Sam P. de Visser

Two-state reactivity mechanisms of hydroxylation and epoxidation by cytochrome P-450 revealed by theory.

Recent computational studies of alkane hydroxylation and alkene epoxidation by a model active species of the enzyme cytochrome P-450 reveal a two-state reactivity (TSR) scenario in which the information content of the product distribution is determined jointly by two states. TSR is used to reconcile the dilemma of the consensus rebound mechanism of alkane hydroxylation, which emerged from experimental studies of ultra-fast radical clocks. The dilemma, stated succinctly as radicals are both present and absent and the rebound mechanism is both right and wrong, is simply understood once one is cognizant that the mechanism operates by two states, one low-spin (LS) the other high-spin (HS). In both states, bond activation proceeds in a manner akin to the rebound mechanism, but the LS mechanism is effectively concerted, whereas the HS is stepwise with incursion of radical intermediates.

The 'push' effect of the thiolate ligand in cytochrome P450: a theoretical gauging

The push effect of the thiolate ligand in cytochrome P450 is investigated using density functional calculations. Theory supports Dawsons postulate that the push effect is crucial for the heterolytic O-O bond cleavage of ferric-peroxide, as well as for controlling the Fe(III)/Fe(II) redox process and gating the catalytic cycle. Two energetic factors that contribute to the push effect are revealed. The dominant one is the field factor (DeltaE(field)=54-103 kcal/mol) that accounts for the classical electrostatic repulsion with the negative charge of thiolate. The smaller factor is a quantum mechanical effect (DeltaE(QM)(sigma)=39 kcal/mol, DeltaE(QM)(pi)=4 kcal/mol), which is associated with the sigma- and pi-donor capabilities of thiolate. The effects of ligand replacement, changes in hydrogen bonding and dielectric screening are discussed in term of these quantities. In an environment with a dielectric constant of 5.7, the total push effect is reduced to 29-33 kcal/mol. Manifestations of the push effect on other properties of thiolate enzymes are discussed.

Iron-containing enzymes : versatile catalysts of hydroxylation reactions in nature

Nonheme iron(IV)-oxo oxidants in enzymes: Spectroscopic properties and reactivity patterns Heme iron(IV)-oxo oxidants in enzymes: Spectroscopic properties and reactivity patterns Mechanism and function of taurine/ -ketoglutarate dioxygenase enzymes, an update Mechanism and function of cysteine dioxygenase enzymes Mechanism and function of heme peroxidase enzymes Mechanism and function of cytochrome P450 enzymes Biomimetic studies of mononuclear nonheme iron containing oxidants Biomimetic studies of mononuclear porphyrin containing oxidants Density functional calibration studies on iron-containing systems Density functional theory studies on isomerisation reactions catalyzed by cytochrome P450 enzymes Quantum mechanics/molecular mechanics studies of peroxidase enzymes Theoretical modelling of nonheme iron containing oxidants

One oxidant, many pathways: A theoretical perspective of monooxygenation mechanisms by cytochrome P450 enzymes

Density functional theoretical studies of monooxygenation reactivity of the high-valent oxoiron(IV) porphyrin cation-radical compound of cytochrome P450, the so-called Compound I, and of its precursor, the ferric(III)-hydroperoxide species, are described. The degeneracy of the spin states of Compound I, its electron deficiency, and dense orbital manifold lead to two-state and multi-state reactivity scenarios and may thereby create reactivity patterns as though belonging to two or more different oxidants. Most of the controversies in the experimental data are reconciled using Compound I as the sole competent oxidant. Theory finds ferric(III)-hydroperoxide to be a very sluggish oxidant, noncompetitive with Compound I. If and when Compound I is absent, P450 oxidation will logically proceed by another form, but this has to be more reactive than ferric(III)-hydroperoxide. Theoretical studies are conducted to pinpoint such an oxidant for P450.

What Is the Difference between the Manganese Porphyrin and Corrole Analogues of Cytochrome P450's Compound I?

Density functional calculations on oxo-manganese complexes of corrole (1) and porphyrin (2 and 3) show a fundamental difference. The ground state of I is the singlet manganese(V) state, 1A(MnV), in which corrole is a closed shell. In contrast, 2 and 3 have high-spin manganese(IV) states, 1A1u and 3A2u respectively. This difference and the state ordering for each system are rationalized based on the competition between the intrinsic tendency of manganese to prefer high-spin electronic configurations, vis-à-vis the general tendency to prefer double occupancy in the low-lying orbitals. The outcome of this competition is determined primarily by the identity of the macrocycle, corrole versus porphyrin. Corrole with a small cavity holds the MnO moiety with a high off-plane displacement, and thereby prefers the low-spin state. On the other hand, porphyrin with the wider cavity holds the MnO moiety closer to the plane, and thereby prefers high-spin states.

How Does Ethene Inactivate Cytochrome P450 En Route to Its Epoxidation? A Density Functional Study

The suicidal complex 4 2, which inactivates cytochrome P450 during olefin epoxidation, was shown by density functional calculations to be formed from the same high-spin intermediate (4 1-III) that leads to stereochemical scrambling.

