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)
[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://
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
made.
ChemistryOpen 2016, 5, 192 – 196
192
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim