Infrared spectroscopy reveals metal-independent
carbonic anhydrase activity in crotonyl-CoA
carboxylase/reductase†
Aharon Gomez, ‡§
a
Matthias Tinzl,§
b
Gabriele Stoffel,
b
Hendrik Westedt,
b
Helmut Grubmüller,
c
Tobias J. Erb,
bd
Esteban Vöhringer-Martinez *
a
and Sven T. Stripp *
ef
The conversion of CO
2
by enzymes such as carbonic anhydrase or carboxylases plays a crucial role in many
biological processes. However, in situ methods following the microscopic details of CO
2
conversion at the
active site are limited. Here, we used infrared spectroscopy to study the interaction of CO
2
, water,
bicarbonate, and other reactants with b-carbonic anhydrase from Escherichia coli (EcCA) and crotonyl-
CoA carboxylase/reductase from Kitasatospora setae (KsCcr), two of the fastest CO
2
-converting
enzymes in nature. Our data reveal that KsCcr possesses a so far unknown metal-independent CA-like
activity. Site-directed mutagenesis of conserved active site residues combined with molecular dynamics
simulations tracing CO
2
distributions in the active site of KsCCr identify an ‘activated’water molecule
forming the hydroxyl anion that attacks CO
2
and yields bicarbonate (HCO
3
−
). Computer simulations also
explain why substrate binding inhibits the anhydrase activity. Altogether, we demonstrate how in situ
infrared spectroscopy combined with molecular dynamics simulations provides a simple yet powerful
new approach to investigate the atomistic reaction mechanisms of different enzymes with CO
2
.
Introduction
Developing catalytic strategies for the capture and conversion of
carbon dioxide (CO
2
) is key to increased mitigation, utilization,
and sequestration of this critical greenhouse gas. While still
being a challenge for synthetic chemistry enzymes provide
a natural blueprint for efficient CO
2
-converting catalysts.
1
Several enzymes are known that interact with CO
2
and/or
bicarbonate (HCO
3
−
) during catalysis, in particular carbonic
anhydrases (CAs) and carboxylases.
CAs catalyze the reversible conversion of CO
2
,H
2
O, and
bicarbonate (HCO
3
−
) with rate enhancements of close to 8 ×
10
6
compared to the reaction in aqueous solution (eqn (1)). This
makes them one of the most effective CO
2
-converting catalysts
in nature.
2
CAs are present in all three domains of life and have
been classied in eight families.
3
Almost all known CAs feature
a zinc cation (Zn
2+
) as active site cofactor,
4
which plays a central
role in catalysis as Zn
2+
coordinates a hydroxide anion (OH
−
)
that attacks CO
2
as a nucleophile to form HCO
3
−
(Scheme 1).
The OH
−
species itself is generated through proton abstraction
from a zinc-bound water molecule to a nearby base, which is
usually a histidine.
5–8
CO
2
+2H
2
O4HCO
3
−
+H
3
O
+
(1)
Carboxylases catalyze the addition of CO
2
to an acceptor
substrate with the family of enoyl-CoA carboxylases/reductases
(ECRs) encompassing some of the most efficient CO
2
-xing
enzymes found in nature.
9
ECRs catalyze the reductive carbox-
ylation of a,b-unsaturated enoyl-CoAs with the reduced form of
nicotinamide adenine dinucleotide phosphate (NADPH) as
cofactor. Hydride transfer from NADPH to the enoyl-CoA
substrate generates a reactive enolate species, which acts as
a nucleophile that attacks a CO
2
molecule bound at the active
site.
10,11
At the example of crotonyl-CoA carboxylase/reductase
from Kitasatospora setae (KsCcr), Fig. 1 illustrates how CO
2
-
a
Departamento de F´
ısico Qu´
ımica, Facultad de Ciencias Qu´
ımicas, Universidad de
Concepci´
on, Concepci´
b
Department of Biochemistry and Synthetic Metabolism, Max-Planck-Institute for
Terrestrial Microbiology, Karl-von-Frisch-Str. 10, D-35043 Marburg, Germany
c
Department of Theoretical and Computational Biophysics, Max-Planck-Institute for
Multidisciplinary Sciences, Am Fassberg 11, 37077 Göttingen, Germany
d
Center for Synthetic Microbiology (SYNMIKRO), Germany
e
Freie Universit¨
at Berlin, Experimental Molecular Biophysics, Arnimallee 14, 14195
Berlin, Germany
f
Technische Universit¨
at Berlin, Division of Physical Chemistry, Strasse des 17. Juni 124,
10623 Berlin, Germany. E-mail: s.stripp@tu-berlin.de
†Electronic supplementary information (ESI) available. See DOI:
https://doi.org/10.1039/d3sc04208a
‡Present address: Departamento de Ciencias Biol´
ogicas y Qu´
ımicas, Facultad de
Medicina y Ciencia, Universidad San Sebasti´
an, Concepci´
on, Chile.