The experimentally elusive oxidant of cytochrome P450: A theoretical "trapping" defining more closely the "real" species

Cytochrome P450 is a vital enzyme in oxidative biotransformations, responsible for the detoxification of biological systems and for the synthesis of sex hormones. Recent experimental results demonstrate that, despite previous reports, 4] the active species of the enzyme, compound I (Cpd I, 1; Scheme 1), might have never been detected since it does not seem to accumulate during the catalytic cycle even at low temperature. To date, the only characterized Cpd I of a cysteinate enzyme belongs to chloroperoxidase (CPO). However, here too the geometry of the species is unknown, and the precise identity of the ground state is still debated. 6] Thus, a key species of one of the most important enzymes of biological systems is known to exist, but eludes detection. We present here theoretical calculations of the so far most extensive and most realistic Cpd I model, 8] with an account of the interaction types exerted by the apoprotein environment. We assign the ground state of Cpd I (1) as A2u, thereby settling previous theoretical disagreements and hopefully contributing toward an eventual resolution of the experimental controversy. The calculations project the unusual nature of this Cpd I that behaves as a chameleon species by adopting its electronic and geometric features to the protein environment to which it has to accomodate. Our benchmark system 2 (Scheme 1) involves octamethyl porphyrin and an axial cysteinato ligand. From an electronic point of view, methyl substituents are good representations of the side chains in 1, while avoiding complications due to internal rotations of the long side chains. Noncovalent interactions revealed by mimetic systems, mutation studies, and X-ray crystal structures of P450 enzymes 11] were taken into account as follows: a) Embedding of 2 in a polarizing medium of a low dielectric constant (ex885.7) serves to mimic the effect of polarization by the dipoles of the protein pocket near Cys 357 (using the numbering system in P450cam). b) An internal NH ́ ́ ́ S [1] S. R. Adams, R. Y. Tsien, Annu. Rev. Physiol. 1993, 35, 755 ± 784. [2] D. W. J. Cruickshank, J. R. Helliwell, L. N. Johnson, Time-Resolved Macromolecular Crystallography, Oxford University Press, Oxford, 1992. [3] I. Schlichting, Acc. Chem. Res. 2000, 33, 532 ± 538; M. H. B. Stowell, T. M. McPhillips, D. C. Rees, S. M. Soltis, E. Abresch, G. Feher, Science 1997, 276, 812 ± 816; B. Perman, V. Srajer, Z. Ren, T. Y. Teng, C. Pradervand, T. Ursby, D. Bourgeois, F. Schotte, M. Wulff, R. Kort, K. Hellingwerf, K. Moffat, Science 1998, 279, 1946 ± 1950. [4] I. Schlichting, S. C. Almo, G. Rapp, K. Wilson, K. Petratos, A. Lentfer, A. Wittinghofer, W. Kabsch, E. F. Pai, G. Petsko, R. S. Goody, Nature 1990, 345, 309 ± 315; B. L. Stoddard, P. Koenigs, N. Porter, K. Petratos, G. A. Petsko, D. Ringe, Proc. Natl. Acad. Sci. USA 1991, 88, 5503 ± 5507; B. L. Stoddard, B. E. Cohen, M. Brubaker, A. D. Mesecar, D. E. Koshland, Nat. Struct. Biol. 1998, 5, 891 ± 897. [5] J. M. Bolduc, D. H. Dyer, W. G. Scott, P. Singer, R. M. Sweet, D. E. Koshland, B. L. Stoddard, Science 1995, 268, 1312 ± 1318. [6] J. Hajdu, I. Andersson, Annu. Rev. Biophys. Biomol. Struct. 1993, 22, 467 ± 498. [7] G. Rapp, Methods Enzymol. 1998, 291, 202 ± 222. [8] I. Schlichting, R. S. Goody, Methods Enzymol. 1997, 277, 467 ± 490; M. Weik, R. B. G. Ravelli, G. Kryger, S. McSweeney, M. L. Raves, M. Harel, P. Gros, I. Silman, J. Kroon, J. L. Sussman, Proc. Natl. Acad. Sci. USA 2000, 97, 623 ± 628. [9] K. Moffat, R. Henderson, Curr. Opin. Struct. Biol. 1995, 5, 656 ± 663. [10] A. J. Scheidig, C. Burmester, R. S. Goody, Structure 1999, 7, 1311 ± 1324. [11] T. Y. Teng, K. Moffat, J. Appl. Crystallogr. 1998, 31, 252 ± 257. [12] L. Peng, I. Silman, J. L. Sussman, M. Goeldner, Biochemistry 1996, 35, 10 854 ± 10 861; L. Peng, M. Goeldner, Methods Enzymol. 1998, 291, 265 ± 278. [13] A. Ostermann, R. Waschipky, F. G. Parak, G. U. Nienhaus, Nature 2000, 404, 205 ± 208. [14] L. Peng, F. Nachon, J. Wirz, M. Goeldner, Angew. Chem. 1998, 110, 2838 ± 2840; Angew. Chem. Int. Ed. 1998, 37, 2691 ± 2693. [15] J. M. Walker, G. P. Reid, J. A. McCray, D. R. Trentham, J. Am. Chem. Soc. 1988, 110, 7170 ± 7177. [16] I. R. Dunkin, J. Gebicki, M. Kiszka, D. Sanin-Leira, Spectrochim. Acta Part A 1997, 53, 2553 ± 2557. [17] J. L. Sussman, M. Harel, F. Frolow, C. Oefner, A. Goldman, L. Toker, I. Silman, Science 1991, 253, 872 ± 879. [18] G. Koellner, M. Weik, A. Specht, L. Peng, M. Harel, D. Bourgeois, I. Silman, J. Kroon, M. Goeldner, J. Sussman, unpublished results. [19] T. Ursby, M. Weik, E. Fioravanti, M. Delarue, M. Goeldner, D. Bourgeois, unpublished results.

Computational Approaches to Cytochrome P450 Function

This chapter describes computational strategies for investigating the species in the catalytic cycle of the enzyme cytochrome P450, and the mechanisms of its main processes: alkane hydroxylation, alkene epoxidation, arene hydroxylation, and sulfoxidation. The methods reviewed are molecular mechanical (MM)-based approaches (used e.g., to study substrate docking), quantum mechanical (QM) and QM/MM calculations (used to study electronic structure and mechanism).