QM/MM Thiel paper by Janus Eriksen

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QM/MM Thiel paper by Janus Eriksen

  1. 1. Outline Introduction 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)
  2. 2. Outline Introduction to cytochrome P450 Previous cytochrome P450cam studies The present cytochrome P450cam study Results1 Introduction to cytochrome P450 Cytochrome P450 - general features Cytochrome P450 - enzymatic mechanism2 Previous cytochrome P450cam studies Cpd1 formation and proton relay channels Site-directed mutagenesis studies3 The present cytochrome P450cam study The agenda Computational methods4 Results Comparison of the two proton relay channels Janus Juul Eriksen Altarsha; Benighaus; Kumar and Thiel (2010)
  3. 3. Outline Introduction to cytochrome P450 Cytochrome P450 - general features Previous cytochrome P450cam studies Cytochrome P450 - enzymatic mechanism The present cytochrome P450cam study ResultsCytochrome P450 - general features 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)]. Janus Juul Eriksen Altarsha; Benighaus; Kumar and Thiel (2010)
  4. 4. Outline Introduction to cytochrome P450 Cytochrome P450 - general features Previous cytochrome P450cam studies Cytochrome P450 - enzymatic mechanism The present cytochrome P450cam study ResultsCytochrome P450 - enzymatic mechanism 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 protonatedFigure: The P450 twice by local transfer from surroundingcatalytic 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)]. Janus Juul Eriksen Altarsha; Benighaus; Kumar and Thiel (2010)
  5. 5. Outline Introduction to cytochrome P450 Cpd1 formation and proton relay channels Previous cytochrome P450cam studies Site-directed mutagenesis studies The present cytochrome P450cam study ResultsCpd1 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 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)]. Janus Juul Eriksen Altarsha; Benighaus; Kumar and Thiel (2010)
  6. 6. Outline Introduction to cytochrome P450 Cpd1 formation and proton relay channels Previous cytochrome P450cam studies Site-directed mutagenesis studies The present cytochrome P450cam study ResultsSite-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)]. Janus Juul Eriksen Altarsha; Benighaus; Kumar and Thiel (2010)
  7. 7. Outline Introduction to cytochrome P450 The agenda Previous cytochrome P450cam studies Computational methods The present cytochrome P450cam study ResultsThe 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)]. Janus Juul Eriksen Altarsha; Benighaus; Kumar and Thiel (2010)
  8. 8. Outline Introduction to cytochrome P450 The agenda Previous cytochrome P450cam studies Computational methods The present cytochrome P450cam study ResultsMD 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. Janus Juul Eriksen Altarsha; Benighaus; Kumar and Thiel (2010)
  9. 9. Outline Introduction to cytochrome P450 The agenda Previous cytochrome P450cam studies Computational methods The present cytochrome P450cam study ResultsQM 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 Janus Juul Eriksen Altarsha; Benighaus; Kumar and Thiel (2010)
  10. 10. Outline Introduction to cytochrome P450 The agenda Previous cytochrome P450cam studies Computational methods The present cytochrome P450cam study ResultsQM/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. Janus Juul Eriksen Altarsha; Benighaus; Kumar and Thiel (2010)
  11. 11. Outline Introduction to cytochrome P450 The agenda Previous cytochrome P450cam studies Computational methods The present cytochrome P450cam study ResultsQM/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)]. Janus Juul Eriksen Altarsha; Benighaus; Kumar and Thiel (2010)
  12. 12. Outline Introduction to cytochrome P450 Previous cytochrome P450cam studies Comparison of the two proton relay channels The present cytochrome P450cam study ResultsComparison 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. Janus Juul Eriksen Altarsha; Benighaus; Kumar and Thiel (2010)
  13. 13. Outline Introduction to cytochrome P450 Previous cytochrome P450cam studies Comparison of the two proton relay channelsThe present cytochrome P450cam study 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 Janus Juul Eriksen Altarsha; Benighaus; Kumar and Thiel (2010)
  14. 14. Outline Introduction to cytochrome P450 Previous cytochrome P450cam studies Comparison of the two proton relay channelsThe present cytochrome P450cam study 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 Janus Juul Eriksen Altarsha; Benighaus; Kumar and Thiel (2010)

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