Compound I in heme thiolate enzymes: a comparative QM/MM study

This study directly compares the active species of heme enzymes, so-called Compound I (Cpd I), across the heme-thiolate enzyme family. Thus, sixty-four different Cpd I structures are calculated by hybrid quantum mechanical/molecular mechanical (QM/MM) methods using four different cysteine-ligated heme enzymes (P450(cam), the mutant P450(cam)-L358P, CPO and NOS) with varying QM region sizes in two multiplicities each. The overall result is that these Cpd I species are similar to each other with regard to many characteristic features. Hence, using the more stable CPO Cpd I as a model for P450 Cpd I in experiments should be a reasonable approach. However, systematic differences were also observed, and it is shown that NOS stands out in most comparisons. By analyzing the electrical field generated by the enzyme on the QM region, one can see that (a) the protein exerts a large influence and modifies all the Cpd I species compared with the gas-phase situation and (b) in NOS this field is approximately planar to the heme plane, whereas it is approximately perpendicular in the other enzymes, explaining the deviating results on NOS. The calculations on the P450(cam) mutant L358P show that the effects of removing the hydrogen bond between the heme sulfur and L358 are small at the Cpd I stage. Finally, Mossbauer parameters are calculated for the different Cpd I species, enabling future comparisons with experiments. These results are discussed in the broader context of recent findings of Cpd I species that exhibit large variations in the electronic structure due to the presence of the substrate.     

Quantum mechanical/molecular mechanical study of mechanisms of heme degradation by the enzyme heme oxygenase: the strategic function of the water cluster

Heme degradation by heme oxygenase (HO) enzymes is important in maintaining iron homeostasis and prevention of oxidative stress, etc. In response to mechanistic uncertainties, we performed quantum mechanical/molecular mechanical investigations of the heme hydroxylation by HO, in the native route and with the oxygen surrogate donor H2O2. It is demonstrated that H2O2 cannot be deprotonated to yield Fe(III)OOH, and hence the surrogate reaction starts from the FeHOOH complex. The calculations show that, when starting from either Fe(III)OOH or Fe(III)HOOH, the fully concerted mechanism involving O-O bond breakage and O-C(meso) bond formation is highly disfavored. The low-energy mechanism involves a nonsynchronous, effectively concerted pathway, in which the active species undergoes first O-O bond homolysis followed by a barrier-free (small with Fe(III)HOOH) hydroxyl radical attack on the meso position of the porphyrin. During the reaction of Fe(III)HOOH, formation of the Por+*FeIV=O species, compound I, competes with heme hydroxylation, thereby reducing the efficiency of the surrogate route. All these conclusions are in accord with experimental findings (Chu, G. C.; Katakura, K.; Zhang, X.; Yoshida, T.; Ikeda-Saito, M. J. Biol. Chem. 1999, 274, 21319). The study highlights the role of the water cluster in the distal pocket in creating "function" for the enzyme; this cluster affects the O-O cleavage and the O-Cmeso formation, but more so it is responsible for the orientation of the hydroxyl radical and for the observed alpha-meso regioselectivity of hydroxylation (Ortiz de Montellano, P. R. Acc. Chem. Res. 1998, 31, 543). Differences/similarities with P450 and HRP are discussed.     

Searching for the second oxidant in the catalytic cycle of cytochrome P450: a theoretical investigation of the iron(III)-hydroperoxo species and its epoxidation pathways

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.     

The high-valent iron-oxo species of polyoxometalate, if it can be made, will be a highly potent catalyst for C-H hydroxylation and double-bond epoxidation

This study uses density functional theory (DFT) calculations to explore the reactivity of the putative high-valent iron-oxo reagent of the iron-substituted polyoxometalate (POM-FeO4-), derived from the Keggin species, PW12O40(3-). It is shown that POM-FeO4- is in principle capable of C-H hydroxylation and C=C epoxidation and that it should be a powerful oxidant, even more so than the Compound I species of cytochrome P450. The calculations indicate that in a solvent, the barriers, and especially those for epoxidation, become sufficiently small that one may expect an extremely fast reaction. An experimental investigation (by R.N. and A.M.K.) shows, however, that the formation of POM-FeO4- using the oxygen donor, F5PhI-O, leads to a persistent adduct, POM-FeO-I-PhF5(4-), which does not decompose to POM-FeO4- + F5Ph-I at the working temperature and exhibits sluggish reactivity, in accord with previous experimental results (Hill, C. L.; Brown, R. B., Jr. J. Am. Chem. Soc. 1986, 108, 536 and Mansuy, D.; Bartoli, J.-F.; Battioni, P.; Lyon, D. K.; Finke, R. G. J. Am. Chem. Soc. 1991, 113, 7222). Subsequent calculations indeed reveal that the gas-phase binding energy of F5PhI to POM-FeO4- is high (ca. 20 kcal/mol) compared to the corresponding binding energy of propene (ca. 2-3 kcal/mol). As such, the POM-FeO-I-PhF5(4-) complex is expected to be persistent toward the displacement of F5PhI by a substrate like propene, leading thereby to sluggish oxidative reactivity. According to theory, overcoming this technical difficulty may turn out to be very rewarding. The question is, can POM-FeO4- be made?     

Density functional theory investigation of the active site of [Fe]-hydrogenases: effects of redox state and ligand characteristics on structural,electronic, and reactivity properties of complexes related to the [2Fe]H subcluster

The effects of redox state and ligand characteristics on structural, electronic, and reactivity properties of complexes related to the [2Fe](H) subcluster of [Fe]-hydrogenases have been investigated by DFT calculations and compared with experimental and theoretical data obtained investigating both the enzyme and synthetic model complexes. Our results show that Fe(II)Fe(II) species characterized by OH or H(2)O groups terminally coordinated to the iron atom distal to the terminal sulfur ligand (Fe(d)) are less stable than corresponding mu-OH or mu-H(2)O species, suggesting that the latter are destabilized or kinetically inaccessible in the enzyme. In addition, results obtained investigating Fe(I)Fe(I) and Fe(II)Fe(I) complexes show that structure and relative stability of species characterized by a mu-CO group are significantly affected by the electronic properties of the ligands coordinated to the iron atoms. The investigation of reaction pathways for H(2) activation confirms and extends a previous hypothesis indicating that H(2) can be cleaved on Fe(II)Fe(II) species. In particular, even though [Fe]-hydrogenases are proposed to bind and activate H(2) at a single iron center, the comparison of our data with experimental results obtained studying synthetic complexes (Zhao, X.; Georgakaki, I. P.; Miller, M. L.; Mejia-Rodriguez, R.; Chiang, C.-Y.; Darensbourg, M. Y. Inorg. Chem. 2002, 41, 3917) suggests that activation paths involving both metal ions are also possible. Moreover, mu-H Fe(II)Fe(I) complexes are predicted to correspond to stable species and might be formed in the enzyme catalytic cycle.     

The elusive oxidant species of cytochrome P450 enzymes: characterization by combined quantum mechanical/molecular mechanical (QM/MM) calculations

The primary oxidant of cytochrome P450 enzymes, Compound I, is hard to detect experimentally; in the case of cytochrome P450(cam), this intermediate does not accumulate in solution during the catalytic cycle even at temperatures as low as 200 K (ref 4). Theory can play an important role in characterizing such elusive species. We present here combined quantum mechanical/molecular mechanical (QM/MM) calculations of Compound I of cytochrome P450(cam) in the full enzyme environment as well as density functional studies of the isolated QM region. The calculations assign the ground state of the species, quantify the effect of polarization and hydrogen bonding on its properties, and show that the protein environment and its specific hydrogen bonding to the cysteinate ligand are crucial for sustaining the Fe-S bond and for preventing the full oxidation of the sulfur.     

Structural studies of nonclassical cyclobutylmethyl cations by the ab initio method

Ab initio calculations at the MP2/cc-pVTZ level show that the cyclobutylmethyl cation is a nonclassical sigma-delocalized species, which is distinct from the global minimum C2-symmetric cyclopentyl cation (Schleyer, P. v. R.; Carneiro, J. W. de M.; Koch, W.; Raghavachari, K. J. Am. Chem. Soc. 1989, 111, 5475). Relatively lower level DFT calculations, on the other hand, show that the primary cyclobutylmethyl cation spontaneously collapses into the cyclopentyl cation (Prakash, G. K. S.; Reddy, V. P.; Rasul, G.; Casanova, J.; Olah, G. A. J. Am. Chem. Soc. 1998, 120, 13362). The secondary 1-cyclobutylethyl cation is also a nonclassical carbocation, as shown by calculations at the MP2/cc-pVTZ level. Two structures having energy minima are identified for the latter cation on the potential energy surface. The conformer in which the methyl group is in the exo orientation is a global minimum and is favored over the corresponding endo conformer by 1.2 kcal/mol at the MP2/cc-pVTZ//MP2/cc-pVTZ +ZPE level of calculations. The tertiary 1-cyclobutyl-1-methylethyl cation, at this level of calculations, also involves substantial nonclassical sigma-delocalization, showing that the nonclassical stabilization is more important for cyclobutylmethyl cations relative to the cyclopropylmethyl cations. The 13C NMR chemical shifts obtained from GIAO-CCSD(T)/tzp/dz calculations further substantiate the nonclassical structures for these carbocations.     

An efficient proton-coupled electron-transfer process during oxidation of ferulic acid by horseradish peroxidase: coming full cycle

Quantum mechanics/molecular mechanics calculations were utilized to study the process of oxidation of a native substrate (ferulic acid) by the active species of horseradish peroxidase (Dunford, H. B. Heme Peroxidases; Wiley-VCH: New York, 1999), Compound I and Compound II, and the manner by which the enzyme returns to its resting state. The results match experimental findings and reveal additional novel features. The calculations demonstrate that both oxidation processes are initiated by a proton-coupled electron-transfer (PCET) step, in which the active species of the enzyme participate only as electron-transfer partners, while the entire proton-transfer event is being relayed from the substrate to and from the His42 residue by a water molecule (W402). The reason for the observed (Henriksen, A; Smith, A. T.; Gajhede, M. J. Biol. Chem. 1999, 274, 35005-35011) similar reactivities of Compound I and Compound II toward ferulic acid is that the reactive isomer of Compound II is the, hitherto unobserved, Por(*)(+)Fe(III)OH isomer that resembles Compound I. The PCET mechanism reveals that His42 and W402 are crucial moieties and they determine the function of the HRP enzyme and account for its ability to perform substrate oxidation (Poulos, T. L. Peroxidases and Cytochrome P450. In The Porphyrin Handbook; Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Academic Press: New York, 2000; Vol. 4, pp 189). In view of the results, the possibility of manipulating substrate oxidation by magnetic fields is an intriguing possibility.     

New features in the catalytic cycle of cytochrome P450 during the formation of compound I from compound 0

Density functional theory (DFT) is applied to the dark section of the catalytic cycle of the enzyme cytochrome P450, namely, the formation of the active species, Compound I (Cpd I), from the ferric-hydroperoxide species (Cpd 0) by a protonation-assisted mechanism. The chosen 96-atom model includes the key functionalities deduced from experiment: Asp(251), Thr(252), Glu(366), and the water channels that relay the protons. The DFT model calculations show that (a) Cpd I is not formed spontaneously from Cpd 0 by direct protonation, nor is the process very exothermic. The process is virtually thermoneutral and involves a significant barrier such that formation of Cpd I is not facile on this route. (b) Along the protonation pathway, there exists an intermediate, a protonated Cpd 0, which is a potent oxidant since it is a ferric complex of water oxide. Preliminary quantum mechanical/molecular mechanical calculations confirm that Cpd 0 and Cpd I are of similar energy for the chosen model and that protonated Cpd 0 may exist as an unstable intermediate. The paper also addresses the essential role of Thr(252) as a hydrogen-bond acceptor (in accord with mutation studies of the OH group to OMe).     

Theoretical study of N-demethylation of substituted N,N-dimethylanilines by cytochrome P450: the mechanistic significance of kinetic isotope effect profiles

The mechanism of N-demethylation of N,N-dimethylanilines (DMAs) by cytochrome P450, a highly debated topic in mechanistic bioinorganic chemistry (Karki, S. B.; Dinnocenczo, J. P.; Jones, J. P.; Korzekwa, K. R. J. Am. Chem. Soc. 1995, 117, 3657), is studied here using DFT calculations of the reactions of the active species of the enzyme, Compound I (Cpd I), with four para-(H, Cl, CN, NO2) substituted DMAs. The calculations resolve mechanistic controversies, offer a consistent mechanistic view, and reveal the following features: (a) the reaction pathways involve C-H hydroxylation by Cpd I followed by a nonenzymatic carbinolamine decomposition. (b) C-H hydroxylation is initiated by a hydrogen atom transfer (HAT) step that possesses a "polar" character. As such, the HAT energy barriers correlate with the energy level of the HOMO of the DMAs. (c) The series exhibits a switch from spin-selective reactivity for DMA and p-Cl-DMA to two-state reactivity, with low- and high-spin states, for p-CN-DMA and p-NO2-DMA. (d) The computed kinetic isotope effect profiles (KIEPs) for these scenarios match the experimentally determined KIEPs. Theory further shows that the KIEs and TS structures vary in a manner predicted by the Melander-Westheimer postulate: as the substituent becomes more electron withdrawing, the TS is shifted to a later position along the H-transfer coordinate and the corresponding KIEs increases. (e) The generated carbinolaniline can readily dissociate from the heme and decomposes in a nonenzymatic environment, which involves water assisted proton shift.     

Better understanding of the ring-cleavage process of cyanocyclopropyl anionic derivatives. A theoretical study based on the electron localization function

[reaction: see text] Theoretical calculations at the B3LYP/6-31+G(d), MP2/6-31+G(d), and G3(MP2) levels have been carried out to understand the alternative reaction pathways (the cyclopropyl ring cleavage (RC) and the retrocycloaddition reaction (rCA)) of a constrained tricyanocyclopropyl anionic derivative. The more energetically favorable path is found to be the RC process, a formally "forbidden" rearrangement (Leiviers, M.; Tam, I.; Groves, K.; Leung, D.; Xie, Y.; Breslow, R. Org. Lett. 2003, 5, 19, 3407) yielding an allylic anion system via a concerted transition structure, in agreement with experimental outcomes. rCA is more energetically favorable along a two-stage mechanism, via an intermediate, than a synchronous concerted process. By using isodesmic reactions, we have found that B3LYP presents limitations when it calculates carbon-carbon bond-breaking processes along the present rCA reaction. A detailed analysis of the nature of the topology of the reactive potential energy surface for the RC process points out the presence of a valley-ridge inflection point in the uphill part. An explanation for the low-energy barrier associated with RC is furnished on the analysis of the evolution of the twisting (dis-/conrotatory) motions of cyano substituents in the cyclopropyl ring as well as on the number and type of electron pairs provided by the electron localization function (ELF).     

Coordination chemistry at the molybdenum site of sulfite oxidase: redox-induced structural changes in the cysteine 207 to serine mutant

The redox chemistry of the molybdenum site of the C207S mutant of recombinant human sulfite oxidase has been studied via potentiometric titrations employing both electron paramagnetic resonance (EPR) spectroscopy and X-ray absorption spectroscopy (XAS) as probes of the active site structure. In earlier EXAFS studies, oxidized Cys207Ser enzyme has been shown to possess a novel tri-oxo active site, in which Ser207 does not appear to be a ligand to Mo [George, G. N.; Garrett, R. M.; Prince, R. C.; Rajagopalan, K. V. J. Am. Chem. Soc. 1996, 118, 8588-8592]. Redox titrations show that the active site is modified under reducing conditions to a mono-oxo Mo(IV) species, probably with Ser207 ligated to the metal. The Mo(IV) species can be reoxidized to a mono-oxo Mo(V) species still coordinated to Ser207, which in turn can be further reoxidized to yield the initial tri-oxo Mo(VI) structure with loss of Ser207 ligation.     

