Rational Design of Dual Active Sites in a Single Protein Scaffold: A Case Study of Heme Protein in Myoglobin

Article abstract of DOI:10.1002/open.201500224

Rational protein design has been proven to be a powerful tool for creating functional artificial proteins. Although many artificial metalloproteins with a single active site have been successfully created, those with dual active sites in a single protein scaffold are still relatively rare. In this study, we rationally designed dual active sites in a single heme protein scaffold, myoglobin (Mb), by retaining the native heme site and creating a copper-binding site remotely through a single mutation of Arg118 to His or Met. Isothermal titration calorimetry (ITC) and electron paramagnetic resonance (EPR) studies confirmed that a copper-binding site of [3-His] or [2-His-1-Met] motif was successfully created in the single mutant of R118H Mb and R118M Mb, respectively. UV/Vis kinetic spectroscopy and EPR studies further revealed that both the heme site and the designed copper site exhibited nitrite reductase activity. This study presents a new example for rational protein design with multiple active sites in a single protein scaffold, which also lays the groundwork for further investigation of the structure and function relationship of heme/non-heme proteins.

Full text of DOI:10.1002/open.201500224

DOI: 10.1002/open.201500224
Rational Design of Dual Active Sites in a Single Protein
Scaffold: A Case Study of Heme Protein in Myoglobin
Xiao-Gang Shu,[a] Ji-Hu Su,[b] Ke-Jie Du,[a] Yong You,[c] Shu-Qin Gao,[c] Ge-Bo Wen,[c] Xiangshi Tan,[d] and Ying-
Wu Lin*[a, c]
Rational protein design has been proven to be a powerful tool
for creating functional artificial proteins. Although many artifi-
cial metalloproteins with a single active site have been suc-
cessfully created, those with dual active sites in a single pro-
tein scaffold are still relatively rare. In this study, we rationally
designed dual active sites in a single heme protein scaffold,
myoglobin (Mb), by retaining the native heme site and creat-
ing a copper-binding site remotely through a single mutation
of Arg118 to His or Met. Isothermal titration calorimetry (ITC)
and electron paramagnetic resonance (EPR) studies confirmed
that a copper-binding site of [3-His] or [2-His-1-Met] motif was
successfully created in the single mutant of R118H Mb and
R118M Mb, respectively. UV/Vis kinetic spectroscopy and EPR
studies further revealed that both the heme site and the de-
signed copper site exhibited nitrite reductase activity. This
study presents a new example for rational protein design with
multiple active sites in a single protein scaffold, which also lays
the groundwork for further investigation of the structure and
function relationship of heme/non-heme proteins.
enzyme, MauG, possesses two hemes c in distinct spin states
with a long-range charge-resonance model.[3] It is thus attrac-
tive to create functional artificial proteins with multiple active
sites for more efficient biocatalysis. Rational protein design has
been proven to be a powerful tool, based on the scaffold of
either natural proteins or de novo proteins.[4] Although many
artificial metalloproteins with a single active site have been
successfully created,[4] those with dual active sites in the same
protein are still relatively rare. Based on a protein dimer,
Roelfes and co-workers designed an artificial metalloenzyme
with dual copper sites capable of catalyzing the Diels–Alder re-
action.[5] Using domain swapping for horse heart myoglobin
(Mb), Hirota and co-workers designed a heterodimeric protein
with two different heme active sites, which exhibits distinct
ligand binding properties.[6] These artificial metalloproteins
were designed using two proteins by dimerization. Alternative-
ly, Dutton and co-workers designed 4-helix bundles that can
accommodate two heme groups or one heme and one other
cofactor such as zinc chlorin.[7] Moreover, dual Fe–S clusters or
metal-binding sites for Zn and Hg can be created in 3-helix
bundles.[8] Therefore, development of other methods to design
functional proteins with dual active sites in a single protein
scaffold will broaden the variety of artificial enzymes. Note that
although simply fusing two enzymes together might be
a straightforward method,[9] chemical modifications as com-
monly used for fusion, as well as protein–protein interactions,
should be considered to minimize the side effects on the
active sites.
