QM/MM Thiel paper by Janus Eriksen

OutlineIntroduction to cytochrome P450

Previous cytochrome P450cam studiesThe present cytochrome P450cam study

Results

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

Janus Juul Eriksen

March 11, 2011

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



Results

1 Introduction to cytochrome P450Cytochrome P450 - general featuresCytochrome P450 - enzymatic mechanism

2 Previous cytochrome P450cam studiesCpd1 formation and proton relay channelsSite-directed mutagenesis studies

3 The present cytochrome P450cam studyThe agendaComputational methods

4 ResultsComparison of the two proton relay channels




Results

Cytochrome P450 - general featuresCytochrome P450 - enzymatic mechanism

Cytochrome P450 - general features

Cytochrome P450Cam (CYP101) belong to the CYP superfamily which arereknowned for being remarkable reactive and promiscuous towards a broadspectrum 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)].




Results

Cytochrome P450 - general featuresCytochrome P450 - enzymatic mechanism

Cytochrome P450 - enzymatic mechanism

Figure: The P450catalytic cycle[Meunier(1991)]

1: The substrate binds to the active site ofthe enzyme changing the state of the hemeiron from low-spin to high-spin[Meunier(1991), Poulos(1987)].

2: The change in the electronic state of theactive site favours the transfer of anelectron reducing the ferric heme iron(Fe(III)) to the ferrous state (Fe(II)).

3: O2 binds covalently to the distal axialcoordination position of the heme iron.The Cys ligand acts as e− donor and theoxygen is thus activated, sometimesallowing the Fe-O bond to dissociatecausing the uncoupling reaction.

4: A second electron is transferred via thee−-transport system, reducing the O2

adduct to a negatively charged peroxogroup.

5: The peroxo group is rapidly protonated

twice by local transfer from surrounding

amino-acid side chains, releasing one mole

of water, and forming a highly reactive

Fe(V)-oxo species [Meunier(1991),

Ortiz de Montellano(2005)].Janus Juul Eriksen Altarsha; Benighaus; Kumar and Thiel (2010)



Results

Cpd1 formation and proton relay channelsSite-directed mutagenesis studies

Cpd1 formation and the importance and appearance of the protonrelay channels

The reaction in step 5 contains a proton relay, i.e. a chemical species that acts asboth 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 relaychannels were investigated.The COOH side chain of Glu366 is linked to the OH group of Thr252 by a chain ofwater 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)].




Results

Cpd1 formation and proton relay channelsSite-directed mutagenesis studies

Site-directed mutagenesis studies

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)].




Results

The agendaComputational methods

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)].




Results


MD simulations

The initial coordinates for the Cpd0 system were taken from the X-ray crystalstructure 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 andMD simulations using CHARMM22 as implemented in charmm.

The heme unit along with the Cys357 and the OOH ligands and the outer 8 A ofsolvent layer were kept fixed during the initial runs.

The protonation states were determined according to the hbuild procedure ofcharmm at pH = 7, previously published protonation states based onPoisson-Boltzmann calculations and visual inspection of the environment ofcharged amino acids and histidine residues[Schoneboom(2002), Schoneboom(2004)].

A water layer of 16 A thickness was constructed around the enzyme using theinsight ii (2000) software and the inner 8 A of the solvent layer were equilibrated(3ps at 300K) while keeping the outer 8 A fixed as mentioned above. More watermolecules were added to the solvent (3 times) to account for low water densityafter 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.




Results


QM region

In both system (the Glu366 and Asp251 channel) the QM region consisted of: ironporphine (without heme side chains), the sulfur atom of Cys357, the axial OOHmoiety, and Meo-Thr (represented by CH3OCH2CH3).In the case of the Asp251 channel, the QM region furthermore housed Wat901 andAsp251 (represented by CH3COOH).The Glu366 channel system contained Wat523, Wat566, Wat687, and Wat902 inaddition to Glu366 (represented by CH3COOH).

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[Schoneboom(2004)].




Results


QM/MM method - 1

Due to the three different spin states present in Cpd0, the QM method is bound toincorporate not only dynamic but also static electron correlation effects. Sinceab 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 itoffers a reasonable ammount (20 %) of HF exchange to handle the possible spincontamination from the quartet and sextet state.

With respect to the basis sets, calculations have been carried out employing a) thesmall-core effective core potential and the associated LACVP basis of a double ζquality on Fe and 6-31G on the remaining atoms for geometry optimizations andb) the Turbomole-TZVP basis set on all atoms for single-point calculations. a) isdenoted B1 in the paper while b) is denoted B2.

The MM part is at all times described by the CHARMM22 force field within thedl-poly program.

The heme parameters in charmm were determined for a Fe(II) containing hemegroup 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.




Results


QM/MM method - 2

Minimized snapshots from the MD trajectories were taken as initial structures forQM/MM optimizations.

The boundary conditions are treated by satisfying the free valencies at the QMboundary with hydrogen link atoms which are included in the SCF treatment ofthe 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-electronHamiltonian of the QM calculations, thus allowing QM polarization, and theQM/MM electrostatic interactions were evaluated from the QM electrostaticpotential (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)].




Results

Comparison of the two proton relay channels


The computed QM/MM barriers indicate that uncoupling is unfavorable in thecase of the Thr252MeO-Thr mutant, whereas there are two energetically feasibleproton transfer pathways for coupling; these are Mechanism I: homolytic OO bondcleavage followed by coupled protonelectron transfer and Mechanism II:proton-assisted heterolytic OO bond cleavage. The study shows Mechanism I to befavorable.

The corresponding rate-limiting barriers for the formation of Compound I arehigher in the mutant than in the wild-type enzyme. These findings are consistentwith the experimental observations that the Thr252MeO-Thr mutant forms thealcohol product exclusively (via Cpd1), but at lower reaction rates compared withthe 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.




Results


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Results


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QM/MM Thiel paper by Janus Eriksen

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