A proton-shuttle mechanism mediated by the porphyrin in benzene hydroxylation by cytochrome p450 enzymes

Benzene hydroxylation is a fundamental process in chemical catalysis. In nature, this reaction is catalyzed by the enzyme cytochrome P450 via oxygen transfer in a still debated mechanism of considerable complexity. The paper uses hybrid density functional calculations to elucidate the mechanisms by which benzene is converted to phenol, benzene oxide, and ketone, by the active species of the enzyme, the high-valent iron-oxo porphyrin species. The effects of the protein polarity and hydrogen-bonding donation to the active species are mimicked, as before (Ogliaro, F.; Cohen, S.; de Visser, S. P.; Shaik, S. J. Am. Chem. Soc. 2000, 122, 12892-12893). It is verified that the reaction does not proceed either by hydrogen abstraction or by initial electron transfer (Ortiz de Montellano, P. R. In Cytochrome P450: Structure, Mechanism and Biochemistry, 2nd ed.; Ortiz de Montellano, P. R., Ed.; Plenum Press: New York, 1995; Chapter 8, pp 245-303). In accord with the latest experimental conclusions, the theoretical calculations show that the reactivity is an interplay of electrophilic and radicalar pathways, which involve an initial attack on the pi-system of the benzene to produce sigma-complexes (Korzekwa, K. R.; Swinney, D. C.; Trager, W. T. Biochemistry 1989, 28, 9019-9027). The dominant reaction channel is electrophilic and proceeds via the cationic sigma-complex,( 2)3, that involves an internal ion pair made from a cationic benzene moiety and an anionic iron porphyrin. The minor channel proceeds by intermediacy of the radical sigma-complex, (2)2, in which the benzene moiety is radicalar and the iron-porphyrin moiety is neutral. Ring closure in these intermediates produces the benzene oxide product ((2)4), which does not rearrange to phenol ((2)7) or cyclohexenone ((2)6). While such a rearrangement can occur post-enzymatically under physiological conditions by acid catalysis, the computations reveal a novel mechanism whereby the active species of the enzyme catalyzes directly the production of phenol and cyclohexenone. This enzymatic mechanism involves proton shuttles mediated by the porphyrin ring through the N-protonated intermediate, (2)5, which relays the proton either to the oxygen atom to form phenol ((2)7) or to the ortho-carbon atom to produce cyclohexenone product ((2)6). The formation of the phenol via this proton-shuttle mechanism will be competitive with the nonenzymatic conversion of benzene oxide to phenol by external acid catalysis. With the assumption that (2)5 is not fully thermalized, this novel mechanism would account also for the observation that there is a partial skeletal retention of the original hydrogen of the activated C-H bond, due to migration of the hydrogen from the site of hydroxylation to the adjacent carbon (so-called "NIH shift" (Jerina, D. M.; Daly, J. W. Science 1974, 185, 573-582)). Thus, in general, the computationally discovered mechanism of a porphyrin proton shuttle suggests thatthere is an enzymatic pathway that converts benzene directly to a phenol and ketone, in addition to nonenzymatic production of these species by conversion of arene oxide to phenol and ketone. The potential generality of protonated porphyrin intermediates in P450 chemistry is discussed in the light of the H/D exchange observed during some olefin epoxidation reactions (Groves, J. T.; Avaria-Neisser, G. E.; Fish, K. M.; Imachi, M.; Kuczkowski, R. J. Am. Chem. Soc. 1986, 108, 3837-3838) and the general observation of heme alkylation products (Kunze, K. L.; Mangold, B. L. K.; Wheeler, C.; Beilan, H. S.; Ortiz de Montellano, P. R. J. Biol. Chem. 1983, 258, 4202-4207). The competition, similarities, and differences between benzene oxidation viz. olefin epoxidation and alkanyl C-H hydroxylation are discussed, and comparison is made with relevant experimental and computational data. The dominance of low-spin reactivity in benzene hydroxylation viz. two-state reactivity (Shaik, S.; de Visser, S. P.; Ogliaro, F.; Schwarz, H.; Schr?der, D. Curr. Opin. Chem. Biol. 2002, 6, 556-567) in olefin epoxidation and alkane hydroxylation is traced to the loss of benzene resonance energy during the bond activation step.     

