QM/MM Thiel paper by Janus Eriksen

Outline
Introduction to cytochrome P450
Previous cytochrome P450cam studies
The present cytochrome P450cam study
Results

Altarsha; Benighaus; Kumar and Thiel (2010)
- Coupling and uncoupling mechanisms in the methoxythreonine mutant of
cytochrome P450cam: A quantum mechanical/molecular mechanical study.

Janus Juul Eriksen

March 11, 2011

Janus Juul Eriksen Altarsha; Benighaus; Kumar and Thiel (2010)

Outline
Results

1 Introduction to cytochrome P450
Cytochrome P450 - general features
Cytochrome P450 - enzymatic mechanism

2 Previous cytochrome P450cam studies
Cpd1 formation and proton relay channels
Site-directed mutagenesis studies

3 The present cytochrome P450cam study
The agenda
Computational methods

4 Results
Comparison of the two proton relay channels


Outline
Results

Cytochrome P450Cam (CYP101) belong to the CYP superfamily which are
reknowned for being remarkable reactive and promiscuous towards a broad
spectrum of substrates in activating inert C-H bonds.
The enzyme superfamily catalyze the addition of molecular oxygen to nonactivated
hydrocarbons at physiological temperature a reaction that requires high
temperature to proceed in the absence of a catalyst.

Figure: Cytochrome P450cam [Lee(2010)].


Outline
Results

1: The substrate binds to the active site of
the enzyme changing the state of the heme
iron from low-spin to high-spin
[Meunier(1991), Poulos(1987)].
2: The change in the electronic state of the
active site favours the transfer of an
electron reducing the ferric heme iron
(Fe(III)) to the ferrous state (Fe(II)).
3: O2 binds covalently to the distal axial
coordination position of the heme iron.
The Cys ligand acts as e− donor and the
oxygen is thus activated, sometimes
allowing the Fe-O bond to dissociate
causing the uncoupling reaction.
4: A second electron is transferred via the
e− -transport system, reducing the O2
adduct to a negatively charged peroxo
group.
5: The peroxo group is rapidly protonated
Figure: The P450
twice by local transfer from surrounding
catalytic cycle
[Meunier(1991)] amino-acid side chains, releasing one mole
of water, and forming a highly reactive
Fe(V)-oxo species [Meunier(1991),
Ortiz de Montellano(2005)].

Outline
Results

Cpd1 formation and the importance and appearance of the proton
relay channels
The reaction in step 5 contains a proton relay, i.e. a chemical species that acts as
both Brønsted base and Brønsted acid during the course of the reaction.
In previous studies by W. Thiel et al. [Thiel(2006)], the two possible proton relay
channels were investigated.
The COOH side chain of Glu366 is linked to the OH group of Thr252 by a chain of
water molecules while Asp251 is connected to the protein surface by a salt bridge.
The COOH group of Asp251 points away from the heme but on the basis of free
energy proﬁles from classical MD simulations and adaptive umbrella sampling
techniques [Torrie(1977)] it is shown that Asp251 may become linked via Wat901
to the OH group of Thr252 by an internal rotation of reasonable amplitude.

Figure: The Glu366 and Asp251 channels [Thiel(2006)].

Outline
Results


In other previous studies by W. Thiel et al. [Thiel(2009)], the ratio between the
coupling and uncoupling processes is investigated with respect to mutation of the
Thr252 residue (Thr252X, X ∈ {Ser, Val, Ala, Gly}) and the mutations are found
to favor the uncoupling of O2 as an additional water becomes stable in the Asp251
channel. This is in sync with general assumptions of site-directed Thr252X
mutagenesis in P450 enzymes [Kimata(1995), Newcomb(2000), Vaz(1998)].

Figure: Coupling and uncoupling mechanisms [Thiel(2009)].


Outline
The agenda
Results

The agenda

The study is a direct extension of the previous mutagenesis studies performed by
the group [Thiel(2009)] as they want to consider the eﬀect of the Thr252MeO-Thr
mutation in terms of disruptions of the proton relay channels (Asp251 and Glu366)
which are essential prerequisites for the conversion of Cpd0 into Cpd1.

Figure: The Glu366 and Asp251 channels with MeO-Thr present [Thiel(2010)].


