Hole Hopping through Tryptophan in Cytochrome P450

Electron-transfer kinetics have been measured in four conjugates of cytochrome P450 with surface-bound Ru-photosensitizers. The conjugates are constructed with enzymes from Bacillus megaterium (CYP102A1) and Sulfolobus acidocaldarius (CYP119). A W96 residue lies in the path between Ru and the heme in CYP102A1, whereas H76 is present at the analogous location in CYP119. Two additional conjugates have been prepared with (CYP102A1)W96H and (CYP119)H76W mutant enzymes. Heme oxidation by photochemically generated Ru^(3+) leads to P450 compound II formation when a tryptophan residue is in the path between Ru and the heme; no heme oxidation is observed when histidine occupies this position. The data indicate that heme oxidation proceeds via two-step tunneling through a tryptophan radical intermediate. In contrast, heme reduction by photochemically generated Ru+ proceeds in a single electron tunneling step with closely similar rate constants for all four conjugates

Quantum mechanics/molecular mechanics modeling of drug metabolism:Mexiletine N-hydroxylation by cytochrome P450 1A2

The mechanism of cytochrome P450­(CYP)-catalyzed hydroxylation of primary amines is currently unclear and is relevant to drug metabolism; previous small model calculations have suggested two possible mechanisms: direct N-oxidation and H-abstraction/rebound. We have modeled the N-hydroxylation of (<i>R</i>)-mexiletine in CYP1A2 with hybrid quantum mechanics/molecular mechanics (QM/MM) methods, providing a more detailed and realistic model. Multiple reaction barriers have been calculated at the QM­(B3LYP-D)/MM­(CHARMM27) level for the direct N-oxidation and H-abstraction/rebound mechanisms. Our calculated barriers indicate that the direct N-oxidation mechanism is preferred and proceeds via the doublet spin state of Compound I. Molecular dynamics simulations indicate that the presence of an ordered water molecule in the active site assists in the binding of mexiletine in the active site, but this is not a prerequisite for reaction via either mechanism. Several active site residues play a role in the binding of mexiletine in the active site, including Thr124 and Phe226. This work reveals key details of the N-hydroxylation of mexiletine and further demonstrates that mechanistic studies using QM/MM methods are useful for understanding drug metabolism

Fragment-Based Approaches to the Development of Mycobacterium tuberculosis CYP121 Inhibitors.

The essential enzyme CYP121 is a target for drug development against antibiotic resistant strains of Mycobacterium tuberculosis. A triazol-1-yl phenol fragment 1 was identified to bind to CYP121 using a cascade of biophysical assays. Synthetic merging and optimization of 1 produced a 100-fold improvement in binding affinity, yielding lead compound 2 (KD = 15 μM). Deconstruction of 2 into its component retrofragments allowed the group efficiency of structural motifs to be assessed, the identification of more LE scaffolds for optimization and highlighted binding affinity hotspots. Structure-guided addition of a metal-binding pharmacophore onto LE retrofragment scaffolds produced low nanomolar (KD = 15 nM) CYP121 ligands. Elaboration of these compounds to target binding hotspots in the distal active site afforded compounds with excellent selectivity against human drug-metabolizing P450s. Analysis of the factors governing ligand potency and selectivity using X-ray crystallography, UV-vis spectroscopy, and native mass spectrometry provides insight for subsequent drug development.MEK was supported by a Commonwealth (University of Cambridge) Scholarship awarded in conjunction with the Cambridge Commonwealth Trust and Cambridge Overseas Trust. AGC and KJM were supported by grants from the BBSRC (Grant No: BB/I019669/1 and BB/I019227/1). GGJ received funding from the Ogden Trust and the Isaac Newton Trust administered through the University of Cambridge Bursary Scheme. DSCH was supported by a Croucher Cambridge International Scholarship awarded in conjunction between the Croucher Foundation and the Cambridge Overseas Trust. SAH was supported by an Oliphant Cambridge Australia Scholarship (App No: 10132070) awarded by the Cambridge Commonwealth Trust. The contributions of LBM and LPSC were supported by funds from the Francis Crick Institute, which receives its core funding principally from Wellcome Trust, Cancer Research UK, and the UK Medical Research Council (to LPSC - MC_UP_A253_1111) and funds from FAPESP, CNPq and CAPES-PDSE (to LBM - 2011/21232-1, 140079/2013-0, 99999.003125/2014-09).This is the final version of the article. It first appeared from the American Chemical Society via http://dx.doi.org/10.1021/acs.jmedchem.6b0000

