
matically enhanced oxidase activity, from 0.30 s-1 to 52 s-1,
making a designed metalloenzyme with activity comparable
to its target native enzymes.
Construction, expression and purification of G65Y-
CuBMb(+6) were carried out using a protocol reported previ-
ously.4 The mutations were confirmed by DNA sequencing,
and the corresponding protein containing the mutations was
corroborated by mass spectrometry (calculated MW: 17477;
observed MW: 17476). The electronic absorption spectra in
the ultraviolet and visible region (UV-vis) of G65Y-
CuBMb(+6) in both ferric and ferrous forms are similar to
those of G65Y-CuBMb (Figure S2a), and the EPR spectrum of
Figure 2. ET studies between cyt b5 and G65Y-CuBMb and
G65YCuBMb(+6). (a) Representative stopped-flow UV-visible
spectra of cyt b5 oxidation catalyzed by G65Y-CuBMb(+6) and
time trace at characteristic wavelength (inset). (b) Time trace
of cyt b5 oxidation represented by absorption at 556 nm,
overlaid with global spectroscopic fitting (red lines).
To confirm that the dramatic increase in the oxidase activ-
ity of G65Y-CuBMb(+6) is due to the enhanced ET, we first
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repeated the above experiment by systematically removing
like in native oxidases. Finally, 50 nM G65Y-CuBMb(+6) was
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each protein component involved in the O2 reduction. As
shown in Figure S5, minimal O2 reduction occurred in the
absence of either NADH-cyt b5 reductase, cyt b5, or G65Y-
CuBMb(+6), suggesting all three proteins are essential for the
enzymatic activity. Furthermore, we monitored the oxida-
tion of cyt b5 in the presence of either G65Y-CuBMb or G65Y-
CuBMb(+6) through stopped-flow UV-vis spectroscopy, in
order to estimate the ET rate between cyt b5 and the two Mb
variants (Figure 2 and Figure S6). We first found that the
pseudo first order ET rate constants between 5 μM cyt b5 and
G65Y-CuBMb(+6) increased linearly as the concentration of
G65Y-CuB(+6) increased (Figure S7),13 consistent with bimo-
lecular reactions as observed previously. Then we used the
same concentration of cyt b5 (0.5 μM) as in the oxidase activi-
ty assay, but we increased concentration of Mb variants from
50 nM to 0.5 μM, which is still a fraction of cyt b5. The pseu-
do first order rate constant between cyt b5 and G65Y-
CuBMb(+6) was determined as (1.2 ± 0.2) x 101 s-1, which is
~400 fold faster than that of G65Y-CuBMb ((3.0 ± 0.5) x 10-2 s-
1). This 400-fold increase is in the same order of magnitude
as those reported by Hoffman and coworkers (180-fold in-
crease).13 The slight difference is attributable to the differ-
ences in the Mb mutations and redox cofactors (heme vs. Zn
porphyrin) used in the two studies. More importantly, this
ET rate enhancement is similar to the rate enhancement of
O2 reduction, which strongly suggests that the enhanced ET
rate is responsible for the increased oxidase activity. The
reduction potentials of G65Y-CuBMb(+6) and G65Y-CuBMb
were determined to be (115 ± 11) and (129 ± 5) mV by spec-
troelectrochemical methods (Figure S8). Since the reduction
potentials of both mutants are similar, these results suggest
that the major contributor to the increased ET rate is the
enhanced electrostatic interaction between the negatively
charged cyt b5 and positively charged G65Y CuBMb(+6), not
due to a difference in ET driving force.
able to reduce 220 μM of O2 in the reaction system (Figure
1b), corresponding to > 4,400 turnovers.
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Figure 3. (a) Measurement of O2 reduction and H2O2 pro-
duction in the O2 reduction reaction. (b) Representative ki-
netic UV-visible spectra of reaction solution with 100 μM
NADH, sampled at 0.5 s interval over 100 s. NADH oxidation
and oxygen reduction catalyzed by G65Y-CuBMb(+6) (inset).
Rational design of proteins that mimic native enzymes
both structurally and functionally is both exciting and chal-
lenging because nature has millions of years to evolve en-
zymes to carry out complex reactions like O2 reduction.