Outline
The agenda
Results

MD simulations

The initial coordinates for the Cpd0 system were taken from the X-ray crystal
structure IDZ8 (Fe-O2 complex) [Schlichting(2000)].
The system consists of 24,988 atoms including 5,891 TIP3P water molecules.
The solvated systems were relaxed by performing MM energy minimizations and
MD simulations using CHARMM22 as implemented in charmm.
The heme unit along with the Cys357 and the OOH ligands and the outer 8 ˚ of
A
solvent layer were kept ﬁxed during the initial runs.
The protonation states were determined according to the hbuild procedure of
charmm at pH = 7, previously published protonation states based on
Poisson-Boltzmann calculations and visual inspection of the environment of
charged amino acids and histidine residues
[Sch¨neboom(2002), Sch¨neboom(2004)].
o o
˚
A water layer of 16 A thickness was constructed around the enzyme using the
insight ii (2000) software and the inner 8 ˚ of the solvent layer were equilibrated
A
(3ps at 300K) while keeping the outer 8 ˚ ﬁxed as mentioned above. More water
A
molecules were added to the solvent (3 times) to account for low water density
after relaxation af the inner water molecules.
In the present study this implies protonated Glu366 and deprotonated Asp251 in
the Glu366 channel and vice versa in the Asp251 channel.


Outline
The agenda
Results

QM region
In both system (the Glu366 and Asp251 channel) the QM region consisted of: iron
porphine (without heme side chains), the sulfur atom of Cys357, the axial OOH
moiety, and Meo-Thr (represented by CH3 OCH2 CH3 ).
In the case of the Asp251 channel, the QM region furthermore housed Wat901 and
Asp251 (represented by CH3 COOH).
The Glu366 channel system contained Wat523, Wat566, Wat687, and Wat902 in
addition to Glu366 (represented by CH3 COOH).
the Cpd0 Fe(III) complex can exist in three diﬀerent states; a doublet, a quartet,
and a sextet state, of which the doublet ground state lies 8.3 and 9.0 kcal/mol
below the quartet and the sextet, respectively, and thus only the doublet state is
taken into account in the present study.

Figure: Schematic representation of the doublet, quartet and sextet state of Cpd0
[Sch¨neboom(2004)].
o


Outline
The agenda
Results

QM/MM method - 1

Due to the three different spin states present in Cpd0, the QM method is bound to
incorporate not only dynamic but also static electron correlation effects. Since
ab inito multi-reference methods are too expensive for the size of the QM region,
the unrestricted Kohn-Sham formalism is prefered in the form of UB3LYP as it
offers a reasonable ammount (20 %) of HF exchange to handle the possible spin
contamination from the quartet and sextet state.
With respect to the basis sets, calculations have been carried out employing a) the
small-core effective core potential and the associated LACVP basis of a double ζ
quality on Fe and 6-31G on the remaining atoms for geometry optimizations and
b) the Turbomole-TZVP basis set on all atoms for single-point calculations. a) is
denoted B1 in the paper while b) is denoted B2.
The MM part is at all times described by the CHARMM22 force field within the
dl-poly program.
The heme parameters in charmm were determined for a Fe(II) containing heme
group but with but with modified charges on some of the atoms.
For camphor, the parameters were assigned according to charmm conventions with
some of the charges derived from QM calculations (B3LYP/6-31G(d)). In addition,
three internal coordinates were represented by special parameters as they had no
counterparts within the charmm library.


Outline
The agenda
Results

QM/MM method - 2
Minimized snapshots from the MD trajectories were taken as initial structures for
QM/MM optimizations.
The boundary conditions are treated by satisfying the free valencies at the QM
boundary with hydrogen link atoms which are included in the SCF treatment of
the QM fragment and the link atoms thus describe (in an approximate manner)
the ’missing’ charge density as an inhibit part of the QM region
[Thiel(1996), Thiel(2005)].
An electronic embedding scheme was adopted in the QM/MM calculations, i.e.
interactions with MM charges were incorporated into the LCAO-MO one-electron
Hamiltonian of the QM calculations, thus allowing QM polarization, and the
QM/MM electrostatic interactions were evaluated from the QM electrostatic
potential (at HF/6-31G(d) [RESP] level) and the MM partial charges.
No cutoﬀs were introduced for the non-bonding MM and QM/MM interactions.
The turbomole program was used for the QM treatment in the QM/MM as well
as in the pure QM calculations. The CHARMM22 force ﬁeld was run through the
dl-poly program to handle the MM part of the systems. The QM/MM
calculations were performed with the chemshell package that integrates the
turbomole and dl-poly programs and also performs geometry optimization with
the hybrid internal coordinate optimizer, hdlc. The transition states (and
intermediate states) were obtained employing the partitioned rational function
optimizer (p-rfo) module of hdlc which uses an explicit Hessian matrix updated
via the Broyden-Fletcher-Goldfarb-Shanno (BFGS) algorithm (quasi-Newton
optimization) [Thiel(2010)].


