The structure of 4-hydroxylphenylpyruvate dioxygenase complexed with 4-hydroxylphenylpyruvic acid reveals an unexpected inhibition mechanism

Article abstract of DOI:10.1016/j.cclet.2021.02.041

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is an important target for both drug and pesticide discovery. As a typical Fe(II)-dependent dioxygenase, HPPD catalyzes the complicated transformation of 4-hydroxyphenylpyruvic acid (HPPA) to homogentisic acid (HGA). The binding mode of HPPA in the catalytic pocket of HPPD is a focus of research interests. Recently, we reported the crystal structure of Arabidopsis thaliana HPPD (AtHPPD) complexed with HPPA and a cobalt ion, which was supposed to mimic the pre-reactive structure of AtHPPD-HPPA-Fe(II). Unexpectedly, the present study shows that the restored AtHPPD-HPPA-Fe(II) complex is still nonreactive toward the bound dioxygen. QM/MM and QM calculations reveal that the HPPA resists the electrophilic attacking of the bound dioxygen by the trim of its phenyl ring, and the residue Phe381 plays a key role in orienting the phenyl ring. Kinetic study on the F381A mutant reveals that the HPPD-HPPA complex observed in the crystal structure should be an intermediate of the substrate transportation instead of the pre-reactive complex. More importantly, the binding mode of the HPPA in this complex is shared with several well-known HPPD inhibitors, suggesting that these inhibitors resist the association of dioxygen (and exert their inhibitory roles) in the same way as the HPPA. The present study provides insights into the inhibition mechanism of HPPD inhibitors.

Full text of DOI:10.1016/j.cclet.2021.02.041

G Model
CCLET-6170; No. of Pages 5
Chinese Chemical Letters xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Communication
The structure of 4-hydroxylphenylpyruvate dioxygenase complexed
with 4-hydroxylphenylpyruvic acid reveals an unexpected inhibition
mechanism
Xiaoning Wanga,1, Hongyan Linb,1, Junjun Liuc, Xinyun Zhaoa, Xi Chena,
,
*
b,
d
Wenchao Yangb, , Guangfu Yang , Chang-guo Zhan
*
*
a
College of Chemistry and Material Science, South-Central University for Nationalities, Wuhan 430074, China
b
c
Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, China
School of Pharmacy, Tongji Medical College of Huazhong University of Science & Technology, Wuhan 430030, China
d
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536, United States
A R T I C L E I N F O
A B S T R A C T
Article history:
4-Hydroxyphenylpyruvate dioxygenase (HPPD) is an important target for both drug and pesticide
discovery. As a typical Fe(II)-dependent dioxygenase, HPPD catalyzes the complicated transformation of
4-hydroxyphenylpyruvic acid (HPPA) to homogentisic acid (HGA). The binding mode of HPPA in the
catalytic pocket of HPPD is a focus of research interests. Recently, we reported the crystal structure of
Arabidopsis thaliana HPPD (AtHPPD) complexed with HPPA and a cobalt ion, which was supposed to
mimic the pre-reactive structure of AtHPPD-HPPA-Fe(II). Unexpectedly, the present study shows that the
restored AtHPPD-HPPA-Fe(II) complex is still nonreactive toward the bound dioxygen. QM/MM and QM
calculations reveal that the HPPA resists the electrophilic attacking of the bound dioxygen by the trim of
its phenyl ring, and the residue Phe381 plays a key role in orienting the phenyl ring. Kinetic study on the
F381A mutant reveals that the HPPD-HPPA complex observed in the crystal structure should be an
intermediate of the substrate transportation instead of the pre-reactive complex. More importantly, the
binding mode of the HPPA in this complex is shared with several well-known HPPD inhibitors, suggesting
that these inhibitors resist the association of dioxygen (and exert their inhibitory roles) in the same way
as the HPPA. The present study provides insights into the inhibition mechanism of HPPD inhibitors.
© 2021 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Received 16 January 2021
Received in revised form 17 February 2021
Accepted 18 February 2021
Available online xxx
Keywords:
4-Hydroxyphenylpyruvate dioxygenase
QM/MM calculation
Potential surface scan
Substrate self-inhibition
4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a member of
Fe(II)-dependent α-keto dioxygenase that catalyzes the conversion
of 4-hydroxyphenylpyruvic acid (HPPA) to homogentisic acid
(HGA). HPPD can also catalyze the conversion of other α-keto acid,
such as phenylpyruvate and isocaproate to 2-hydroxyphenylace-
tate and β-hydroxy β-methylbutyrate, respectively [1,2]. Featured
in aerobic forms of life, HPPD participates in the tyrosine
catabolism, which is necessary for the photosynthesis process.