Therefore, any progress in protein design that moves closer
to properties of native enzymes is encouraging, with the ul-
timate goal being to match the activity of native enzymes
while using the model to understand and explore potential
applications. While most biochemical studies of native en-
zymes use site-directed mutagenesis to find residues that
lower the activity of the enzyme, biosynthetic modeling
seeks to uncover residues that result in a gain of function and
increase the activity; the two approaches, therefore, com-
plement each other. For example, while ET is known to play
a critical role in oxidase activity, the exact mechanism is not
well understood. A report by Brunori and coworkers indicat-
ed the internal ET rate accounts for the turnover number,
and both ET rate and turnover number display the same pH
and temperature dependence.19 Another report by Fabian
and coworkers provided evidence that the rate-limiting step
is the initial electron transfer to the catalytic site.20 Our re-
sults here using biosynthetic models strongly suggest that ET
rate into the active site plays a critical role in increasing en-
zymatic activity in a mechanism like that of a native oxidase.
A related and exciting area of research is to immobilize na-
tive enzymes and their variants onto electrodes to under-
stand the protein-protein and protein-electrode interactions
To investigate whether the O2 reduction in the Mb vari-
ant is a 4e- process, as in native oxidases, we measured H2O2
produced from the reaction using an amperometric H2O2
sensor. Under the same conditions as in the O2 reduction
experiment shown in Figure 1b, the G65Y-CuBMb(+6) gener-
ates peroxide at 3.5 s-1 (Figure 3a), or 7% of total O2 reduced,
while the G65Y-CuBMb produced 22% of peroxide (Figure
S1).4 Furthermore, since the NADH used in the O2 reduction
experiment is a two-electron reductant, the ratio of the rate
of NADH consumed in the reaction over that of the O2 re-
duced can be used to determine whether the O2 reduction is
a 4e- process, as demonstrated in other heme enzymes.18 The
NADH oxidation was monitored by absorption at 352 nm
(see Figure 3b).18 The rate of the reaction is compared to that
of O2 reduction using an O2-selectvie electrode under the
same conditions as in Figure 3b. As shown in Figure 3b (in-
set), the ratio between the rate of NADH oxidation and that
of O2 reduction is (1.91 ± 0.05) for G65Y-CuBMb(+6). This
result is consistent with the low percentage of H2O2 directly
probed by the above electroanalytical method. Together,
both results strongly suggest that the O2 reduction carried
out by the G65Y-CuBMb(+6) is a 4e- process producing H2O
1m, 21
for efficient ET in applications such as biofuel cells.
Since our biosynthetic models are much smaller and more
robust that native oxidases and their variants, being able to
design biosynthetic models that match the O2 reduction
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2588; (h) Dürrenberger, M.; Ward, T. R., Curr. Opin. Chem. Biol.
activity of native enzymes may open a new avenue to explore
application in biofuel cells.
2014, 19, 99-106; (i) Petrik, I. D.; Liu, J.; Lu, Y., Curr. Opin. Chem. Biol.
2014, 19, 67-75; (j) Zastrow, M. L.; Peacock, A. F.; Stuckey, J. A.;
Pecoraro, V. L., Nature Chem. 2012, 4, 118-23; (k) Joh, N. H.; Wang,
T.; Bhate, M. P.; Acharya, R.; Wu, Y.; Grabe, M.; Hong, M.;
Grigoryan, G.; DeGrado, W. F., Science 2014, 346, 1520-1524; (l)
Kleingardner, J. G.; Kandemir, B.; Bren, K. L., J. Am. Chem. Soc. 2014,
136, 4-7; (m) Bachmeier, A.; Armstrong, F., Curr. Opin. Chem. Biol.
2015, 25, 141-151; (n) Ray, K.; Heims, F.; Schwalbe, M.; Nam, W., Curr.
Opin. Chem. Biol. 2015, 25, 159-171.
(2) (a) Fabian, M.; Skultety, L.; Jancura, D.; Palmer, G., Biochim.
Biophys. Acta 2004, 1655, 298-305; (b) Brzezinski, P.; Gennis, R. B., J.
Bioenerg. Biomembr. 2008, 40, 521-31; (c) Wikstrom, M., Biochim.
Biophys. Acta 2012, 1817, 468-75; (d) Yoshikawa, S.; Shimada, A.,
Chem. Rev. 2015, 115, 1936-1989.