QM/MM study of the active species of the human cytochrome P450 3A4, and the influence thereof of the multiple substrate binding

Cytochrome P450 3A4 is involved in the metabolism of 50% of all swallowed drugs. The enzyme functions by means of a high-valent iron-oxo species, called compound I (Cpd I), which is formed after entrance of the substrate to the active site. We explored the features of Cpd I using hybrid quantum mechanical/molecular mechanical calculations on various models that are either substrate-free or containing one and two molecules of diazepam as a substrate. M?ssbauer parameters of Cpd I were computed. Our major finding shows that without the substrate, Cpd I tends to elongate its Fe-S bond, localize the radical on the sulfur, and form hydrogen bonds with A305 and T309, which may hypothetically lead to Cpd I consumption by H-abstraction. However, the positioning of diazepam close to Cpd I, as enforced by the effector molecule, was found to strengthen the NH...S interactions of the conserved I443 and G444 residues with the proximal cysteinate ligand. These interactions are known to stabilize the Fe-S bond, and as such, the presence of the substrate leads to a shorter Fe-S bond and it prevents the localization of the radical on the sulfur. This diazepam-Cpd I stabilization was manifested in the 1W0E conformer. The effector substrate did not influence Cpd I directly but rather by positioning the active substrate close to Cpd I, thus displacing the hydrogen bonds with A305 and T309, and thereby giving preference to substrate oxidation. It is hypothesized that these effects on Cpd I, promoted by the restrained substrate, may be behind the special metabolic behavior observed in cases of multiple substrate binding (also called cooperative binding). This restraint constitutes a mechanism whereby substrates stabilize Cpd I sufficiently long to affect monooxygenation by P450s at the expense of Cpd I destruction by the protein residues.     

Amphiphilic 1-deoxynojirimycin derivatives through click strategies for chemical chaperoning in N370S Gaucher cells

In Gaucher disease (GD), mutant β-glucocerebrosidases (β-GCase) that are misfolded are recognized by the quality control machinery of the endoplasmic reticulum (ER) and degraded proteolytically. Hydrophobic iminosugars can be used as pharmacological chaperones to provide an improvement in the folding of the enzyme and promote trafficking from the ER. We have developed here an efficient click procedure to tether hydrophobic substituents to N-azidopropyl-1-deoxynojirimycin. A set of 14 original iminosugars was designed and evaluated for inhibition of commercially available glucosidases. Most of the compounds were micromolar inhibitors of those enzymes. In vitro inhibition assays with the N370S β-GCase revealed that the sublibrary containing the derivatives with aromatic aglycons displayed the highest inhibitory potency. Chaperone activity of the whole set of synthetic compounds was also explored in mutant Gaucher cells. The most active compound gave a nearly 2-fold increase in enzyme activity at 20 μM, a significantly higher value than the 1.33-fold recorded for the reference compound N-nonyl-1-deoxynojirimycin (N-nonyl-DNJ). As previously reported with bicyclic sp(2)-iminosugars (Luan, Z.; Higaki, K.; Aguilar-Moncayo, M.; Ninomiya, H.; Ohno, K.; Garci?a-Moreno, M. I.; Ortiz Mellet, C.; Garci?a Ferna?ndez, J. M.; Suzuki, Y. ChemBioChem 2009, 10, 2780), in vitro inhibition of β-GCase measured for the compounds did not correlate with the cellular chaperone activity. The potency of new iminosugar chaperones is therefore not predictable from structure-activity relationships studies based on the in vitro β-GCase inhibition.     