Outline
Previous cytochrome P450cam studies Comparison of the two proton relay channels
Results

Comparison of the two proton relay channels
The computed QM/MM barriers indicate that uncoupling is unfavorable in the
case of the Thr252MeO-Thr mutant, whereas there are two energetically feasible
proton transfer pathways for coupling; these are Mechanism I: homolytic OO bond
cleavage followed by coupled protonelectron transfer and Mechanism II:
proton-assisted heterolytic OO bond cleavage. The study shows Mechanism I to be
favorable.
The corresponding rate-limiting barriers for the formation of Compound I are
higher in the mutant than in the wild-type enzyme. These findings are consistent
with the experimental observations that the Thr252MeO-Thr mutant forms the
alcohol product exclusively (via Cpd1), but at lower reaction rates compared with
the wild-type enzyme.
With respect to the two different channels, the rate-limiting barriers are somewhat
lower in the Glu366 channel than in the Asp251 channel. The Asp251 channel is in
contact with bulk water, though, so it should be rather facile to reprotonate
Asp251 after each coupling reaction that involves proton transfer in the Asp251
channel. This is not true for Glu366, which resides in a hydrophobic pocket and is
thus difficult to reprotonate.


Outline
Results

Lee, Y. T.; Wilson, R. F.; Rupniewski, I.; Goodin, D. B Biochemistry 2010,
49, 3412
Meunier, B.; de Visser, S. P.; Shaik, S. Chem. Rev. 2004, 104, 3947

Poulos, T. L.; Finzel, B. C.; Howard, A. J. J. Mol. Biol. 1987, 195, 687

Ortiz de Montellano, P. R.
Cytochrome P450: Structure, Mechanism, and Biochemistry 2005, 3rd Ed.,
Kluwer Academic/Plenum Publishers, New York, USA
Zheng, J.; Wang, D.; Thiel, W.; Shaik, S. J. Am. Chem. Soc. 2006, 128,
13204
Torrie, G. M.; Valleau, J. P. J. Comput. Phys. 1977, 23, 187

Altarsha, M.; Benighaus, T.; Kumar, D.; Thiel, W. J. Am. Chem. Soc. 2009,
131, 4755
Kimata, Y.; Shimata, H.; Hirose, T. Biochem. Biophys. Res. Commun.
1995, 208, 96
Newcomb, M.; Toy, P. H. Acc. Chem. Res. 2000, 33, 449


Outline
Results

Vaz, A. D. N.; McGinnity, D. F.; Coon, M. J. Proc. Natl. Acad. Sci. USA
1998, 95, 3555
Altarsha, M.; Benighaus, T.; Kumar, D.; Thiel, W. J. Biol. Inorg. Chem.
2010, 15, 361
Schlichting, I.; Berendzen, J.; Chu, K.; Stock, A. M.; Maves, S. A.;
Benson, D. E.; Sweet, R. M.; Ringe, D.; Petsko, G. A.; Sligar, S. G. Science
2000, 287, 1615
Sch¨neboom, J. C.; Lin, H.; Reuter, N.; Thiel, W.; Cohen, S.; Ogliaro, F.;
o
Shaik, S. J. Am. Chem. Soc. 2002, 124, 8142
Sch¨neboom, J. C.; Thiel, W. J. Phys. Chem. B 2004, 108, 7468
o

Bakowies, D.; Thiel, W. J. Phys. Chem. 1996, 100, 10580

Altun, A.; Thiel, W. J. Phys. Chem. B 2005, 109, 1268


QM/MM Thiel paper by Janus Eriksen

Recommandé

Recommandé

Contenu connexe

Tendances

Tendances (20)

En vedette

En vedette (8)

Similaire à QM/MM Thiel paper by Janus Eriksen

Similaire à QM/MM Thiel paper by Janus Eriksen (20)

Plus de molmodbasics

Plus de molmodbasics (20)

QM/MM Thiel paper by Janus Eriksen