Metalloproteins with multiple active sites usually perform bio-
logical functions effectively. For example, in cytochrome c ox-
idase (CcO), heme a delivers electrons to the heme a3:CuB site,
where O2 is reduced to H2O,[1] and in copper nitrite reductase
(NIR), type-1 copper delivers electrons to type-2 copper, the
site of nitrite binding and reduction.[2] Moreover, a di-heme
Due to the small size (153 amino acids) with a single heme
group, the easy-to-produce Mb has been favored as a protein
model for heme protein design.[10] By redesigning the heme
active site, the O2 carrier Mb has been converted successfully
into various artificial enzymes.[4a,d–h,10–11] Copper (CuB) and iron
(FeB) binding sites can also be designed close to the heme
iron, mimicking the heteronuclear metal center in CcO and
nitric oxide reductase (NOR), respectively.[12] With these advan-
ces, we are interested in design of dual active sites remote
from each other using Mb as a model protein to create multi-
ple metal centers in the same protein scaffold, which potential-
ly produces artificial enzymes more efficient than those with
a single active site.
[a] X.-G. Shu, Dr. K.-J. Du, Prof. Dr. Y.-W. Lin
School of Chemistry and Chemical Engineering
University of South China Hengyang 421001 (P. R. China)
E-mail: linlinying@hotmail.com
ywlin@usc.edu.cn
[b] Dr. J.-H. Su
Department of Modern Physics
University of Science and Technology of China, Hefei 230026 (P. R. China)
[c] Y. You, S.-Q. Gao, Prof. Dr. . D. G.-B. Wen, Prof. Dr. Y.-W. Lin
Laboratory of Protein Structure and Function
University of South China, Hengyang 421001 (P. R. China)
[d] Prof. Dr. X. Tan
Department of Chemistry, Shanghai Key Lab of Chemical Biology for Protein
Research & Institute of Biomedical Science
Fudan University, Shanghai 200433 (P. R. China)
To this end, we adopted a new approach of retaining the
native heme and creating another metal-binding site remotely
from the heme site (Figure 1). The previous experience of de-
signing a CuB site in the heme pocket of Mb suggests that it is
essential to create a metal-binding motif, such as a motif of
three histidine,[3-His], as found for the type-2 copper site in
NIR.[2] By a close inspection of the crystal structure of sperm
Supporting information for this article can be found under http://
dx.doi.org/10.1002/open.201500224.
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This is an open access article under the terms of the Creative Commons
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from the heme iron) is coordinated by both His24 (2.03 Š) and
His119 (1.67 Š), as well as a water molecule (3.69 Š). We hy-
pothesize that this Cu1-binding site containing two His resi-
dues is more stable than the other three copper-binding sites
on the protein surface. Moreover, the CD atom of Arg118 is
close to Cu1 with a distance of 3.79 Š, suggesting that a His or
Met introduced at this position may serve as an additional
ligand for the copper. These observations encouraged us to
construct both R118H Mb and R118M Mb single mutants.
R118H Mb and R118M Mb mutants were expressed and puri-
fied using the procedure reported previously.[12a] The protein
was confirmed by matrix-assisted laser desorption time-of-fight
(MALDI-TOF) mass spectrometry studies (Figure S2 in the Sup-
porting Information). To investigate whether any copper binds
to the designed metal-binding site, we performed isothermal
titration calorimetry (ITC) studies for the two single mutants,
with WT Mb as a control. Titration of CuII into the solution of
WT Mb caused only a slight change in binding enthalpy. In
contrast, major changes were observed when the CuII was ti-
trated into a solution containing R118M Mb or R118H Mb
under the same condition (Figure 2). These results strongly
suggest that introducing the R118H or R118M mutation in-
creases the CuII-binding significantly over that of WT Mb. To
obtain quantitative information of the binding, we used
a single-site binding model to fit the data, yielding a dissocia-
tion constant (Kd) value of ~81 and ~11 mm, and a stoichiome-
try of CuII to protein of 0.83Æ0.31 and 1.02Æ0.05, for R118M
Mb and R118H Mb, respectively. Note that due to the limitation
of ITC technique as well as data fitting, these results should be
treated as approximate values. Moreover, slight changes in
binding enthalpy were still observed for the two mutants with
CuII/Mb ratio higher than 2 (Figure 2B,C), which suggests the
existence of a weak binding site or nonspecific binding to the
protein surface. The data fitting for WT Mb showed a larger Kd
Figure 1. Design of dual active sites in a single protein scaffold of Mb.
