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Physical chemistry Z 0225 Electrostatic Basis for Enzyme Catalysis -[247 refs.]. -(WARSHEL*, A.; SHARMA, P. K.; KATO, M.; XIANG, Y.; LIU, H.; OLSSON, M. H. M.; Chem. Rev. (Washington, D. C.) 106 (2006) 8, 3210-3235; Dep. Chem., Univ. South. Calif., Los Angeles, CA 90089, USA; Eng.) -Lindner 42-255

Phosphoryl transfer plays key roles in signaling, energy transduction, protein synthesis, and maintaining the integrity of the genetic material. On the surface, it would appear to be a simple nucleophile displacement reaction. However, this simplicity is deceptive, as, even in aqueous solution, the low-lying d-orbitals on the phosphorus atom allow for eight distinct mechanistic possibilities, before even introducing the complexities of the enzyme catalyzed reactions. To further complicate matters, while powerful, traditional experimental techniques such as the use of linear free-energy relationships (LFER) or measuring isotope effects cannot make unique distinctions between different potential mechanisms. A quarter of a century has passed since Westheimer wrote his seminal review, ‘Why Nature Chose Phosphate’ (Science 235 (1987), 1173), and a lot has changed in the field since then. The present review revisits this biologically crucial issue, exploring both relevant enzymatic systems as well as the corresponding chemistry in aqueous solution, and demonstrating that the only way key questions in this field are likely to be resolved is through careful theoretical studies (which of course should be able to reproduce all relevant experimental data). Finally, we demonstrate that the reason that naturereallychose phosphate is due to interplay between two counteracting effects: on the one hand, phosphates are negatively charged and the resulting charge-charge repulsion with the attacking nucleophile contributes to the very high barrier for hydrolysis, making phosphate esters among the most inert compounds known. However, biology is not only about reducing the barrier to unfavorable chemical reactions. That is, the same charge-charge repulsion that makes phosphate ester hydrolysis so unfavorable also makes it possible to regulate, by exploiting the electrostatics. This means that phosphate ester hydrolysis can not only be turned on, but also be turned off, by fine tuning the electrostatic environment and the present review demonstrates numerous examples where this is the case. Without this capacity for regulation, it would be impossible to have for instance a signaling or metabolic cascade, where the action of each participant is determined by the fine-tuned activity of the previous piece in the production line. This makes phosphate esters the ideal compounds to facilitate life as we know it.

Epoxidation (C=C) vis-à-vis allylic hydroxylation (C-H) reactions of propene with a model compound I (Cpd I) of the enzyme cytochrome P450 were studied using B3LYP density functional theory. Potential energy profiles and kinetic isotope effects (KIE) were calculated. The interactions in the protein pocket were mimicked by adding two external NH- - -S hydrogen bonds to the thiolate ligand and by introducing a nonpolar medium (with a dielectric constant, epsilon = 5.7) that can exert a polarization effect on the reacting species. A two-state reactivity (TSR) with high-spin (HS) and low-spin (LS) states were located for both processes (Ogliaro, F.; Harris, N.; Cohen, S.; Filatov, M.; de Visser, S. P.; Shaik, S. J. Am. Chem. Soc. 2000, 122, 8977-8989. de Visser, S. P.; Ogilaro, F.; Harris, N.; Shaik, S. J. Am. Chem. Soc. 2001, 123, 3037-3047). The HS processes were found to be stepwise, whereas the LS processes were characterized as nonsynchronous but effectively concerted pathways. The computed KIE for C-H hydroxylation with and without tunneling corrections are large (>7), and they support the assignment of the corresponding transition states as hydrogen-abstraction species (Groves, J. T.; Han, Y.-Z. In Cytochrome P450: Structures, Mechanism and Biochemistry, 2nd ed.; Ortiz de Montellano, P. R., Ed.; Plenum Press: New York, 1995; Chapter 1; pp 3-48). In the gas phase, epoxidation is energetically favorable by 3.4 kcal mol(-1). Inclusion of zero-point energy reduces this difference but still predicts C=C/C-H > 1. Environmental effects were found to have major impact on the C=C/C-H ratio as well as on the stereoselectivity of the processes. Thus, two NH- - -S hydrogen bonds away from the reaction center reverse the regioselectivity and prefer hydroxylation, namely, C=C/C-H <1. The polarity of the medium further accentuates the trend and leads to a change by 2 orders of magnitude in the regioselectivity, C=C/C-H << 1. Furthermore, since the environmental interactions prefer the LS over the HS reactions, both hydroxylation and epoxidation processes are rendered more stereoselective, again by 2 orders of magnitude. It follows, therefore, that Cpd I is a chameleon oxidant (Ogliaro, F.; Cohen, S.; de Visser, S. P.; Shaik, S. J. Am. Chem. Soc. 2000, 122, 12892-12893; Ogliaro, F.; de Visser, S. P.; Cohen, S.; Kaneti, J.; Shaik, S. Chembiochem. 2001, 2, 848-851; Ogliaro, F.; de Visser, S. P.; Groves, J. T.; Shaik, S. Angew. Chem., Int. Ed. 2001, 40, 2874-2878) that tunes its reactivity and selectivity patterns in response to the protein environment in which it is accommodated. A valence bond (VB) model, akin to "redox mesomerism" (Bernadou, J.; Fabiano, A.-S.; Robert, A.; Meunier, B. J. Am. Chem. Soc. 1994, 116, 9375-9376), is constructed and enables the description of a chameleon transition state. It shows that the good donor ability of the thiolate ligand and the acceptor ability of the iron porphyrin create mixed-valent situations that endow the transition state with a great sensitivity to external perturbations as in the...