As an effective target for herbicides [3–5] and promising target for
the treatment of type I tyrosinemia and alkaptonuria [6], HPPD
attracts much research interests.
Numerous studies were performed to unveil the catalytic
mechanism of HPPD [7–11] as well as other α-keto acid
dioxygenases. Different to mechanism of majority α-keto dioxy-
genases that have three substrates (Scheme S1 in Supporting
information), HPPD has only two substrates, i.e., HPPA and
dioxygen, to accomplish all the chemistry that common to this
family. The commonly accepted mechanism is that the catalysis is
initiated by the bidentate binding of HPPA with the ferrous center
in the HPPD active site, followed by the oxygen addition, keto acid
decarboxylation, phenyl ring hydroxylation, side chain migration,
and dissociation of the product HGA (Scheme 1). The reported
catalytic constants (kcat) for the conversion of HPPA catalyzed by
wild-type HPPD range from 1.1 sÀ1 to 7.8 s-1 [10,12–14]. According
to the transition state theory, the activation free energy for this
reaction is about 16À17 kcal/mol. These studies also showed that
among those steps the phenyl ring hydroxylation step should be
rate-determining [9,11,15].
* Corresponding authors.
E-mail addresses: chen@mail.scuec.edu.cn (X. Chen),
tomyang@mail.ccnu.edu.cn (W. Yang), gfyang@mail.ccnu.edu.cn (G. Yang).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.cclet.2021.02.041
1001-8417/© 2021 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article as: X. Wang, H. Lin, J. Liu et al., The structure of 4-hydroxylphenylpyruvate dioxygenase complexed with 4-
hydroxylphenylpyruvic acid reveals an unexpected inhibition mechanism, Chin. Chem. Lett., https://doi.org/10.1016/j.cclet.2021.02.041

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pocket that can accommodate more substrate conformations, and
the potential role of Gln293 in the connection of H-bond network
of Ser267-Asn282-Gln307.
In our previous works the bound HPPA was proven to be in keto
form by hybrid quantum mechanics/molecular mechanics
(QM/MM) calculations. These calculations also showed that
structure of the restored HPPD-HPPA-Fe(II) complex resembled
the crystal complex structure with RMSD value 0.073 Å for the key
geometrical parameters in the metal centers [12]. It was thought
that the latter mimicked the pre-reactive HPPD-HPPA complex.
However, the HPPA binding mode in this complex differs
significantly to the proposed ones [20–22], in which the keto
and carboxylic groups of HPPA lie respectively trans to the residue
Glu394 and His226, and the phenyl ring flipps away from the
residue Glu394. These differences prompted us to re-investigate
the HPPD-HPPA-Co(II) crystal complex in order to find out the
nature of this structure.
In this study, the cobalt ion in the crystal structure is replaced
with a ferrous ion to construct a AtHPPD-HPPA-Fe(II) complex.
Since there are unpaired electrons in the catalytic site of the
restored complex, it is necessary to determine the spin state for
this complex. Previous studies showed that different density
functionals provide qualitatively different predictions [23] on the
relative stability for the intermediate species in different spin
states. The benchmark CCSD(T) calculations supported the B3LYP
energetic [23].
Scheme 1. The common catalytic mechanism for the conversion of HPPA catalyzed
by HPPD.
Collected in Table 1 are the selected geometrical parameters,
atomic charges and spin densities for selected atoms, and the
relative Gibbs free energy for the HPPD-HPPA-O2 complex with
different spin multiplicity. Part parameters are also shown in
Fig. S1 (Supporting information) for comparison. In spin states
such as the singlet, triplet and quintet state, the dioxygen is
attached to the ferrous ion with interatomic Fe-O distances of 1.7–
2.0 Å. However, in septet state the dioxygen is separated from the
ferrous center with interatomic Fe-O distance of 3.6 Å. The
optimized Fe-O distances in triplet, quintet and septet state are
consistent with the results obtained in previous studies on
α-ketoglutarate dependent oxygenase [24,25]. According to the
relative energies for the geometries obtained in different spin
states (Table 1), the quintet, triplet, open-shell singlet, and singlet
states lie about 0.5, 11.8, 24.1 and 28.4 kcal/mol above the ground
septet state, which are thought to be a consequence of tighter
binding of O2 to Fe [23]. Population analysis on septet state shows
that there are respectively 4 and 2 unpaired alpha-electrons on the
ferrous ion and the dioxygen, implying a strong Pauli repulsive
interaction between them. The repulsive force should also resist
the approaching of the dioxygen to the nearby HPPA α-KG group.
Hence, the septet state should not be favorable for the upcoming
dioxygen addition reaction. In quintet state, the electrons from the
dioxygen are partially counter balanced by that of the iron leading
to a net spin density of 0.55, consistent with the predictions of
previous studies [23]. The quintet state is only ꢀ0.5 kcal/mol less
stable than the septet, and is by far more stable than the other
three states by ꢀ11À23 kcal/mol. Population analysis also shows
that in quintet state the bound dioxygen is negatively charged and
is in doublet spin state, which is thought to be a reactive species
toward the α-KG group. Above discussions suggest that the quintet
should be the most favorable spin state for the dioxygen addition
reaction. This conclusion is consistent with the experimental
observations [26,27] and previous theoretical studies on the O2-
activation steps of α-KG dioxygenases [9,23], which are also most
likely to be run on the quintet potential energy surface (PES).
Analysis mentioned above suggests that quintet state is most
favorable for the reaction of bound dioxygen with HPPA. According
to the accepted mechanism of HPPD catalysis described in
Scheme 1, the negatively charged oxygen atom from the dioxygen,
Similar to other α-keto dioxygenases, the HPPD shows ordered
substrate/inhibitor binding. The association of HPPA or HPPD
inhibitor is always prior to the binding of the dioxygen. It was
observed that the association of the HPPA greatly induced the
reactivity toward dioxygen [16]. Most HPPD inhibitors mimic
substrate HPPA. However, experimental studies indicated that the
HPPD-inhibitor-Fe(II) complexes performed poorly in associating
the molecule dioxygen [17].
Recently, we reported the crystal structure of Arabidopsis
thaliana HPPD (AtHPPD) complexed with the substrate HPPA [12]
and a cobalt ion (Fig. 1). In this complex, the pyruvate moiety of
HPPA in a bidentate chelation with the cobalt ion leads to an
octahedral coordination geometry involving the facial triad. The
phenolic hydroxyl forms a hydrogen bond with the side chain of
Asn423. The benzene ring of HPPA shapes a T-p interaction with
Phe381. Compared to the other reported AtHPPD structures (PDB
ID: 1SQD, 1SP9 and 1TG5) [18,19], the HPPD-HPPA-Co(II) structure
mainly shows the following differences: 1) a conformational
alteration for residue Phe428 on the C-terminal α-helix, 2) β-sheet
fragment (Phe250-Phe253) rotates ꢀ 30ꢁ transforming into a loop,
and 3) a conformation change for Gln293 upon the binding of
HPPA. These differences reflect the flexibility of the HPPD binding
Fig. 1. (A) Full view and (B) close view of the binding of HPPA at the catalytic site of
the AtHPPD-HPPA crystal complex. Values are in the unit of angstrom.
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Table 1
Key interatomic distances, atomic charges, spin densities and relative QM/MM Gibbs free energy of the ternary HPPD-HPPA-O2 complex calculated at different spin states.a
Spin states
Distance (Å)
Fe-Ob/Oa-C2'
Charges/spin densities (a.u.)
Grel (kcal/mol)
Fe-O3'/Fe-O4'
Fe
Oa
Ob
C2'
Singlet
Open singlet
Triplet
Quintet
Septet
1.74/3.04
1.84/3.13
1.91/3.12
2.00/3.33
3.62/5.93
2.07/1.93
2.08/1.94
2.09/1.92
2.23/2.06
2.35/2.05
0.81/
À0.29/
À0.03/
0.44/
28.4
24.1
11.8
0.5
0.85/1.08
1.07/1.22
0.81/4.40
0.39/4.05
À0.24/-0.61
À0.28/0.60
À0.24/-0.44
0.06/0.95
À0.01/-0.42
À0.16/0.31
À0.03/-0.11
À0.08/1.01
0.42/0.01
0.42/-0.01
0.44/0.00
0.46/0.00
0.0
a
The single-point QM/MM energy for each spin states is calculated at the B3LYP/6À311++G(2d,2p) level by using geometries optimized at the B3LYP/6À31+G(d) level. The
Gibbs free energy is calculated by the single-point energy plus the thermal dynamic correction calculated at the B3LYP/6À31+G(d) level.
namely Oa, will attack the carbonyl carbon C2' of the α-KG group,
affording an oxidized intermediate, which should be a local
minimum of the potential energy surface (PES) along the reaction
coordinate. During these calculations the RC2'-C3' – ROa-C2' is set as
the reaction coordinate. However, the QM/MM minimum energy
path scan on the PES fails to locate this local minimum. In fact,
these calculations show that with the approaching of the Oa to the
C2' atom (Fig. 2) the total energy of the complex structure keeps
rising, demonstrating that the nucleophilic attack of the bound
dioxygen is hold back by certain factors. Thus, in this ternary
complex structure the bound HPPA behaves like an HPPD inhibitor
by resisting the association of dioxygen.
In order to locate the possible factors that hinder the
approaching of the dioxygen to the α-KG group of HPPA, we
perform reaction coordinate calculations on a QM-cluster con-
structed from the crystal complex. Surprisingly, calculations on
this QM-cluster show that the dioxygen associates successfully
with the HPPA (Figs. S2 and S3 in Supporting information). This
suggests that the protein environment surrounding the catalytic
triad should play important role in affecting the interaction of
dioxygen with HPPA. By comparing the pre-reactive structure of
the QM-cluster with the QM region of the HPPD-HPPA-O2 complex,
it is found that the orientation of the phenyl ring profoundly affects
the electrophilic attacking of the bound dioxygen. NCIPLOTs
analysis (Fig. 3B) on one snapshot from the QM/MM PES scan
shows that there is a significant repulsive interaction (brown
region) between the incoming dioxygen and the phenyl carbon
Fig. 3. (A) Residues around the phenyl ring of HPPA in the optimized structure of
ternary HPPD-HPPA-O2 complex. The value is in the unit of angstrom. (B) NCIPLOTs
of non-bonded interactions among the attacking dioxygen, 4-hydroxyl group, and
pyruvate group. The green area illustrates attractive interaction and the brown area
represents repulsive interaction. The color scale is -0.04 <
r < 0.04 a. u.
atoms, and a weak attractive interaction (blue region) between the
dioxygen and the benzene hydrogen. Obviously, both interactions
should hold back the nucleophilic attacking of the bound dioxygen
on the α-KG group. On the other hand, the orientation of the phenyl
ring is fixed by sidechains of the residue Gln379, Phe381, and
Asn423 and buried water (Fig. 3A). Among these residues, the
Phe381 should play a key role in restraining the rotation of the
phenyl ring. B-factor analysis on the AtHPPD-HPPA crystal
structure shows low temperature values for these residues
(Fig. S8 in Supporting information). Hence, these aromatic side
chains are not likely to rotate upon thermal fluctuations.
To verify the assumption mentioned above, QM/MM geometri-
cal optimization is performed on a F381A mutant, followed by PES
scan along the reaction coordinate RC2'-C3' – ROa-C2' (Fig. 4). As can
be seen from this figure, in F381A mutant the Oa from the bound
dioxygen gradually approaches C2' of the α-KG group and
successfully covalently bond with the latter. During the forming
of the Oa-C2' bond, the C3' atom departs from the C2' atom, leading
to the release of a carbon dioxide. Meanwhile, the HPPA phenyl ring
rotates ꢀ40ꢁ, which is three times it does in the wild-type enzyme
(Fig. S4 in Supporting information). Noteworthily, the mechanism
described here is different to the one obtained from the QM-cluster
calculations (Fig. S2 in Supporting information), in which addition
of the dioxygen and departure of the carbon dioxide are taken
place in a stepwise manner. This suggests that the position of the
Fig. 2. Relative QM/MM energy profile for the attack of the bound dioxygen on the
carbonyl carbon of HPPA in the ternary HPPD-HPPA-O2 complex in quintet state. The
relative energy is determined at the B3LYP/6-311++G(2d,2p):AMBER level.
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and Phe424. The overall binding mode of HPPA is quite like the
binding modes of these three inhibitors, except that HPPA phenyl
ring only forms a T-
p stacking against the phenyl sidechain of
Phe381. The minimum substructures of these HPPD inhibitors
mimic the α-keto acid moiety of HPPA. The orientation of their
aromatic rings resembles the HPPA phenyl ring. The QM/MM
calculations combined with QM calculations predict that the free
energy change for the binding of dioxygen to the iron is only
-1.5 kcal/mol, suggesting a very weak negative binding affinity
between them. As illustrated in previous section (Fig. 3), the phenyl
ring will block the further association of dioxygen to the ketone
carbon, hence the bound HPPA will behave like an inhibitor due to
the failure of associating the O2. The aromatic rings of the HPPD
inhibitors show similar shape and orientation to the HPPA phenyl
ring. Following the story of the latter, these aromatic rings will also
repel the association of dioxygen to the adjacent ketone carbons.
Thus, these inhibitors exert their roles by preventing the addition
of O2 and suppressing the oxidation of the active site metal ion.
This inference is supported by the experimental observation that
the HPPD does not bind O2 in the presence of its inhibitors.
In our experiments on the conversion reaction of HPPA
catalyzed by AtHPPD, significant substrate-inhibition effect was
Fig. 4. Relative energy profile and geometries of the stationary points for the
dioxygen addition step in F398A mutant. The total charge and multiplicity for the
QM region are set to be
0 and quintet, respectively. The relative energy is
determined at the B3LYP/6-311++G(2d,2p):AMBER level.
phenyl ring may also affect the shape of potential energy profile for
the reaction of the bound dioxygen with the α-KG group. Free
energy barrier and free energy change for this step are respectively
calculated to be 18.8 and -40.3 kcal/mol, demonstrating that the
dioxygen will readily be associated with the substrate HPPA.
It is necessary to address the role of the residue Phe381 during
the catalysis in order to characterize the HPPD-HPPA complex
observed in the crystal. Kinetic experiments [12] showed that the
also observed with Ksi =226.0 mmol/L and Km =44.4 mmol/L (see
Supporting information for details). The inhibition constants (Ki) of
NTBC [28], sulcotrione and Y13508 against HPPD were reported to
be 6.43, 5.79 and 1.30 nmol/L, respectively. These data imply that
HPPA binds to HPPD with different conformations. Some
conformations lead to the reaction product HGA or the intermedi-
ate HPA. Some others do not react with HPPD leading to substrate
self-inhibition. In this study, we prove that the substrate
conformation observed in the AtHPPD-HPPA crystal structure is
an inactive form, which contributes to the substrate self-inhibition
of HPPD.
Km for F381A mutant was 9.1
mmol/L, whereas the one for the
wild-type HPPD was 1.9 mol/L. The F381A mutant had similar kcat
m
(0.92 sÀ1) to that of the wild-type (1.08 sÀ1). These data suggest
that Phe381 may not directly participate the catalytic reaction.
Hence, HPPA observed in the crystal structure should be a
temporary intermediate during the substate transportations,
instead of the previously assumed pre-reactive configuration. As
HPPA is transported into pre-reactive configuration, it will be
readily converted to HGA and released to the environment, leaving
an empty catalytic pocket. As evidenced by the crystal structure
reporting the bound HPPA (Fig. S11 in Supporting information),
three out of four subunits are vacant, implying that most
conversion reactions in these places have completed. The present
study demonstrates that the HPPA binding mode observed in the
crystal structure is not the reactive mode. Despite various binding
modes advanced so far, more proofs are still needed to validate
them. So far, it is still an open question for the determine the
reactive binding mode of HPPA.
Declaration of competing interest
The authors report no declarations of interest.
Acknowledgments
The work was supported by the National Key R&D Program (No.
2018YFD0200100) and National Natural Science Foundation of
China (Nos. 21837001, 21273089 and 22007035, U20A2038), the
Open Project Fund of the Key Laboratory of the Pesticides and
Chemical Biology of Central China Normal University (No. 2018-
A01), the Fundamental Research Funds for the South-Central
University for Nationalities (No. CZW20020), the Fundamental
Research Funds for the Central Universities (No. KJ02072020-
0657), and Hubei Province Natural Science Foundation (No.
2020CFB487).
It is interesting to compare the HPPA binding mode in this study
to the ones of known HPPD inhibitors [28] NTBC (PDB ID: 5CTO),
sulcotrione (PDB ID: 5YWG) and Y13508 according to the crystal
structures (Fig. 5). For each inhibitor in Fig. 5, two carbonyl groups
form chelation interaction with the ferrous ion and its aromatic
ring forms a
p-p stacking interaction with side chains of Phe381
Appendix A. Supplementary data
Supplementarymaterialrelatedtothisarticlecanbefound, inthe
online version, at doi:https://doi.org/10.1016/j.cclet.2021.02.041.