whale Mb (PDB code 1JP6[13]), we found that a single mutation
of Arg118 to His would create a [3-His] copper-binding motif,
since the other two His residues (His24 and His119) are avail-
able in the native Mb with a proper conformation to form the
motif (Figure 1). Similarly, mutation to Met would likely create
a [2-His-1-Met] copper-binding site, as in the active site of pep-
tidylglycine monooxygenase (PHM).[14]
The choice of Arg118 as the target for mutation to build the
metal-binding motif was further supported by a crystallograph-
ic study of the F33Y CuBMb mutant in the presence of CuII.[15]
The structure revealed that, when the protein crystal was
soaked with an excess of copper ions (10 equiv CuSO4), in ad-
dition to the copper in the designed CuB site, there were four
other copper-binding sites in the crystal structure (Figure S1 in
the Supporting Information). Notably, a copper (Cu1, ~20 Š
Figure 2. ITC data for titration of WT Mb (A), R118M Mb (B), and R118H Mb (C) with CuII at 258C. Top, raw data. Bottom, plot of integrated heats versus CuII/
Mb ratio.
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value of ~752 mm, with a stoichiometry of 1.73Æ0.28, likely
due to the binding of CuII to other sites such as the Cu2 site
(Figure S1 in the Supporting Information). These results dem-
onstrate that the [3-His] and [2-His-1-Met] copper-binding sites
were successfully constructed in Mb by a single mutation, with
both sites exhibiting an affinity for CuII much higher (~68- and
~9-fold, respectively) than that for WT Mb, although another
weak binding site may also exist on the protein surface of the
mutants.
Table 1. Comparison of spin Hamiltonian parameters deduced from the
simulation of the EPR spectra.
Copper-binding site
gx
gy
gz
WT Mb (Site-1)
WT Mb (Site-2)
2.047
2.052
2.047
2.047
2.043
2.045
2.076
2.050
2.052
2.048
2.050
2.070
2.071
2.076
2.263
2.305
2.250
2.263
2.263
2.280
2.358
R118H Mb ([3-His])
R118M Mb ([2-His-1-Met])
PHM ([3-His])
PHM ([2-His-1-Met])
NIR (Type-2 Cu site)
To further confirm the CuII binding to the designed metal-
binding site, we collected electron paramagnetic resonance
(EPR) spectra. As shown in Figure 3A, the EPR spectra of R118H
Mb: myoglobin, PHM: peptidylglycine monooxygenase, NIR: nitrite reduc-
tase
Figure 3. A) EPR spectra of R118H Mb (i), R118M Mb (ii), and WT Mb (iii)
(0.3 mm) in the presence of 1 equiv CuII and free CuII in buffer (iv), collected
at 30 K, 2 mW power, and 9.43 GHz. B) Expanded view comparison of the
parallel hyperfine region of the spectra in (A).
Figure 4. A) EPR spectra for CuII (1, 2, 3, 5, and 10 equiv) titration of WT Mb
(0.18 mm) and 5 equiv free CuII in the same buffer solution (the arrow indi-
cates the free CuII and CuII bound to site-2, respectively). B) EPR spectra com-
parison of R118H Mb, R118M Mb, and WT Mb in the presence of 5 equiv CuII
under the same conditions (CuII binding sites are indicated by arrows).
