Coupling and uncoupling mechanisms in the methoxythreonine mutant of cytochrome P450cam: a quantum mechanical/molecular mechanical study

The Thr252 residue plays a vital role in the catalytic cycle of cytochrome P450cam during the formation of the active species (Compound I) from its precursor (Compound 0). We investigate the effect of replacing Thr252 by methoxythreonine (MeO-Thr) on this protonation reaction (coupling) and on the competing formation of the ferric resting state and H2O2 (uncoupling) by combined quantum mechanical/molecular mechanical (QM/MM) methods. For each reaction, two possible mechanisms are studied, and for each of these the residues Asp251 and Glu366 are considered as proton sources. 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. 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 Compound I), but at lower reaction rates compared with the wild-type enzyme

Two distinct electrophilic oxidants effects hydroxylation in cytochrome P-450-catalyzed reactions [20]

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Cytochrome P450-catalyzed hydroxylation of mechanistic probes that distinguish between radicals and cations. Evidence for cationic but not for radical intermediates

Oxidation of the mechanistic probes trans,trans-2-methoxy-3- phenylmethylcyclopropane and methylcubane by six cytochrome P450 isozymes has been studied. The probes differentiate between radical and cationic species in that different structural rearrangements occur for the two types of intermediates. The P450 isozymes are the phenobarbital-inducible hepatic isozymes P450 2B1 (from rat) and P450 2B4 (from rabbit), the expressed truncated isozymes P450 Δ2B4 and P450 Δ2E1 (ethanol-inducible, from rabbit), and mutants of the latter two in which an active site threonine was replaced with alanine, Δ2B4 T302A, and Δ2E1 T303A. Cationic rearrangement products were found from both probes. Oxidations of trans,trans-2-methoxy-3- phenylmethylcyclopropane gave small amounts of radical-derived rearrangement products indicating that hydroxylation occurs via insertion reactions with transition state lifetimes in the 80-200 fs range. A mechanistic description of cytochrome P450-catalyzed hydroxylations that is in accord with the present and previous radical probe results is presented. This description incorporates the recent demonstrations that two electrophilic oxidants are produced in the natural course of P450 oxidation reactions and that both electrophilic oxidant forms can effect hydroxylation reactions. Following production of a peroxo-iron species, protonation gives a hydroperoxo-iron species. Protonation of the hydroperoxo-iron species gives an iron-oxo species and water. Hydroxylations by both the hydroperoxo-iron and iron-oxo species occur by insertion reactions. The hydroperoxo-iron species inserts the elements of OH+ producing protonated alcohol products that can react in solvolysis-type reactions to give cationic rearrangement products. The iron- oxo species reacts by insertion of an oxygen atom.link_to_subscribed_fulltex

Quantitative analysis of therapeutic drugs in dried blood spot samples by paper spray mass spectrometry: an avenue to therapeutic drug monitoring

A method is presented for the direct quantitative analysis of therapeutic drugs from dried blood spot samples by mass spectrometry. The method, paper spray mass spectrometry, generates gas phase ions directly from the blood card paper used to store dried blood samples without the need for complex sample preparation and separation; the entire time for preparation and analysis of blood samples is around 30 s. Limits of detection were investigated for a chemically diverse set of some 15 therapeutic drugs; hydrophobic and weakly basic drugs, such as sunitinib, citalopram, and verapamil, were found to be routinely detectable at approximately 1 ng/mL. Samples were prepared by addition of the drug to whole blood. Drug concentrations were measured quantitatively over several orders of magnitude, with accuracies within 10% of the expected value and relative standard deviation (RSD) of around 10% by prespotting an internal standard solution onto the paper prior to application of the blood sample. We have demonstrated that paper spray mass spectrometry can be used to quantitatively measure drug concentrations over the entire therapeutic range for a wide variety of drugs. The high quality analytical data obtained indicate that the technique may be a viable option for therapeutic drug monitorin

Geometric and electronic structure and reactivity of a mononuclear 'side-on' nickel(III)-peroxo complex

Metal-dioxygen adducts, such as metal-superoxo and -peroxo species, are key intermediates often detected in the catalytic cycles of dioxygen activation by metalloenzymes and biomimetic compounds. The synthesis and spectroscopic characterization of an end-on nickel(II)-superoxo complex with a 14-membered macrocyclic ligand was reported previously. Here we report the isolation, spectroscopic characterization, and high-resolution crystal structure of a mononuclear side-on nickel(III)-peroxo complex with a 12-membered macrocyclic ligand, [Ni(12-TMC)(O(2))](+) (1) (12-TMC = 1,4,7,10-tetramethyl-1,4,7,10-tetraazacyclododecane). In contrast to the end-on nickel(II)-superoxo complex, the nickel(III)-peroxo complex is not reactive in electrophilic reactions, but is capable of conducting nucleophilic reactions. The nickel(III)-peroxo complex transfers the bound dioxygen to manganese(II) complexes, thus affording the corresponding nickel(II) and manganese(III)peroxo complexes. Our results demonstrate the significance of supporting ligands in tuning the geometric and electronic structures and reactivities of metal-O(2) intermediates that have been shown to have biological as well as synthetic usefulness in biomimetic reactions

Asymmetric reduction of activated alkenes using an enoate reductase from Gluconobacter oxydans

Richter N, Gröger H, Hummel W. Asymmetric reduction of activated alkenes using an enoate reductase from Gluconobacter oxydans. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 2011;89(1):79-89.A recombinant enoate reductase from Gluconobacter oxydans was heterologously expressed, purified, characterised and applied in the asymmetric reduction of activated alkenes. In addition to the determination of the kinetic properties, the major focus of this work was to utilise the enzyme in the biotransformation of different interesting compounds such as 3,5,5-trimethyl-2-cyclohexen-1,4-dione (ketoisophorone) and (E/Z)-3,7-dimethyl-2,6-octadienal (citral). The reaction proceeded with excellent stereoselectivities (>99% ee) as well as absolute chemo-and regioselectivity, only the activated C-C bond of citral was reduced by the enoate reductase, while non-activated C-C bond and carbonyl moiety remained untouched. The described strategy can be used for the production of enantiomerically pure building blocks, which are difficult to prepare by chemical means. In general, the results show that the investigated enoate reductase is a promising catalyst for the use in asymmetric C-C bond reductions