Iron(III)-hydroperoxo, [Por(CysS)Fe(III)-OOH](-), a key species in the catalytic cycle of cytochrome P450, was recently identified by EPR/ENDOR spectroscopies (Davydov, R.; Makris, T. M.; Kofman, V.; Werst, D. E.; Sligar, S. G.; Hoffman, B. M. J. Am. Chem. Soc. 2001, 123, 1403-1415). It constitutes the last station of the preparative steps of the enzyme before oxidation of an organic compound and is implicated as the second oxidant capable of olefin epoxidation (Vaz, A. D. N.; McGinnity, D. F.; Coon, M. J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3555-3560), in addition to the penultimate active species, Compound I (Groves, J. T.; Han, Y.-Z. In Cytochrome P450: Structure, Mechanism and Biochemistry, 2nd ed.; Ortiz de Montellano, P. R., Ed.; Plenum Press: New York, 1995; pp 3-48). In response, we present a density functional study of a model species and its ethylene epoxidation pathways. The study characterizes a variety of properties of iron(III)-hydroperoxo, such as the O-O bonding, the Fe-S bonding, Fe-O and Fe-S stretching frequencies, its electron attachment, and ionization energies. Wherever possible these properties are compared with those of Compound I. The proton affinities for protonation on the proximal and distal oxygen atoms of iron(III)-hydroperoxo, and the effect of the thiolate ligand thereof, are determined. In accordance with previous results (Harris, D. L.; Loew, G. H. J. Am. Chem. Soc. 1998, 120, 8941-8948), iron(III)-hydroperoxo is a strong base (as compared with water), and its distal protonation leads to a barrier-free formation of Compound I. The origins of this barrier-free process are discussed using a valence bond approach. It is shown that the presence of the thiolate is essential for this process, in line with the "push effect" deduced by experimentalists (Sono, M.; Roach, M. P.; Coulter, E. D.; Dawson, J. H. Chem. Rev. 1996, 96, 2841-2887). Finally, four epoxidation pathways of iron(III)-hydroxperoxo are located, in which the species transfers oxygen to ethylene either from the proximal or from the distal sites, in both concerted and stepwise manners. The barriers for the four mechanisms are 37-53 kcal/mol, in comparison with 14 kcal/mol for epoxidation by Compound I. It is therefore concluded that iron(III)-hydroperoxo, as such, cannot be a second oxidant, in line with its significant basicity and poor electron-accepting capability. Possible versions of a second oxidant are discussed.

C−H hydroxylation by the enzyme cytochrome P450 is one of Nature’s important and most ubiquitous processes. There is strong evidence that the mechanism proceeds by initial hydrogen abstraction, from the alkane, by the high‐valent iron−oxo species of the enzyme, followed by a rebound of the alkyl radical to form the ferric−alcohol product complex (the Groves “rebound” mechanism). Nevertheless, the “rebound” mechanism is still controversial due to the ultrashort radical lifetimes deduced from radical‐clock experiments. This review describes the main elements of the controversy and its updated resolution by theory, with an emphasis on the controversial rebound step. The theoretically derived model for alkane hydroxylation is found to involve two‐state reactivity (TSR). In TSR, radicals are produced on two different spin‐state surfaces, and thereafter they react differently; on the low‐spin surface, the rebound proceeds with no product rearrangement and the lifetime of the radicals is ultrashort (or zero), while on the high‐spin surface the barrier for rebound is substantial and the lifetime of the radical is sufficiently long that rearrangement may compete with product formation by rebound. A new valence‐bond model is developed to model the rebound barrier on the high‐spin surface and conceptualize its dependence on the nature of the alkyl radical. The possible intermediacy of carbocationic intermediates alongside radicals is discussed. It is shown that the TSR scenario provides a satisfactory rationale for the controversial findings in the field, and makes verifiable predictions. One of the predictions of TSR is that the ratio of unrearranged to rearranged products, [U/R], will be subject to an intrinsic isotope effect that is substrate‐dependent. This prediction and its possible verification by experiment are discussed. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

The active site of HRP Compound I (Cpd I) is modeled using hybrid density functional theory (UB3LYP). The effects of neighboring amino acids and of environmental polarity are included. The low-lying states have porphyrin radical cationic species (Por(*)(+)). However, since the Por(*)(+) species is a very good electron acceptor, other species, which can be either the ligand or side chain amino acid residues, may participate in electron donation to the Por(*)(+) moiety, thereby making Cpd I behave like a chemical chameleon. Thus, this behavior that was noted before for Cpd I of P450 is apparently much more wide ranging than initially appreciated. Since chemical chameleonic behavior property was found to be expressed not only in the properties of Cpd I itself, but also in its reactivity, the roots of this phenomenon are generalized. A comparative discussion of Cpd I species follows for the enzymes HRP, CcP, APX, CAT (catalase), and P450.