§These authors contributed equally.
Cite this: Chem. Sci.,2024,15, 4960
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Received 11th August 2023
Accepted 27th February 2024
DOI: 10.1039/d3sc04208a
rsc.li/chemical-science
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binding is achieved through four amino acid residues and one
conserved water molecule that is coordinated by an aspartate
E171 and a histidine H365 (mW).
12
All molecular species involved in the above described CO
2
-
conversions (H
2
O, CO
2
, HCO
3
−
) show characteristic absorbance
between 4000–1000 cm
−1
, which makes them available to
Fourier-transform infrared (FTIR) spectroscopy.
13–16
In a protein
sample, however, these signals are overlaid by the intense
absorbance of bulk water and the amide bands of the protein
backbone.
17
This limitation can be overcome by FTIR difference
spectroscopy, which provides the means to distinguish between
protein sample background signals and the signature of a given
reaction upon a specic trigger.
18
We developed a FTIR
difference spectroscopy-based setup in which catalysis can be
triggered via the gas phase.
19
Compared to the conventional
transmission conguration a protein lm is formed on top of
the silicon crystal of an attenuated total reection (ATR) optical
cell,
20
which makes the protein amendable to changes in the gas
phase, e.g., by switching from a inert carrier gas (100% N
2
or Ar,
dening the background signal) to a ‘reactive’gas mixture (see
ESI†for further details). This specic design allows studying the
reaction of CO
2
-converting enzymes providing the substrate
(i.e., CO
2
)in situ and thus the reaction trigger for these
enzymes.
19
Here, we applied in situ ATR FTIR spectroscopy to study the
interaction of KsCcr with CO
2
. Our results show that the active
site of KsCcr does not only bind CO
2
but surprisingly possesses
a so-far unknown, intrinsic CA-like activity, which enables the
enzyme to catalyze the reversible interconversion of CO
2
,H
2
O,
and HCO
3
−
. Studying the reaction in absence or presence of
substrates or inhibitors with wild-type and ve active site vari-
ants, we identied key residues for the observed CA-like activity
including a cluster of strongly hydrogen-bonded, ‘local’water
molecules. Moreover, computer simulations suggest that
conformational dynamics and substrate binding in KsCcr
modulate CO
2
binding at the active site. Combining experiment
and simulation, we propose a mechanism for the CA-like
activity of KsCcr that involves an ‘activated’water molecule,
which is essential for CO
2
-binding during the CO
2
-xation
reaction of KsCcr but also serves as nucleophilic OH
−
anion in
the enzyme's CA-like reaction.
Results and discussion
Infrared signatures of anhydrase activity
First, we pipetted 1 mlKsCcr solution (200 mM protein in 25 mM
Tris/HCl pH 7.5) on the ATR crystal of the FTIR spectrometer
and monitored water evaporation under dry N
2
gas in situ. Once
sufficiently concentrated, we rehydrated the protein lm under
a stream of aerosol that was created by sending dry N
2
gas (3
L min
−1
) through a wash bottle containing a dilute Tris/HCl
buffer solution (1 mM, pH 7.5). Then, we added 10% CO
2
to
the N
2
carrier gas for 50–100 s and recorded data to calculate
a series of time-resolved in situ ATR FTIR difference spectra that
result from the interaction of KsCcr with CO
2
(Fig. S1†). In
reference experiments with pure water and buffer solution,
25 mM Tris/HCl (pH 8) was found to be sufficiently concen-
trated preventing acidication in the presence of 10% CO
2
(Fig. S2†).
Fig. 2A depicts a ‘CO
2
–N
2
’FTIR difference spectrum recorded
25 s aer addition of 10% CO
2
. The positive band at 2341 cm
−1
corresponds to CO
2
in solution.
13
Further positive bands were
observed at 1618 cm
−1
, 1358 cm
−1
, and 1298 cm
−1
, the latter as
a shoulder. These bands are assigned to bicarbonate in
solution,
14–16
i.e., the asymmetric and symmetric stretching
modes of CO
2
(v
2
,v
3
) and the HCO
3
−
bending mode (v
4
). A
broad negative band at 3000 cm
−1
appeared in an energy regime
corresponding to a strongly hydrogen-bonded network of ‘local’
water molecules,
21–23
indicating that water is consumed during
bicarbonate formation.