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In conclusion, we have succeeded in engineering Mb,
which natively binds O2 reversibly, into an oxidase that dis-
plays the O2 reduction activity of a native oxidase with a sim-
ilar rate and 4e- reduction process. We achieved the goal by
enhancing the ET rates through engineering more favorable
electrostatic interactions between Mb and its redox partner
cyt b5, following the introduction of similar structural ele-
ments (conserved His and Tyr) from the native enzyme into
Mb. By demonstrating our ability to design proteins that
match the activities of native enzymes, we have obtained
deeper understanding of structural features important for
oxidase activity, which may allow engineering artificial en-
zymes for biochemical and biotechnology applications such
as more efficient ORR catalysts for biofuel cells.
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10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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43
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50
51
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53
54
55
56
57
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59
60
(3) Durley, R. C.; Mathews, F. S., Acta Crystallogr., Sect. D: Biol.
Crystallogr. 1996, 52, 65-76.
(4) Miner, K. D.; Mukherjee, A.; Gao, Y. G.; Null, E. L.; Petrik, I. D.;
Zhao, X.; Yeung, N.; Robinson, H.; Lu, Y., Angew. Chem., Int. Ed.
2012, 51, 5589-5592.
(5) Yu, H.; Schulten, K., PLoS Comput. Biol. 2013, 9, e1002892.
(6) Kjaergaard, C. H.; Rossmeisl, J.; Nørskov, J. K., Inorg. Chem. 2010,
49, 3567-3572.
(7) (a) Kim, E.; Chufán, E. E.; Kamaraj, K.; Karlin, K. D., Chem. Rev.
2004, 104, 1077-133; (b) Collman, J. P.; Devaraj, N. K.; Decréau, R. a.;
Yang, Y.; Yan, Y.-L.; Ebina, W.; Eberspacher, T. a.; Chidsey, C. E. D.,
Science 2007, 315, 1565-8; (c) Nagano, Y.; Liu, J.-G.; Naruta, Y.; Ikoma,
T.; Tero-Kubota, S.; Kitagawa, T., J. Am. Chem. Soc. 2006, 128, 14560-
14570.
ASSOCIATED CONTENT
Supporting Information
Experimental detail of protein expression, purification and
spectroscopic characterization is described as supporting
information. This material is available free of charge via the
Internet at http://pubs.acs.org.
(8) Collman, J. P.; Ghosh, S.; Dey, A.; Decréau, R. A.; Yang, Y., J. Am.
Chem. Soc. 2009, 131, 5034-5035.
AUTHOR INFORMATION
(9) (a) Sigman, J. A.; Kim, H. K.; Zhao, X.; Carey, J. R.; Lu, Y., Proc.
Natl. Acad. Sci. U. S. A. 2003, 100, 3629-34; (b) Sigman, J. A.; Kwok, B.
C.; Lu, Y., J. Am. Chem. Soc. 2000, 122, 8192-8196; (c) Wang, N.; Zhao,
X.; Lu, Y., J. Am. Chem. Soc. 2005, 127, 16541-16547; (d) Zhao, X.;
Nilges, M. J.; Lu, Y., Biochemistry 2005, 44, 6559-6564; (e) Zhao, X.;
Yeung, N.; Wang, Z.; Guo, Z.; Lu, Y., Biochemistry 2005, 44, 1210-1214;
(f) Liu, X.; Yu, Y.; Hu, C.; Zhang, W.; Lu, Y.; Wang, J., Angew. Chem.,
Int. Ed. 2012, 51, 4312-4316; (g) Yu, Y.; Mukherjee, A.; Nilges, M. J.;
Hosseinzadeh, P.; Miner, K. D.; Lu, Y., J. Am. Chem. Soc. 2014, 136,
1174-1177; (h) Bhagi-Damodaran, A.; Petrik, I. D.; Marshall, N. M.;
Robinson, H.; Lu, Y., J. Am. Chem. Soc. 2014, 136, 11882-11885; (i) Yu,
Y.; Lv, X.; Li, J.; Zhou, Q.; Cui, C.; Hosseinzadeh, P.; Mukherjee, A.;
Nilges, M. J.; Wang, J.; Lu, Y., J. Am. Chem. Soc. 2015, 137, 4594–4597;
(j) Yu, Y.; Zhou, Q.; Wang, L.; Liu, X.; Zhang, W.; Hu, M.; Dong, J.;
Li, J.; Lv, X.; Ouyang, H.; Li, H.; Gao, F.; Gong, W.; Lu, Y.; Wang, J.,
Chem. Sci. 2015, 6, 3881-3885.
(10) Lee, H. J.; Gennis, R. B.; Ädelroth, P., Proc. Natl. Acad. Sci. U. S.