Gauging the relative oxidative powers of compound I, ferric-hydroperoxide, and the ferric-hydrogen peroxide species of cytochrome P450 toward C-H hydroxylation of a radical clock substrate

Density functional calculations were performed in response to the controversies regarding the identity of the oxidant species in cytochrome P450. The calculations were used to gauge the relative C-H hydroxylation reactivity of three potential oxidant species of the enzyme, the high-valent oxo-iron species Compound I (Cpd I), the ferric hydroperoxide Compound 0 (Cpd 0), and the ferric-hydrogen peroxide complex Fe(H(2)O(2)). The results for the hydroxylation of a radical probe substrate, 1, show the following trends: (a) Cpd I is the most reactive species; in its presence the other two reagents will be silent. (b) In the absence of Cpd I, substrate oxidation by Cpd 0 and Fe(H(2)O(2)) will take place via a stepwise mechanism that involves initial O-O homolysis followed by H-abstraction from 1. (c) Cpd 0 will undergo mostly porphyrin hydroxylation and only approximately 15% of substrate oxidation producing mostly the rearranged alcohol, 3 (Scheme 2). (d) Fe(H(2)O(2)) will generate mostly free hydrogen peroxide (uncoupling). A small fraction will perform substrate oxidation and lead mostly to 3. Reactivity probes for these reagents are kinetic isotope effect (KIE) and the product ratio of unrearranged to rearranged alcohols, [2/3]. Thus, for substrate oxidation by Cpd 0 or Fe(H(2)O(2)) KIE will be small, approximately 2, while Cpd I will have large KIE values. Typically both Cpd 0 and Fe(H(2)O(2)) will lead to a [2/3] ratio < 1, while Cpd I will lead to ratios > 1. In addition, the product isotope effect (KIE(2)/KIE(3) not equal 1) is expected from the reactivity of Cpd I.     

How does product isotope effect prove the operation of a two-state "rebound" mechanism in C-H hydroxylation by cytochrome P450?

C-H hydroxylation is a fundamental process. In Nature it is catalyzed by the enzyme cytochrome P450, in a still-debated mechanism that poses a major intellectual challenge for both experiment and theory; currently, the opinions keep swaying between the original single-state rebound mechanism, a two-oxidant mechanism (where ferric peroxide participates as a second oxidant, in addition to the primary active species, the high-valent iron-oxo species), and two-state reactivity (TSR) mechanism (where two spin states are involved). Recent product isotope effect (PIE) measurements for the trans-2-phenyl-methyl cyclopropane probe (1), led Newcomb and co-workers (Newcomb, M.; Aebisher, D.; Shen, R.; Esala, R.; Chandrasena, P.; Hollenberg, P.; Coon, M. J. J. Am. Chem. Soc. 2003, 125, 6064-6065) to rule out TSR in favor of the two-oxidant scenario, since the direction of the PIE was at odds with the one predicted from calculations on methane hydroxylation. The present report describes a density functional theoretical study of C-H hydroxylation of the Newcomb probe, 1, leading to rearranged (3) and unrearranged (2) products. Our study shows that the reaction occurs via TSR in which the high-spin pathway gives dominant rearranged products, whereas the low-spin pathway favors unrearranged products. The calculated PIE(2/3) values based on TSR are found to be in excellent agreement with the experimental data of Newcomb and co-workers. This match between experiment and theory makes a strong case that the reaction occurs via TSR mechanism.     

Dynamics of terrace formation in a nanostructured thin block copolymer film

We have used dynamic self-consistent field (DSCF) theory to investigate the structural evolution of an ABA block copolymer thin film placed between a solid substrate and a free surface. In line with the few existing theoretical studies for pure homopolymers and mixtures, the free interface is introduced by a void component. In our calculations, the free surface experiences surface roughening and eventually the formation of terraces, as in the experiments. The kinetic pathway of the microstructures was compared to findings of an existing detailed experimental study (Knoll, A.; Lyakhova, K. S.; Horvat, A.; Krausch, G.; Sevink, G. J. A.; Zvelindovsky, A. V.; Magerle, R. Nat. Mater. 2004, 3, 886) and was found to be equivalent in detail. This corroborates our assumption in this earlier work that the pathway due to changing film thickness is similar to a pathway due to changing surface energetics. Moreover, our calculations show for the first time that microstructural transitions are a driving force of polymer/air interface curving and the formation of terraces.     