Scheme 1 Mechanism of CO
2
hydration in carbonic anhydrase. Top
row, left to right: CO
2
enters the active site and replaces a water
molecule (W, black) near the zinc cation (Zn
2+
). In the next step, CO
2
is
converted to HCO
3
−
upon a nucleophilic attack (NA) of the zinc-
bound hydroxide (Zn
2+
–OH
−
). Bottom row, right to left: HCO
3
−
is
replaced by another water molecule (W, blue), the latter which is
activated to OH
−
upon proton transfer (PT) via oriented water mole-
cules W1 and W2 and a conserved histidine (His, blue). In the last step,
this histidine changes its orientation to release the proton into bulk
solvent, and water re-binds (W, black) near the active site.
Fig. 1 Active site of crotonyl CoA carboxylase/reductase. Crystal
structure of KsCcr in complex with reaction product ethylmalonyl
coenzyme A, E-CoA (PDB ID 6OWE). The bond between C3 and C30of
E-CoA is drawn translucent to emphasize the CO
2
binding site. KsCcr
active site residues F170 and N81 interact with E-CoA while H365 and
E171 coordinate a ‘bridging’water molecule, mW (red sphere, distance
to H365 and E171 each 2.8 Å). A local water cluster connects the active
site with bulk solvent (blue spheres, shortest distance to mW 2.9 Å).
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To conrm assignment of the observed bands, we investi-
gated potential isotope effects. Adding
13
CO
2
gas instead of CO
2
resulted in a specic down-shiof the CO
2
band to 2278 cm
−1
(D63), as well as the v
2
and v
3
bands of HCO
3
−
to 1586 cm
−1
and
1322 cm
−1
(D32 and D36, respectively). The isotope effect on the
band at 1258 cm
−1
was rather minor while the broad negative
band at 3000 cm
−1
was not affected by
13
CO
2
, which is in line
with our assignments of CO
2
/HCO
3
−
and ‘local’water. We also
investigated the inuence of solvent isotope effects by
exchanging the hydrated KsCcr protein lm from H
2
OtoD
2
O
(Fig. 2B). In the presence of D
2
O, we observed a large down-shi
of the negative band from 3000 cm
−1
to 2280 cm
−1
(D720)
supporting our assignment of the water cluster. While the H/D
exchange only had an insignicant effect on the v
2
and v
3
bands,
the shoulder at 1298 cm
−1
seemed to disappear in the
deuterated sample. This is due to a ∼300 cm
−1
down-shithat
moves the signal out of the detection window of our FTIR setup
and additionally conrms the COH (v
4
) assignment.
15
Next, we studied the kinetics of the reaction between KsCcr
and CO
2
. The difference spectra were simulated with contri-
butions from CO
2
,H
2
O, and HCO
3
−
and corrected for unspe-
cic changes (Fig. S3†). The resulting ‘peak area’for each
reactant was plotted against time. Fig. 2C shows the changes of
HCO
3
−
(given by the sum of v
2
,v
3
, and v
4
) and ‘local’water
(vOH
−
)inKsCcr upon reaction with CO
2
. We titrated the
enzyme in ve consecutive steps changing the gas atmosphere
to a continuous partial pressure of 1, 3, 10, 30, and 100% CO
2
followed by exposure to 100% N
2
aer each CO
2
step. Qualita-
tively, these data demonstrate that the intensity of the HCO
3
−
and water bands are proportional to the CO
2
concentration in
Fig. 2 Infrared characterization of the reaction of KsCcr with CO
2
. (A) ‘CO
2
–N
2
’ATR FTIR difference spectra for the reaction with
12
CO
2
(black) or
13
CO
2
(red). Positive signals are assigned to CO
2
(2341 or 2278 cm
−1
) and HCO
3
−
. The COH vibration (v
4
) appears as a shoulder at 1298 cm
−1
(*).
(B) ‘CO
2
–N
2
’ATR FTIR difference spectra in the presence of H
2
O (black) or D
2
O (magenta). The broad negative bands are assigned to ‘local’water
with vOH
−
=3000 cm
−1
and vOH
−
=2280 cm
−1
. (C) Evolution of the bands assigned to HCO
3
−
(black) and vOH
−
(blue) over time in the
presence of 1–100% CO
2
or 100% N
2
. Red lines represent linear fits of the first three data points after gas exchange to calculate the initial reaction
velocity v*
1:(D) Plot of the apparent reaction velocity v*
1of the initial HCO
3
−
formation (black) or initial water consumption (blue) in the CO
2
hydration reaction.
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the atmosphere and that the CO
2
/HCO
3
−
conversion is revers-
ible (eqn (1)). The initial velocity of CO
2
hydration ðv*
1Þwas
estimated by linear regression based on the rst three data
points aer changing the atmosphere from N
2
to CO
2
for each
step. We assume that the reaction velocity is not signicantly
affected by the back reaction due to the small build-up of
HCO
3
−
within the rst 15 s. A similar approach was chosen to
quantify the initial velocity of HCO
3
−
dehydration ðv*
1Þinitiated
by removing CO
2
from the gas atmosphere (Fig. S4†). The data
yielded apparent reaction velocities v*
1and v*
1that are specic
for our experimental approach. Earlier, we explored how the
humidity of concentrated protein lms inuences the velocity
of substrate diffusion
24,25
and its overall elastic properties.
26
Now, we show that corresponding observations are made with
KsCcr: when the humidity was reduced from 75% to 35%
(determined via the OH stretching vibrations of H
2
O, see
Fig. S3†) the velocity of CO
2
hydration decreased accordingly
(Fig. S5†). Although the spectroscopically measured velocities
are lower than in solution assays
27
our data facilitates a quanti-
tatively signicant comparison between samples under tightly
controlled steady-state conditions. To demonstrate the catalytic
activity of KsCcr in solution, we performed the ‘colorimetric’
analysis of CO
2
hydration as pioneered by Wilbur and Ander-
son.
28
Here, the injection of a dened amount of CO
2
-saturated
buffer induces an acidication (eqn (1)), which leads to a bleach
of a strong absorbance band of bromothymol blue that can be
followed over time by UV/vis spectroscopy. Our data in Fig. S6†
demonstrate that CO
2
hydration in aqueous solution is much
slower than in the presence of KsCcr or b-type carbonic anhy-
drase from E. coli (EcCA) conrming the observed anhydrase
activity of KsCcr.
29
Fig. 2D shows how the calculated reaction velocities for
HCO
3
−
formation and water consumption (given in absolute
values v*
1to visually aid the comparison) depend linearly on
the CO
2
concentration. The experimental variation has been
determined in repetitions of ve (Fig. S7†). This conrms the
proposed reaction model of pseudo-rst order kinetics for
enzymatic CO
2
hydration in aqueous solution
30
and highlights
the quantitative connection between CO
2
, HCO
3
−
, and water in
the active site. Fig. S8†depicts a quantication of HCO
3
−
based
on Na
2
CO
3
reference samples and an analysis of the exponential
correlation between CO
2
partial pressure and HCO
3
−
concen-
tration in the protein lm. This facilitates the analysis of the
initial velocity of the back reaction ðv*
1Þas a function of
bicarbonate concentration. The data in Fig. S4†suggest a higher
reaction order and overall slower kinetics. We speculate that the
apolar active site of KsCcr (Fig. 1) may slow down HCO
3
−
binding, which would impede the back reaction.
To verify and benchmark the CO
2
/HCO
3
−
conversion by
KsCcr, we repeated the experiments with carbonic anhydrase
EcCA at conditions comparable to the experiments with KsCcr.
The ‘CO
2
–N
2
’FTIR spectrum for EcCA aer 25 s in the presence
of 10% CO
2
(Fig. 3A) is strikingly similar to the one observed for
KsCcr (Fig. 2B) including the positive features for CO
2
and
HCO
3
−
, as well as the negative water band at 3050 cm
−1
(2300 cm
−1
in D
2
O). However, Fig. 3B shows that EcCA catalyses
the CO
2
/HCO
3
−
conversion nearly four times faster than KsCcr
(v*
1z0:22 s1and 0:06 s1
;respectively). The superior activity
of EcCA is observed in solution as well (Fig. S6†). For compar-
ison unspecicCO
2
conversion by bovine serum albumin (BSA,
v*
1z0:016 s1) is plotted in Fig. 3B. Note the lack of a negative
band at 3000 cm
−1
The CO
2
conversion kinetics of KsCcr in Fig. 2 are in the
range of uncatalyzed CO
2
hydration in solution (Fig. S6†).
Therefore, we probed background CO
2
conversion in (i) water,
(ii) buffer, and (iii) BSA as a generic biological crowder.
31
No
signicant HCO
3
−
formation was observed with pure water,
presumably due to the acidication in unbuffered solution
(Fig. S2†). When recording ‘CO
2
–N
2
’difference spectra on
a drop of Tris/HCl buffer (2 ml, 25 mM, pH 8) approximately 10%
HCO
3
−
formation was observed within the same time frame as
in the KsCcr experiments (Fig. S2†) although much more CO
2
is
dissolved in the drop compared to KsCcr or EcCA (Fig. S9†). As
Fig. 3 Infrared characterization of the reaction of EcCA with CO
2
. (A)
‘CO
2
–N
2
’ATR FTIR difference spectra in the presence of H
2
O (black)
or D
2
O (magenta). These data show that EcCA and KsCcr (Fig. 2) react
very similar with CO
2
. (B) Plotting the evolution of spectral traces for
HCO
3
−
(closed symbols) and vOH
−
(open symbols) against time, the
superior reaction velocity of EcCA (red) over KsCcr (black) and BSA
background (blue) becomes evident.
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argued above, data based on liquid sample cannot be compared
directly, therefore we formed a BSA protein lm to probe
uncatalyzed CO
2
hydration under conditions comparable to the
experiments with KsCcr or EcCA. At pH 7–8, BSA shows up to
30% of the bicarbonate formation activity observed with KsCcr
and a HCO
3
−
/CO
2
ratio similar to KsCcr or EcCA. However, the
unique water feature of KsCcr and EcCA is shied to 3320 cm
−1
,
indicative of uncatalyzed CO
2
hydration from bulk water
(Fig. S9†). Accordingly, when the BSA solution was adjusted to
pH values between 5–9, the observed HCO
3
−
formation resem-
bles the pH prole of the CO
2
/HCO
3
−
couple in the absence of
enzyme. Note that this is not the case for KsCcr: in the same pH
range, this enzyme shows largely unchanged CO
2
hydration
activity (Fig. S9†). Overall, these controls demonstrated that
KsCcr possesses a CA-like activity similar to the reaction of ‘true’
CAs.
Substrate binding and hydrophilic residues inuence
anhydrase activity
In the next step, we investigated the CO
2
/HCO
3
−
conversion
activity of KsCcr in the presence of NADPH, NADP
+
, native
substrate crotonyl-coenzyme A (C-CoA), and side product
butyryl-coenzyme A (B-CoA).
9–11
We tested six different combi-
nations: (i) KsCcr only, (ii) KsCcr + 10 mM NADP
+
, (iii) KsCcr +
10 mM NADPH, (iv) KsCcr + 10 mM NADPH + 1 mM C-CoA, (v)
KsCcr + 1 mM C-CoA, and (vi) KsCcr + 1 mM B-CoA. Fig. 4A
shows the HCO
3
−
peak area observed aer 60 s in the FTIR
difference experiments (i.e., upon saturation of the signals, see
Fig. S9†), normalized to wild-type KsCcr, which denes ‘100%’
bicarbonate formation. The experimental variation has been
determined in repetitions of ve (Fig. S7†). In these experi-
ments, neither NADPH nor NADP
+
affected bicarbonate
formation, while the presence of C-CoA or B-CoA decreased
band intensity down to 33% and 17%, respectively. Based on
our reference experiments (Fig. S9†), we note that about 30%
CO
2
hydration can be considered as background activity, which
is indicated by the dashed line in Fig. 4A. These data suggest
that HCO
3
−
formation and carboxylation are mutually exclusive
indicating that the CO
2
/HCO
3
−
conversion occurs only at the
substrate-free active site of KsCcr. We speculate that the supe-
rior inhibition activity of B-CoA is related to the structural
exibility of this catalytic side product, which has been shown
to t the active site of KsCcr smoothly.
32
These properties might
affect unspecic binding site as well pushing the activity below
the threshold.
We have shown previously that four amino acids play a key
role in CO
2
binding at the active site of KsCcr: histidine H365,
glutamate E171, asparagine N81, and phenylalanine F170.
12
H365 and E171 are involved in coordinating a conserved water
molecule in bridging position (mW), which is in hydrogen-
bonding contact with water molecules that connect the active
site with bulk water (Fig. 1). Asparagine N81 orients CO
2
in the
active site for the carboxylation reaction, and F170 shields the
pocket from water.
To understand the molecular basis of CO
2
/HCO
3
−
conver-
sion in KsCcr, we tested ve active site variants. Qualitatively,
the ‘CO
2
–N
2
’FTIR difference spectra of single point mutants
N81L, F170A, E171A, and H365N were similar to wild-type
KsCcr. However, while N81L and F170A showed full conver-
sion, bicarbonate formation of variants E171A and H365N was
reduced by ca. 50% (Fig. 4A). Compared to H365N variant E171A
showed slightly slower bicarbonate formation. In the KsCcr
E171A/H365N double mutant both conversion and reaction
velocity were found to be reduced even further (Fig. S10†).
Fig. 4B highlights an interesting detail in the difference spectra
of wild-type KsCcr, E171A, H365N, and E171A/H365N: the water
band shis from 3000 cm
−1
to 3030 cm
−1
in the single point
mutants and all the way down to 3320 cm
−1
in the double
Fig. 4 Bicarbonate formation activity of wild-type KsCcr and variants.
(A) The HCO
3
−
peak area after 60 s in the presence of 10% CO
2
is set to
100% for wild-type KsCcr and compared for different conditions (left
panel) and active site variants (right panel) as annotated in the bar plot.
About 30% CO
2
hydration can be considered as unspecific back-
ground activity, which is indicated by the dashed line. (B) ‘CO
2
–N
2
’
FTIR difference spectra after 60 s highlight quantitative differences
(i.e., the intensity of the bicarbonate bands) and qualitative differences
between wild-type KsCcr and variants: the inset shows an up-shift of
the water band (scaled), most clearly visible for KsCcr double variant
E171A/H365N (magenta).
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mutant. This indicates that KsCcr E171A/H365N has lost its CA-
like activity and exhibits only unspecicCO
2
hydration much
like BSA that showed a similar ‘CO
2
–N
2
’FTIR difference spec-
trum (Fig. S9†) and in agreement with the reduced CO
2
hydra-
tion activity reported in Fig. 4A. In summary, these experiments
suggest that the CA-like activity depends on the active site of
KsCcr, and likely involves E171 and H365.
Computer simulations reveal conformation-dependent CO
2
binding and explain substrate inhibition
To rationalize the observed inhibition of CA-like activity
through substrate binding, we performed atomistic MD simu-
lations in the presence of CO
2
using the X-ray crystal structure
of KsCcr that binds both NADPH and side product B-CoA
(ternary complex, PDB ID 6NA4).
32
Note that the enzyme is
a tetramer that shows half-site reactivity, i.e., exists as dimer of
open and closed subunits (colored orange and green in Fig. 5).
Compared to the open subunits the closed subunits contain the
substrate and represent the catalytically active sites in the
ternary complex. For our simulations, we replaced B-CoA by C-
CoA and dened a specic volume box (Fig. 5) to calculate the
CO
2
binding free energy in the open and closed subunit.
Notably, active site residues H365 and E171, which we associate
with CA-like activity, adopted different geometries in the open
and closed conformations. Compared to the open subunit, the
distance between E171 and H365 was 3 Å shorter in the closed
subunit and the conserved water molecule mW was hydrogen-
bonded between the two residues (Fig. 5). Additionally, we
studied the X-ray structure without substrate (binary complex,
PDB ID 6NA4) that presents similar geometries of both residues
in the closed and open subunits.
32
We presume that H365 can act as base in its neutral,
monoprotonated state initiating proton abstraction from mW
and thus forming the hydroxyl ion for subsequent CO
2
hydra-
tion. To determine the protonation state of H365 in different
subunits of the binary and ternary complexes, we calculated the
pK
a
shi(Table S1†).
33
For the open active site, the pK
a
shiis
negative (DpK
a
=−0.9 ±0.1) indicating that H365 rather adopts
a monoprotonated state. For the closed active site without
substrate (binary complex), we also obtained a negative shi
(DpK
a
=−0.6 ±0.1) while the pK
a
shiwas positive in the
presence of the substrate (DpK
a
=+1.2 ±0.1), likely because of
favorable interactions of the H365 with the negatively charged
phosphate groups of C-CoA. Thus, H365 is monoprotonated in
the empty, closed active site and capable of initiating proton
abstraction from mW. In the presence of substrate H365 most
likely changes its protonation state, thus suppressing CA
activity.
In addition, CO
2
binding to the active site plays an important
role. To understand the inuence of conformational changes
and presence of the substrate on CO
2
, we carried out extensive
MD simulations. From the ratio of local CO
2
concentration in
the active site volume (black box in Fig. 5) and the concentration
in the bulk we calculated the CO
2
binding free energy to the
active site volume for the open and closed subunits of the binary
and ternary complexes of KsCcr (DGbind ¼kBTlncact:site
cbulk ;
see ESI†). Our calculations show that DG
bind
of the closed
subunit with substrate (ternary complex) is positive whereas the
closed subunit in the binary complex without substrate presents
the highest CO
2
affinity which makes it more than two times
more probable to nd a CO
2
molecule in the active site than in
the bulk (Fig. 6A). The open subunit in the ternary complex also
shows a signicantly increased CO
2
affinity but the distance
between E171 and H365 is larger in the open active site, and no
mW molecule is observed suggesting a diminished catalytic
activity. We then addressed the binding sites of CO
2
in the
closed active site without substrate where the affinity is highest.
The binding sites connect active site interior and solvent, and
the most buried ones are very close to H365, water molecule mW,
Fig. 5 Computational model. Crystal structure of KsCcr (PDB ID 6NA4) in a dimer-of-dimers configuration with open subunits (orange, left
panel) and closed subunits (green, right panel). A close-up to the active site for the open and closed subunit shows residues N81, F170, E171, and
H365. Note that the E171/H365 distance shrinks from 9 Å to 6 Å in the closed subunit. A black box encloses the volume of the active site used to
analyze the local CO
2
concentration. NADPH is shown in cyan sticks, C-CoA is shown in magenta sticks. The later is exclusively found in the
closed subunit (green, right panel).
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and E171 (Fig. 6B). Notably, the substrate in the ternary complex
occupies the same positions as the CO
2
binding sites (Fig. 6C).
In summary, our computer simulations show that the binary
complex has a higher CO
2
binding affinity compared to the
ternary complex. The CO
2
binding sites in the closed active site
are next to H365 and the conserved water molecule mW, so that
the monoprotonated form of H365 will be able to abstract
a proton from mW to form the nucleophilic hydroxyl ion. The
absence of NADPH or NADP
+
is not expected to affect CO
2
binding or H365 protonation because the coenzyme does not
bind directly to the substrate binding site.
32
In contrast, the
presence of C-CoA or B-CoA in the ternary complex increases the
pK
a
of the putative proton acceptor H365 thereby eliminating its
ability to activate mW water by proton transfer, and simulta-
neously diminishes CO
2
binding. This can explain the
experimentally observed reduction of CA-like activity and is in
line with the fact that the active enzyme ternary complex
promotes CO
2
xation,
32
and not CO
2
hydration.
Conclusions
In this study, we applied in situ ATR FTIR difference spectros-
copy and computer simulations to investigate and understand
the interaction of crotonyl-CoA carboxylase/reductase (KsCcr)
with CO
2
. Our results show that KsCcr possesses a carbonic
anhydrase-like activity, i.e., the interconversion of CO
2
and
HCO
3
−
with water. This reaction is strongly suppressed in the
presence of C-CoA, the natural substrate of KsCcr. Extensive MD
simulations revealed how C-CoA suppresses CO
2
binding and
identied H365 as putative proton acceptor during CO
2
hydra-
tion. Compared to wild-type KsCcr variant H365N indeed
showed about 50% reduced anhydrase activity, similar to
variant E171A. Our pK
a
calculations rationalize how either H365
or E171 can serve as ‘base’in the CO
2
hydration reaction
explaining the relatively large anhydrase activity of the single-
residue variants. In contrast, only slow and unspecicCO
2
hydration is observed with double variant E171A/H365N. In
wild-type KsCcr, H365 and E171 form a hydrogen-bonding
complex through an interstitial, bridging water molecule
(mW). The latter is in contact with a chain of water molecules
that facilitate contact with bulk water. Upon CO
2
hydration our
FTIR data reveal the loss of a broad band at 3000 cm
−1
(2280 cm
−1
in D
2
O), which we assign to a strongly hydrogen-
bonded water cluster, most likely including mW.
Notably, we also observed very similar spectra for EcCA,
which we used as a reference to validate the experimental setup
and conrm our interpretation of KsCcr's CA-like activity. EcCA
coordinates a zinc cofactor that catalyses the deprotonation of
a bound water molecule to a hydroxide ligand (Zn
2+
–OH
−
)
promoted by a nearby histidine base (Scheme 1). CO
2
reacts
with the ligand to HCO
3
−
, which is clearly observed in our FTIR
difference spectra as a positive contribution. In the following
HCO
3
−
leaves the active site and is replaced by another water
Fig. 6 CO
2
binding in the active site of KsCcr. (A) Binding free energy (k
B
T)ofCO
2
in the closed and open subunit of the binary complex with
NADPH and the ternary complex with NADPH and C-CoA from MD simulations. (B) Most probable binding sites of CO
2
in the closed active site of
the binary complex (PDB ID 6NA6) represented as red spheres. (C) Representative snapshot of the ternary complex (PDB ID 6NA4), in which the
substrate occupies the position of the CO
2
binding sites. NADPH is shown in cyan, C-CoA is shown in magenta, and key residues are shown in
green sticks.