Mb and R118M Mb mutants in presence of 1 equiv CuII have
features typical of the type-2 copper,[16] which are distinct from
that for the free CuII in solution, indicating the binding of
copper to protein. Although a similar spectrum was observed
for CuII-WT Mb under the same conditions, shoulder peaks ap-
peared in the parallel region, as indicated by arrows (Fig-
ure 3B). EPR simulation suggested that there are two copper-
binding sites with a ratio of ~1:0.7 in WT Mb, whereas site-2
was not obvious for either CuII-R118M Mb or CuII-R118H Mb
(Figure S3 in the Supporting Information and Table 1). It
should be noted that although ITC studies suggested there
were free CuII in WT Mb with 1 equiv CuII due to the large Kd
value, the EPR spectrum collected at a much higher (~10-fold)
concentration and lower temperature than the ITC studies
showed copper binding to the protein. Meanwhile, a free CuII
signal was indeed observed in EPR titration with excess CuII
ions (Figure 4A). EPR titration further showed that additional
CuII would bind to site-2 on the protein surface, and the signal
intensity of site-1 with respect to site-2 is higher for the mu-
tants than that for WT Mb (Figure 4B ). These observations
suggest that upon mutation of Arg118 in site-1, His118 or
Met118 acts as an additional ligand for the copper, resulting in
a higher copper binding affinity.
Note that CuII-R118M Mb has identical g values to the site-
1 of Mb (Table 1), which suggests that coordination of Met118
causes no geometry alteration for the copper. This may be at-
tributed to that fact that Met has the same side chain of CA–C
atoms to that of Arg, and the sulfur atom of Met118 is likely in
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a similar position of the CD atom of Arg118, with a short dis-
tance favorable for direct coordination (Figure S1 in the Sup-
porting Information). Interestingly, the CuII-R118H Mb has a g
value slightly lower than that of CuII-R118M Mb, which is con-
sistent with the prediction that metal-binding sites with more
N donors have lower g values based on Peisach–Blumberg cor-
relations.[17] These findings also agree with a recent study of
CuII binding to engineered PHM mutants with a [3-His] or [2-
His-1-Met] copper-binding site (Table 1).[14] Meanwhile, when
compared with the type-2 copper site in native NIR from Alca-
logenes faecalis (Table 1),[18] large differences in g values were
observed for both CuII-R118H Mb and CuII-R118M Mb, indicat-
ing that the artificially constructed copper site in Mb does not
identically reproduce the geometry of the type-2 copper in
native NIR. These structural differences might have profound
effects on the protein reactivity of nitrite reduction.
bate was observed for WT Mb and its mutants, which may be
due to an auto-oxidation of ascorbate,[20] since the heme iron
remained in the ferric state in these conditions, and no reac-
tion occurred at the heme site. Additionally, the NO product in
this essay was confirmed by addition of the headspace gas in
the reaction cuvette for CuII-R118H Mb to deoxy WT Mb, which
resulted in the formation of the NO-bound form, as shown in
the UV/Vis spectrum (Figure S4 in the Supporting Information).
To verify that the heme site in the CuII-R118H Mb can also
exhibit NIR activity, we increased the concentration of ascor-
bate to quickly reduce not only the CuII, but also the heme
FeIII. As shown in Figure 6A, the Soret band changed over time
After confirming that CuII indeed binds to the single mutants
of Mb, we then tested whether the copper site confers NIR ac-
tivity, as it resembles the active site of native copper NIR,[2]
albeit not identically. We performed an ascorbate–nitrite assay,
which was demonstrated by Pecoraro and co-workers for test-
ing the NIR activity of type-2 copper center designed in a-heli-
cal coiled coils.[19] In this assay, ascorbate acts as a sacrificial re-
ductant for CuII, and the resultant CuI-bound protein catalyti-
cally reduces nitrite to NO. The oxidation of ascorbate was
monitored by the decrease of its absorption at 265 nm (Fig-
ure 5A). Based on the changes of A265nm as a function of time
Figure 6. A) UV/Vis spectral changes for formation of NO–heme iron(II) com-
plex upon nitrite (5 mm) reduction by CuII-R118H Mb (5 mm) with ascorbate
(12 mm). Kinetic traces at 421 nm are shown as an inset. B) EPR spectra of ni-
trite reduction by R118H Mb (0.18 mm) in the presence (i) and absence (ii) of
1 equiv CuII for 5 min.
from 409 to 421 nm. Two visible bands appeared at ~548 and
~580 nm, indicating a conversion of the met form to the NO-
bound ferrous form (NOÀFe2+).[21] No observation of the fer-
rous heme in the reaction further suggested fast reduction of
À
NO2 and binding of NO. With 1 equiv CuII added, the ob-
served rate constant (kobs) of formation of the NOÀFe2+ com-
plex ((8.73Æ0.05)”10À3 sÀ1) is ~1.7-fold higher than the pro-
tein alone (5.19Æ0.03)”10À3 sÀ1). A ~1.4- and ~1.1-fold kobs
was also observed for R118M Mb and WT Mb, respectively,
upon CuII binding (Figure S5 in the Supporting Information).
These results indicate that the NO produced at the Cu site
Figure 5. A) UV/Vis spectra of a solution of sodium ascorbate (45 mm) and
NaNO2 (5 mm) collected each minute, containing R118H Mb (5 mm) and CuII
(5 mm). B) Decreases of [AscÀ] versus time in samples containing R118H Mb,
R118M Mb, or WT Mb in the absence or presence of CuII.
À
may compete with NO2 and rapidly bind to the ferrous heme
center. Since the NO produced at the heme site will also readi-
ly bind to the ferrous heme center in situ, we did not expect
too much for the enhancement of kobs contributed by the Cu
site. Moreover, the NO product was confirmed by EPR studies
(Figure 6B and inset of Figure S5 in the Supporting Informa-
tion), which exhibited characteristic signals of a nitrosyl ferrous
heme complex (g1 ~2.078, g2 ~2.027, g3 ~1.988), similar to pre-
vious observations (g1 ~2.08, g2 ~2.01, g3 ~1.98).[22] The en-
hanced intensity of the signals further indicates that both the
Cu and heme sites are reactive.
(Figure 5B), the initial rate of ascorbate oxidation (vASC,Ox) for
the first 2000 sec was calculated to be (0.90Æ0.05)”
10À5 mmsÀ1 for CuII-R118H Mb. This value is 2.25- and 1.55-fold
of that for CuII-WT Mb ((0.40Æ0.02)”10À5 mmsÀ1) and CuII-
R118M Mb ((0.58Æ0.02)”10À5 mmsÀ1), respectively. These re-
sults suggest that the copper site in Mb with a higher copper
affinity exhibits a higher NIR activity, presumably due to
a higher occupancy of the copper site according to the ITC
studies. The charge of the active site changed upon mutation
of Arg118 might also affect the binding and reduction of ni-
trite. Note that CuII alone did not show catalytic activity in this
assay, as confirmed in previous study.[19] In the absence of CuII
ions, a low oxidation rate ((0.10Æ0.01)”10À5 mmsÀ1) of ascor-
In conclusion, we demonstrated that dual active sites can be
designed in Mb by retaining the native heme site and creating
a remote copper-binding site through a single mutation. This
study presents a new example for rational protein design with
multiple active sites in a single protein scaffold. In addition,
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MALDI-TOF mass spectra, EPR simulation, and extra UV/
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Acknowledgements
The authors gratefully thank Prof. Yi Lu of the University of Illi-
nois at Urbana-Champaign, USA, for critical revising of the
manuscript and helpful suggestions, and Dr. Yu Xiang of Tsing-
hua University, China, for performing MALDI-TOF mass spectrom-
etry studies. EPR spectra were collected at the high magnetic
field laboratory of the Chinese Academy of Science. This work
was supported by the National Science Foundation of China
(31370812, 21472027), Hunan Provincial Natural Science Founda-
tion (2015JJ1012), Scientific Research Fund of Hunan Provincial
Education Department (15A158, 13C817), and the Zhengxiang
Scholar Program of the University of South China (Y.-W. Lin).
Keywords: electron paramagnetic resonance (EPR) · heme
proteins · metal-binding site · nitrite reductase · protein design
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Received: December 28, 2015
Published online on March 8, 2016
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ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim