Boric Acid-Fueled ATP Synthesis by FoF1 ATP Synthase Reconstituted in a Supramolecular Architecture

Article abstract of DOI:10.1002/anie.202016253

Significant strides toward producing biochemical fuels have been achieved by mimicking natural oxidative and photosynthetic phosphorylation. Here, different from these strategies, we explore boric acid as a fuel for tuneable synthesis of energy-storing molecules in a cell-like supramolecular architecture. Specifically, a proton locked in boric acid is released in a modulated fashion by the choice of polyols. As a consequence, controlled proton gradients across the lipid membrane are established to drive ATP synthase embedded in the biomimetic architecture, which facilitates tuneable ATP production. This strategy paves a unique route to achieve highly efficient bioenergy conversion, holding broad applications in synthesis and devices that require biochemical fuels.

Full text of DOI:10.1002/anie.202016253

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Communications
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How to cite: Angew. Chem. Int. Ed. 2021, 60, 7617–7620
International Edition: doi.org/10.1002/anie.202016253
German Edition: doi.org/10.1002/ange.202016253
Supramolecular Chemistry
Hot Paper
Boric Acid-Fueled ATP Synthesis by FoF1 ATP Synthase Reconstituted
in a Supramolecular Architecture
Xia Xu, Jinbo Fei,* Youqian Xu, Guangle Li, Weiguang Dong, Huimin Xue, and Junbai Li*
Abstract: Significant strides toward producing biochemical
fuels have been achieved by mimicking natural oxidative and
photosynthetic phosphorylation. Here, different from these
strategies, we explore boric acid as a fuel for tuneable synthesis
of energy-storing molecules in a cell-like supramolecular
architecture. Specifically, a proton locked in boric acid is
released in a modulated fashion by the choice of polyols. As
a consequence, controlled proton gradients across the lipid
membrane are established to drive ATP synthase embedded in
the biomimetic architecture, which facilitates tuneable ATP
production. This strategy paves a unique route to achieve
highly efficient bioenergy conversion, holding broad applica-
tions in synthesis and devices that require biochemical fuels.
non-redox proton-generated processes could be integrated to
achieve efficient ATP production in a well-defined system.
Boric acid ester, a typical dynamic chemical bond, has
attracted increasing attention due to extensive applications in
asymmetric organic synthesis, smart drug delivery and self-
healing systems.[9] Polyols such as mannitol are utilized to
form boric acid esters. Their physicochemical and biological
properties can be modulated by molecule structures of
polyols and boric acids. Most boric acid ester chemical
processes produce water rather than proton.[10] The one
releasing proton is the case of boric acid and polyols under
ambient condition, although they are usually used for
analytical chemistry.[11] Hence, it can be envisioned that this
boric acid ester chemistry is utilized to generate proton
gradients as the driving force for bioenergy conversion.
In this communication, we develop boric acid ester
chemistry to controllably synthesize bioenergy molecules in
a biomimetic supramolecular architecture by a non-redox
route. The mechanistic basis is shown in Figure 1. Polyol
triggers the release of proton locked in boric acid by
generation of cyclic boronate esters. As a consequence,
boric acid serves as the fuel to establish a proton gradient
across a lipid bilayer supported by polyelectrolyte-assembled
microcapsules. It drives embedded ATP synthase in the
biomimetic architecture to produce biochemical fuel ATP
from adenosine diphosphate (ADP) and inorganic phosphate
(Pi).
To support soft proteoliposomes to form stable biomim-
etic supramolecular architecture, polyelectrolyte microcap-
sules were constructed via layer-by-layer technique by using
microparticles as the removable template. The whole assem-
bly process is revealed in Figure 2a. The polyelectrolytes were
chosen as typical shell materials of microcapsules because of
their assembly ability through electrostatic interactions. In
detail, manganese carbonate microparticles were prepared
through simple chemical precipitation, which was analysed by
employing scanning electron microscopy (SEM) and trans-
mission electron microscopy (TEM). Figure S1 shows the
mean size of manganese carbonate microparticles with rough
surfaces is around 3 mm. Through purification, the charged
polyelectrolytes (Figure S2a) were sequentially deposited on
the surface of manganese carbonate microparticles through
electrostatic interactions. Microcapsules were obtained after
selective removal of manganese carbonate cores by using
ethylenediaminetetraacetic acid disodium salt (EDTA-Na2)
under mild condition. These assembly processes were moni-
tored by detecting the surface potentials (Figure S2b). After
removing the core, the final PEI-(PSS/PAH)3 microcapsules,
with the similar diameter, possess the typical folded charac-
terization (Figure S2c). The result is in good consistence with
B
ioinspired supramolecular chemistry has made a great
impact on synthesizing specific molecules and constructing
functional hierarchical architectures.[1] By mimicking natural
mitochondrion or chloroplast, much attention has been paid
to developing artificial systems for efficient bioenergy con-
version.[2] As a directly-consumable bioenergy currency,
adenosine triphosphate (ATP) plays critical roles in a wide
range of bioactivities including mass transportation, signal
transduction and biochemical synthesis.[3] In nature, it is
mainly produced through oxidative phosphorylation and
photophosphorylation. Up to now, significant advance in
artificial production of ATP has been achieved in mitochond-
rion or chloroplast-like systems.[4] For instance, nanozyme-
catalyzed cascade reactions were developed to mimic mito-
chondrion toward conversion of glucose into ATP.[5] In
addition, photochemical reactions were coupled to yield
ATP in synthetic chloroplasts.[6] However, few reports involve
non-redox processes.[7] In general, a cross-membrane proton
gradient is necessary driving force for ATP synthesis.[8] Thus,
[*] X. Xu, Prof. Dr. J. Fei, Dr. G. Li, W. Dong, H. Xue, Prof. Dr. J. Li
Beijing National Laboratory for Molecular Sciences (BNLMS), CAS
Key Lab of Colloid, Interface and Chemical Thermodynamics,
Institute of Chemistry, Chinese Academy of Sciences
100190 Beijing (China)
E-mail: feijb@iccas.ac.cn
jbli@iccas.ac.cn
X. Xu, Prof. Dr. J. Fei, H. Xue, Prof. Dr. J. Li
University of Chinese Academy of Sciences
100190 Beijing (China)
Prof. Dr. Y. Xu
Third Military Medical University
400038 Chongqing (China)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202016253.
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was identified by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). As revealed in Figure S3a, the
subunits (a, b, g, e, d, a, b, c) in the purified ATP synthase
were present clearly, indicating the structure integrity.[13]
Then, ATP synthase was reconstituted with mixed lipids
(dimyristoyl phosphatidyl choline (DMPC) and dimyristoyl
phosphatidylglycerole (DMPG), shown in Figure S3b) to
form proteoliposomes, according to the reported method.[14]
The activity of ATP synthase-containing proteoliposomes was
detected by using a luciferin-luciferase assay (Figure S3c).
The result suggests that purified ATP synthase is active and
can be used for the next research. In addition, the protein
content (4 mgmLÀ1) was determined by utilizing coomassie
bright blue (shown in Figure S4). Further, ATP synthase-
containing proteoliposomes were co-incubated with disper-
sive microcapsules to construct the biomimetic architecture,
as revealed in Figure 2a. To verify the successful spreading of
the proteoliposomes on the surface of the microcapsules,
confocal laser scanning microscopy (CLSM) was carried out
to achieve visualization of components using green and red
fluorescent probes. The homogeneously distributed and fully
overlapped fluorescence intensity (Figure 2b and Figure S5)
clearly indicates that the proteoliposomes were spread on the
microcapsules surface to assemble the biomimetic micro-
reactor. It results from microcapsule-mediated liposome
fusion.[15]
Cross-membrane proton gradient is the driving force for
ATP synthase to produce ATP. The general chemical reaction
between boric acid and polyol is shown in Figure 3a. Under
mild condition, boric acid is a weak acid (pKa = 5.8 ꢀ 10À8). It
means proton is at a locked state and stored in this molecule.
In the presence of polyol, boric acid can be transformed into
a stronger acid and proton is unlocked to release. To prove
this, mannitol was utilized as a typical model in an unbuffered
1 mM boric acid solution. The product was characterized by
low resolution electrospray ionization mass spectrometry
Figure 1. a) Schematic illustration of the supramolecular architecture,
which is composed of polyelectrolyte microcapsule supporting ATP
synthase (PDB: 6VON)-containing liposomes. With this biomimetic
architecture, in the presence of polyols, a proton locked in boric acid
is released to generate cross-membrane proton gradients, driving ATP
synthase to transform ADP and Pi into ATP in a controlled fashion.
b) The relevant boric acid ester chemical reaction.
Figure 2. a) Schematic diagram of the supramolecular assembly of the
biomimetic architecture. b) CLSM images of FITC-labeled microcap-
sules (left), TRITC-labeled proteoliposome (middle) and overlapping
structure (right). The excitation wavelengths of FITC and TRITC are
405 nm and 561 nm, respectively (scale bars=3 mm).
the previous reports.[12] These microcapsules can serve as the
supporting substrate to enhance the stability of proteolipo-
somes.
After purified from chloroplast in fresh spinach through
sucrose density gradient centrifugation, cFoF1-ATP synthase
Figure 3. a) Chemical equilibria of boric acid with and without polyols.
b) pH change of boric acid (1 mM) in the presence of different
concentrations of mannitol (100, 200, 300, 400 and 500 mM). c) ATP
synthesis by the assembled bioreactor a function of the reaction time
with and without mannitol and d) the relevant ATP production rate.