Scheme 2 Proposed reaction mechanism. Top row, left to right:
interstitial water mW is deprotonated via H365 (*or E171 in the H365N
variant) when the system adopts the closed state. The resulting mOH
−
species is stabilized by hydrogen bonds (dashed lines) and attacks
a bound CO
2
molecule to form bicarbonate HCO
3
−
. Bottom row, right
to left: when the system adopts the open state, HCO
3
−
leaves the
active site and H365 releases a proton toward bulk solvent. Influx of
water and CO
2
primes KsCcr for another round of anhydrase activity.
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molecule. Binding of water ‘re-activates’the cofactor, resulting
in a broad negative band in our FTIR difference spectra, similar
to what we have observed with KsCcr. Reported here for the rst
time, these results establish a unique spectral signature of CA
activity, i.e., the IR bands of bicarbonate and a strongly-
hydrogen bonded cluster of ‘local’water.
The role and importance of a metal ion in CA has been
discussed intensively.
8
However, in 2021 Hirakawa et al. re-
ported metal-free CAs in cyanobacteria and microalgae that
appear to catalyse CO
2
hydration in a purely organic environ-
ment.
34
These observations are in line with our experiments on
KsCcr that also suggest metal-independent CA-like activity.
Based on our combined experimental and theoretical investi-
gation of CO
2
hydration in KsCcr, we propose a mechanism
related to carbonic anhydrase (Scheme 2): (i) Once the enzyme
adopts the closed state, mW is deprotonated to a bridging
hydroxide, mOH
−
, with the neutral H365 residue serving as base
(our pK
a
calculations suggest that E171 may serve as base in
H365 variants, see Table S1†). (ii) The carboxylate side chain of
E171 accepts a hydrogen bond from mOH
−
, which itself is
stabilized via a hydrogen bond from the imidazole side chain of
protonated H365. When CO
2
is present in the active site mOH
−
will form HCO
3
−
via a nucleophilic attack (NA, the reaction may
involve additional water species, see Fig. S11†). (iii) Bicarbonate
leaves the active site –potentially triggered by a transition from
the closed to the open state –and deprotonation of H365 toward
bulk solvent. We speculate that the protonated imidazolium
cation is not stable in the absence of an interstitial water
species. (iv) This transient opening of the hydrogen-bonding
complex will allow intake of water and CO
2
and prime the
system for a new round of CO
2
hydration.
Summing up, in situ ATR FTIR difference spectroscopy
allowed investigating the interaction of different enzymes with
CO
2
, providing a simple yet powerful approach to directly
identify CA activity for a given biological sample. Moreover, our
method is also suited to identify and characterize water clusters
and will serve as an important tool to analyze CO
2
hydration in
biocatalysis,
35–38
homogenous or heterogeneous catalysts,
39–41
and (de-)hydration reactions in general.
42,43
Data availability
Data are available from the authors upon reasonable request.
Author contributions
M. Tinzl, G. Stoffel, and H. Westedt produced and prepared the
enzymes. A. Gomez performed the molecular dynamics simu-
lations and pK
a
calculations. S. T. Stripp designed and per-
formed the spectroscopy experiments. H. Grubmüller, T. J. Erb,
E. Vöhringer-Martinez and S. T. Stripp discussed the data. E.
Vöhringer-Martinez designed the computer simulations. E.
Vöhringer-Martinez and S. T. Stripp wrote the manuscript.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
The authors acknowledge help from Mariafrancesca Greca and
Federico Baserga at Freie Universit¨
at Berlin in buffer exchange
experiments and UV/vis spectroscopy. EVM and AG are thankful
for nancial support provided by the Max-Planck Society (MPS)
through the CONICYT Program of Int. Cooperation with the
Max Planck for Terrestrial Microbiology in Marburg
(MPG190003) and PhD scholarship “Doctorado Nacional”
(21190262) provided by ANID. MT is thankful for a Postdoctoral
Fellowship from the Swiss National Science Foundation
(P500PB 203136). MT and TJE received support from the MPS
the European Research Council (ERC 637675 ‘SYBORG’). STS
thanks funding by the Deutsche ForschungsgemeinschaDFG
(priority program 1927 “Iron-Sulfur for Life”, STR1554/5-1).
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