Hydride transfer catalyzed by xylose isomerase: mechanism and quantum effects

We have applied molecular dynamics umbrella-sampling simulation and ensemble-averaged variational transition state theory with multidimensional tunneling (EA-VTST/MT) to calculate the reaction rate of xylose-to- xylulose isomerization catalyzed by xylose isomerase in the presence of two Mg2+ ions. The calculations include determination of the free energy of activation profile and ensemble averaging in the transmission coefficient. The potential energy function is approximated by a combined QM/MM/SVB method involving PM3 for the quantum mechanical (QM) subsystem, CHARMM22 and TIP3P for the molecular mechanical (MM) environment, and a simple valence bond (SVB) local function of two bond distances for the hydride transfer reaction. The simulation confirms the essential features of a mechanism postulated on the basis of kinetics and X-ray data by Whitlow et al. (Whitlow, M.; Howard, A. J.; Finzel, B. C.; Poulos, T. L.; Winborne, E.; Gilliland, G. L. Proteins 1991, 9, 153) and Ringe, Petsko, and coworkers (Labie, A.; Allen, K.-N.; Petsko, G. A.; Ringe, D. Biochemistry 1994, 33, 5469). This mechanism involves a rate-determining 1,2-hydride shift with prior and post proton transfers. Inclusion of quantum mechanical vibrational energy is important for computing the free energy of activation, and quantum mechanical tunneling effects are essential for computing kinetic isotope effects (KIEs). It is found that 85% of the reaction proceeds by tunneling and 15% by overbarrier events. The computed KIE for the ratio of hydride to deuteride transfer is in good agreement with the experimental results. The molecular dynamics simulations reveal that proton and hydride transfer reactions are assisted by breathing motions of the mobile Mg2+ ion in the active site, providing evidence for concerted motion of Mg2+ during the hydride transfer step.     

Proton-shuffle mechanism of O-O activation for formation of a high-valent oxo-iron species of bleomycin

Bleomycins (BLMs) can utilize H2O2 to cleave DNA in the presence of ferric ions. DFT calculations were used to study the mechanism of O-O bond cleavage in the low-spin FeIII-hydroperoxo complex of BLM. The following alternative hypotheses were investigated using realistic structural models: (a) heterolytic cleavage of the O-O bond, generating a Compound I (Cpd I) like intermediate, formally BLM-FeV=O; (b) homolytic O-O cleavage, leading to a BLM-FeIV=O species and an OH* radical; and (c) a direct O-O cleavage/H-abstraction mechanism by ABLM. The calculations showed that (a) is a facile and viable mechanism; it involves acid-base proton reshuffle mediated by the side-chain linkers of BLM, causing thereby heterolytic cleavage of the O-O bond and generation of Cpd I. Formation of Cpd I is found to involve a barrier of 13.3 kcal/mol, which is lower than the barriers in the alternative mechanisms (b and c) that possess respective barriers of 31 and 17 kcal/mol. The so-formed Cpd I species with a radical on the side-chain linker, methylvalerate (V), adjacent to the BLM-FeIV=O complex, resembles the formation of the active species of cytochrome c peroxidase in the Poulos-Kraut proton-shuffle mechanism in heme peroxidases (Poulos, T. L.; Kraut, J. J. Biol. Chem. 1980, 255, 8199-8205). Experimental data are discussed and shown to be in accord with this proposal. It suggests that the high-valence Cpd I species of BLM participates in the DNA cleavage. This is an alternative mechanistic hypothesis to the exclusive reactivity scenario based on ABLM (FeIII-OOH).