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(LR-ESI-MS). The ion peak at 371 appears in the mass
relationships need to be established to optimize the process.
spectrum (Figure S6), which can be assigned to the existence
of the relevant boric acid ester. Figure 3b demonstrates that
along with the increase of the concentration of mannitol, the
pH value of the mixed solution has a greater decrease. It
means pH change is proportional to the concentration of
mannitol. These findings indicate that the boric acid ester
chemistry can generate tuneable proton gradients by chang-
ing the concentration of mannitol. To be specific, there is pH
decrease of 1.54 unit when the concentration of mannitol is
200 mM. It can be attributed to the ester reaction of mannitol
and boric acid, shifting the equilibrium reaction of disasso-
ciation (Figure 3a). Based on these findings, we envision
mannitol releases the proton bonded within boric acid to
generate cross-membrane proton gradient, which can serve as
the driving force for ATP production. Thus, boric acid and
mannitol was introduced into the assembled architecture to
investigate ATP production as a function of time. ATP was
measured by using a luciferin-luciferase assay after immersion
into a buffer solution with and without mannitol (Figure 3c).
ATP production was obtained by using the standard curve
(shown in Figure S7). It shows accompanying the introduction
of mannitol, the concentration of produced ATP in the system
increases and tends to a plateau of 0.74 mM at 30 s. Based on
the relevant kinetic analyses in Figure 3d, the maximum
average production rate is 0.05 mMsÀ1 at 10 s. In contrast,
there is no obvious ATP production in the absence of
mannitol. These results demonstrate that mannitol enables
boric acid as a proton fuel to drive ATP synthesis.
To confirm the simplicity and generality of this strategy
above, we investigated other polyol-based systems. The
chemical structures of typical polyols are shown in Figure 4a.
As revealed in Figure 4b, after these polyols were introduced
into the solution, there are obvious pH changes. In detail,
after chemical re-equilibrium, for arabinose, the pH value of
the solution is decreased by 0.35 unit, while for talose, the pH
value went down by 1.17 unit. There is the highest decrease
(1.40 unit) for ribose, as shown in Figure 4b. The above results
reveal that polyols can release proton from boric acid as
a proton source. Meanwhile, it seems the chemical structure
of the polyols is a key factor and more structure-effect
LR-ESI-MS results demonstrate the formation of borate ester
between boric acid and these polyols, as shown in Figure S8–
S10. Further, these boric acid ester systems were applied as
proton fuel to produce ATP. To be specific, Figure 4c gives
the relevant ATP production rates. For arabinose, the average
ATP production rate is 0.0025 mMsÀ1. For talose, it is
0.008 mMsÀ1. In particular, it is the highest up to
0.028 mMsÀ1 for ribose, which is comparable to those in
natural oxidative phosphorylation and photophosphorylation.
Moreover, this result is in good consistence of pH change
above (Figure 4b), validating that greater proton gradient
generates higher ATP production rate.
Taken together, these findings above confirm the sim-
plicity, generality and efficiency of this approach. Importantly,
these polyols can be used for producing bioenergy by redox
reactions in nature. This work provides a non-redox route to
achieve their bioenergy conversion. Considering that intrinsic
biocompatibility of these polyols and high efficiency of ATP
production, the assembled bioreactor can be integrated with
ATP-driven cascade reactions for cell-free protein synthesis
and active transports based on motor protein-containing
biodevices.
In summary, boric acid ester chemistry is explored to
modulate bioenergy synthesis in a bioinspired supramolecular
architecture. Polyols remarkably shift the disassociation
equilibrium of boric acid, achieving a locked-to-unlocked
state transformation of proton. Thus, proton release is
triggered to generate a proton gradient across the lipid
membrane, which serves as the driving force for bioenergy
production. The production rate can be tuned by the choice of
polyols. Under optimal conditions, the production rate of
ATP is as high as 0.05 mMsÀ1 when mannitol is used. This
minimally robust supramolecular system integrates artificial
and natural processes to achieve highly efficient biochemical
transformation. This strategy can be extended to performing
ATP-dependent biosynthesis and active transport.
Acknowledgements
The authors gratefully acknowledge the financial support for
this research from the National Nature Science Foundation of
China (No. 21961142022, 21872150 and 22072160). J.F.
particularly thanks the Youth Innovation Promotion Associ-
ation of CAS (No. 2016032) and Institute of Chemistry, CAS
(No. Y6290512B1).
Conflict of interest
The authors declare no conflict of interest.
Keywords: biochemical fuel · biomimetic architectures ·
biosynthesis · proton gradients · supramolecular assembly
Figure 4. a) Chemical structures of typical polyols. b) pH changes after
boric acid reacts with arabinose, talose and ribose. c) The relevant
average ATP production rates.
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Accepted manuscript online: December 27, 2020
Version of record online: February 25, 2021
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