A. 2011, 108, 17661-17666.
(11) Ferguson-Miller, S.; Babcock, G. T., Chem. Rev. 1996, 96, 2889-
2908.
(12) (a) Decreau, R. A.; Collman, J. P.; Hosseini, A., Chem. Soc. Rev.
2010, 39, 1291-1301; (b) Sengupta, K.; Chatterjee, S.; Samanta, S.; Dey,
A., Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 8431-8436.
(13) (a) Nocek, J. M.; Knutson, A. K.; Xiong, P.; Co, N. P.; Hoffman, B.
M., J. Am. Chem. Soc. 2010, 132, 6165-75; (b) Xiong, P.; Nocek, J. M.;
Vura-Weis, J.; Lockard, J. V.; Wasielewski, M. R.; Hoffman, B. M.,
Science 2010, 330, 1075-8.
(14) Pawate, A. S.; Morgan, J.; Namslauer, A.; Mills, D.; Brzezinski, P.;
Ferguson-Miller, S.; Gennis, R. B., Biochemistry 2002, 41, 13417-13423.
(15) (a) Takesue, S.; Omura, T., J. Biochem. 1970, 67, 267-76; (b)
Livingston, D. J.; McLachlan, S. J.; La Mar, G. N.; Brown, W. D., J.
Biol. Chem. 1985, 260, 15699-707.
(16) Wheeler, K. E.; Nocek, J. M.; Cull, D. A.; Yatsunyk, L. A.;
Rosenzweig, A. C.; Hoffman, B. M., J. Am. Chem. Soc. 2007, 129,
3906-3917.
(17) Hill, J. J.; Alben, J. O.; Gennis, R. B., Proc. Natl. Acad. Sci. U. S. A.
1993, 90, 5863-5867.
(18) Grinkova, Y. V.; Denisov, I. G.; McLean, M. A.; Sligar, S. G.,
Biochem. Biophys. Res. Commun. 2013, 430, 1223-1227.
Corresponding Author
jwang@ibp.ac.cn, yi-lu@illinois.edu.
Author Contributions
‡These authors contributed equally.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We gratefully acknowledge the US National Institutes of
Health (GM06221) to Y.L, Major State Basic Research Pro-
gram of China (2015CB856203), National Science Foundation
of China (21325211, 91313301, and 81302687), Tianjin Municipal
Grant (14ZCZDSY00059, 14JCYBJC43400) and Innovation
Fund For Technology Based Firms (14C26211100178) to J.W..
National Science Foundation of China (31270859), and the
Youth Innovation Promotion Association of Chinese Acade-
my of Sciences to X.L.. We also thank Prof. Robert B. Gennis
and Prof. Alexander Scheeline for helpful discussion, Dr.
Hanlin Ouyang for providing native cyt cbb3 oxidase for con-
trol experiments, and Mr. Evan Mirts for proof reading.
REFERENCES
(1) (a) Reedy, C. J.; Gibney, B. R., Chem. Rev. 2004, 104, 617-650; (b)
Ueno, T.; Abe, S.; Yokoi, N.; Watanabe, Y., Coord. Chem. Rev. 2007,
251, 2717-2731; (c) Das, R.; Baker, D., Annu. Rev. Biochem. 2008, 77,
363-382; (d) Lu, Y.; Yeung, N.; Sieracki, N.; Marshall, N. M., Nature
2009, 460, 855; (e) Heinisch, T.; Ward, T. R., Curr. Opin. Chem. Biol.
2010, 14, 184-199; (f) Kiss, G.; Çelebi-Ölçüm, N.; Moretti, R.; Baker,
D.; Houk, K. N., Angew. Chem., Int. Ed. 2013, 52, 5700-5725; (g)
Zastrow, M. L.; Pecoraro, V. L., Coord. Chem. Rev. 2013, 257, 2565-
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(19) Malatesta, F.; Sarti, P.; Antonini, G.; Vallone, B.; Brunori, M.,
(21) (a) Cracknell, J. A.; Vincent, K. A.; Armstrong, F. A., Chem. Rev.
2008, 108, 2439-2461; (b) Bren, K. L., Interface Focus 2015, 5,
20140091; (c) Fukuzumi, S., Curr. Opin. Chem. Biol. 2015, 25, 18-26.
Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 7410-7413.
(20) Jancura, D.; Antalik, M.; Berka, V.; Palmer, G.; Fabian, M., J. Biol.
Chem. 2006, 281, 26768.
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