Oxygen Activation and Radical Transformations in Heme Proteins and Metalloporphyrins

As a result of the adaptation of life to an aerobic environment, nature has evolved a panoply of metalloproteins for oxidative metabolism and protection against reactive oxygen species. Despite the diverse structures and functions of these proteins, they share common mechanistic grounds. An open-shell transition metal like iron or copper is employed to interact with O_2 and its derived intermediates such as hydrogen peroxide to afford a variety of metal–oxygen intermediates. These reactive intermediates, including metal-superoxo, -(hydro)peroxo, and high-valent metal–oxo species, are the basis for the various biological functions of O_2-utilizing metalloproteins. Collectively, these processes are called oxygen activation. Much of our understanding of the reactivity of these reactive intermediates has come from the study of heme-containing proteins and related metalloporphyrin compounds. These studies not only have deepened our understanding of various functions of heme proteins, such as O2 storage and transport, degradation of reactive oxygen species, redox signaling, and biological oxygenation, etc., but also have driven the development of bioinorganic chemistry and biomimetic catalysis. In this review, we survey the range of O_2 activation processes mediated by heme proteins and model compounds with a focus on recent progress in the characterization and reactivity of important iron–oxygen intermediates. Representative reactions initiated by these reactive intermediates as well as some context from prior decades will also be presented. We will discuss the fundamental mechanistic features of these transformations and delineate the underlying structural and electronic factors that contribute to the spectrum of reactivities that has been observed in nature as well as those that have been invented using these paradigms. Given the recent developments in biocatalysis for non-natural chemistries and the renaissance of radical chemistry in organic synthesis, we envision that new enzymatic and synthetic transformations will emerge based on the radical processes mediated by metalloproteins and their synthetic analogs

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

Altered spin state equilibrium in the T309V mutant of cytochrome P450 2D6: a spectroscopic and computational study

Cytochrome P450 2D6 (CYP2D6) is one of the most important cytochromes P450 in humans. Resonance Raman data from the T309V mutant of CYP2D6 show that the substitution of the conserved I-helix threonine situated in the enzyme’s active site perturbs the heme spin equilibrium in favor of the six-coordinated low-spin species. A mechanistic hypothesis is introduced to explain the experimental observations, and its compatibility with the available structural and spectroscopic data is tested using quantum-mechanical density functional theory calculations on active-site models for both the CYP2D6 wild type and the T309V mutant

Disease concepts and treatment by tribal healers of an Amazonian forest culture

<p>Abstract</p> <p>Background</p> <p>The extensive medicinal plant knowledge of Amazonian tribal peoples is widely recognized in the scientific literature and celebrated in popular lore. Despite this broad interest, the ethnomedical systems and knowledge of disease which guide indigenous utilization of botanical diversity for healing remain poorly characterized and understood. No study, to our knowledge, has attempted to directly examine patterns of actual disease recognition and treatment by healers of an Amazonian indigenous culture.</p> <p>Methods</p> <p>The establishment of traditional medicine clinics, operated and directed by elder tribal shamans in two remote Trio villages of the Suriname rainforest, presented a unique investigational opportunity. Quantitative analysis of clinic records from both villages permitted examination of diseases treated over a continuous period of four years. Cross-cultural comparative translations were articulated of recorded disease conditions through ethnographic interviews of elder Trio shamans and a comprehensive atlas of indigenous anatomical nomenclature was developed.</p> <p>Results</p> <p>20,337 patient visits within the period 2000 to 2004 were analyzed. 75 disease conditions and 127 anatomical terms are presented. Trio concepts of disease and medical practices are broadly examined within the present and historical state of their culture.</p> <p>Conclusion</p> <p>The findings of this investigation support the presence of a comprehensive and highly formalized ethnomedical institution within Trio culture with attendant health policy and conservation implications.</p