References
[1] N.P. Crouch, M.H. Lee, T. Iturriagagoitia-Bueno, et al., Cloning, Expression, and
Purification of Mammalian 4-Hydroxyphenylpyruvate Dioxygenase/α-
Ketoisocaproate Dioxygenase, Academic Press, Salt Lake City, 2000.
[2] M. Kohlmeier, Amino Acids and Nitrogen Compounds, Academic Press, San
Diego, 2015.
[3] H. Ahrens, G. Lange, T. Muller, et al., Angew. Chem. Int. Ed. Eng. 52 (2013) 9388–
9398.
[4] R. Beaudegnies, A.J. Edmunds, T.E. Fraser, et al., Bioorg. Med. Chem. 17 (2009)
4134–4152.
Fig. 5. Binding modes of the HPPA (yellow scheme), Y13508 (green scheme), NTBC
[5] D.W. Wang, H.Y. Lin, B. He, et al., J. Agric. Food Chem. 64 (2016) 8986–8993.
(blue scheme) and sulcotrione (pink scheme) in the catalytic site of HPPD.
4

G Model
CCLET-6170; No. of Pages 5
X. Wang, H. Lin, J. Liu et al.
Chinese Chemical Letters xxx (xxxx) xxx–xxx
[6] D.C. Bartlett, C. Lloyd, P.J. McKiernan, et al., J. Inherit. Metab. Dis. 37 (2014) 745–
752.
[16] K. Johnson-Winters, V.M. Purpero, M. Kavana, et al., Biochemistry 42 (2003)
2072–2080.
[7] D.D. Shah, J.A. Conrad, B. Heinz, et al., Biochemistry 50 (2011) 7694–7704.
[8] D.D. Shah, J.A. Conrad, G.R. Moran, Biochemistry 52 (2013) 6097–6107.
[9] T. Borowski, A. Bassan, P.E. Siegbahn, Biochemistry 43 (2004) 12331–12342.
[10] C. Raspail, M. Graindorge, Y. Moreau, et al., J. Biol. Chem. 286 (2011) 26061–
26070.
[17] G.R. Moran, Arch. Biochem. Biophys. 544 (2014) 58–68.
[18] C. Yang, J.W. Pflugrath, D.L. Camper, et al., Biochemistry 43 (2004) 10414–10423.
[19] I.M. Fritze, L. Linden, J. Freigang, et al., Plant Physiol. 134 (2004) 1388–1400.
[20] J.M. Brownlee, K. Johnson-Winters, D.H.T. Harrison, et al., Biochemistry 43
(2004) 6370–6377.
[11] A. Wójcik, E. Broclawik, P.E.M. Siegbahn, et al., J. Am. Chem. Soc. 136 (2014)
[21] I.M. Fritze, L. Linden, J. Freigang, et al., Plant Physiol. 134 (2004) 1388–1400.
14472–14485.
[22] J. Brownlee, P. He, G.R. Moran, et al., Biochemistry 47 (2008) 2002–2013.
[23] A. Wójcik, M. Radon, T. Borowski, J. Phys. Chem. A 120 (2016) 1261–1274.
[24] T. Borowski, A. Bassan, P.E.M. Siegbahn, Chem. Eur. J. 10 (2004) 1031–1041.
[25] S. Ye, C. Riplinger, A. Hansen, et al., Chemistry 18 (2012) 6555–6567.
[26] M.M. Mbughuni, M. Chakrabarti, J.A. Hayden, et al., Proc. Natl. Acad. Sci. U. S. A.
107 (2010) 16788–16793.
ꢀ
[12] H.Y. Lin, X. Chen, J.N. Chen, et al., Research 2019 (2019)2602414.
[13] J.F. Lin, Y.L. Sheih, T.C. Chang, et al., PLoS One 8 (2013)e69733.
[14] M. Gunsior, J. Ravel, G.L. Challis, et al., Biochemistry 43 (2004) 663–674.
[15] T. Borowski, M. Quesne, M. Szaleniec, QM and QM/MM methods compared:
case studies on reaction mechanisms of metalloenzymes, in: T. Karabencheva-
Christova (Ed.), Advances in Protein Chemistry and Structural Biology,
Academic Press, Salt Lake City, 2015.
[27] J.C. Price, E.W. Barr, B. Tirupati, et al., Biochemistry 42 (2003) 7497–7508.
[28] H.Y. Lin, J.F. Yang, D.W. Wang, et al., FEBS J. 286 (2019) 975–990.
5