Theoretical Investigations On The Metabolic Mechanism Of Different Substrates Mediated By Cytochrome P450Enzymes

As one of the most effective defenders in vivo, cytochrome P450enzymes(CYP450) play a key role in many biochemical reactions. The catalytic role of P450depends on the environment. Under anaerobic conditions, P450s can catalyze somereductive metabolism, such as the reductive dechloridation of carbon tetrachloride.While under aerobic conditions, they can achieve some oxidative metabolism throughactivating oxygen, e.g., the hydroxylation of C-H bond, amine oxidation, etc.However, some details of reaction mechanisms are still unclear. Thus, in the presentwork, density functional theory (DFT) calculations were performed to elaborate themechanisms of oxidation and reduction mediated by P450s, which can provide furthertheoretical insights into the protective role of P450in vivo. The content mainlyincludes the following two parts:1. Theoretical study on the reduction of carbon tetrachloridecatalyzed by cytochrome P450enzymesCarbon tetrachloride (CCl4), as the simplest perhalogenated alkane, is ofconsiderable industrial importance. However, both of its production and usageare declined for the recognition of the toxicity. Experimental studies haveindicated that the reductive metabolism of CCl4to reactive intermediates byP450enzymes is an essential prerequisite for its toxicity. However, themechanistic details remain elusive.The anaerobic metabolism of CCl4by P450enzymes was investigated usingquantum chemical calculations. It was found that under anaerobic conditions,the substrate CCl4might undergo one or two subsequent one-electronreductions to generate different reactive metabolites, trichloromethyl radical (·CCl3) and dichlorocarbene (CCl2) respectively. Meanwhile, it was thereduced ferrous haem complex rather than the unreduced ferric haem complexthat could directly achieve such reductions. Based on the formation of·CCl3, afurther one-electron reduction could take place with the assistance of a protonto yield CCl2, i.e., a further reductive dechloridation of·CCl3could take placevia a novel SE3mechanism. In addition, the·CCl3species was capable ofbinding covalently to the meso-carbon atom of the prosthetic group, leading tothe suicidal destruction of P450enzymes. Whereas, the CCl2species wasinvolved in the CCl4-dependent reversible P450inhibition. It is obvious thatour results are consistent with the experimental findings. Our findings arehelpful to protecting organisms from the halogenated compounds.2. Theoretical studies on the catalytic oxidation by cytochrome P450enzymes(1) The4-hydroxylation of all-trans-retinoic acid mediated by cytochrome P4502C8: A theoretical investigationAll-trans-retinoic acid (atRA) is the most stable metabolite of vitamin A, and hasgreat effects on various physiological processes as a kind of signaling molecule.Experimental results indicate that the4-hydroxylation of atRA catalyzed by CYP2C8is the primary metabolic pathway. However, the binding mode between atRA andCYP2C8as well as the corresponding metabolic mechanism are still unclear.In present work, the binding mode between atRA and CYP2C8was initiallyinvestigated by molecular docking. Then, density functional theory (DFT)calculations were employed to research the reaction mechanism of atRA4-hydroxylation mediated by CYP2C8. Docking results indicate that there are twodifferent binding modes between atRA and CYP2C8. One is there is a salt bridgeinteraction between the anionic carboxylate tail of atRA and the protein environment,while there is no such salt bridge interaction in the other binding mode. Our DFT calculations revealed that such salt bridge interaction has obvious effects on thereaction mechanism of atRA4-hydroxylation. In the former binding mode with theexistence of the salt bridge interaction between atRA and CYP2C8, the C–H bondactivation proceeds via a normal hydrogen atom transfer (HAT) mechanism; in thelatter one without this salt bridge interaction, however, the C–H bond activation isachieved via a stepwise electron transfer and hydrogen atom transfer (ET/HAT)mechanism. These findings enrich the mechanism patterns of C-H bond activationcatalyzed by metalloenzymes and their biomimetics.(2) Oxidation of4-alkyl substituted1,4-dihydropyridines analogs mediated bycytochrome P450: A theoretical investigation1,4-Dihydropyridines (DHPs) have been established as one of the first-line drugsfor a variety of diseases. Experimental findings reveal that the4-alkyl substitutedDHP analogs exhibit inhibitory activity toward certain cytochrome P450enzymes(P450) during their biotransformation by these enzymes, which is calledmechanism-based inactivation. Though much experimental evidence had proved theessentiality of alkyl radical for P450inactivation, the underlying mechanism of suchradical formation remains elusive.In the present study, density functional calculations were employed to investigatethe dealkylation mechanism of4-alkyl substituted DHPs mediated by P450.Interestingly, our results indicate that the initial N-H activation proceeds via aproton-coupled electron transfer (PCET) process, not via the long presumed hydrogenatom transfer (HAT) mechanism or the stepwise electron transfer/proton transfer(ET/PT) one, to form the amino radical and Cpd II complex. Subsequently, homolyticC-C bond cleavage at the4-position occurs to afford the product complex involvingan alkyl radical, an aromatic pyridine derivative. This C-C cleavage step israte-determining for the whole metabolic reaction, to which aromatization contributesas an essential intrinsic driving force. The4-substituent groups induce differences in activation energy barriers and in the transition state structures of hydrogen abstractionprocess. The substrate reactivity correlates well with the stability of the generatedalkyl radical as well as the C-C bond dissociation energy. Understanding themetabolic mechanism of DHP analogues is indeed essential for the related design ofsafer and more efficient drugs. Furthermore, our findings also enrich the mechanisticpicture of amine oxidation catalyzed by P450.(3) Detoxification of1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine bycytochrome P450: A theoretical investigation1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a neurotoxic byproductformed in the chemical synthesis of heroin, is able to induce Parkinson-like syndromein humans and in animals. Experimental results indicate that it can be detoxified bycytochrome P450enzymes (P450s). There are two types of detoxification routes,N-demethylation to form4-phenyl-1,2,3,6-tetrahydropyridine (PTP) and aromatichydroxylation to generate4-(4’-hydroxyphenyl)-1-methyl-1,2,3,6-tetrahydropyridine(MPTP-OH). However, the mechanisms of both detoxification routes are unclear.In the present study, these two detoxification reactions are investigatedtheoretically using hybrid density functional calculations. Quantum chemical resultsreveal that the N-demethylation proceeds via an initial rate-determining hydrogenatom transfer (HAT) step and a subsequent O-rebound to yield the carbinolanilineintermediate. The generated carbionlaniline degrades in a non-enzymatic aqueousenvironment with the assistance of three water molecules to finally form amine andhydrated methanal. While the aromatic hydroxylation proceeds via a rate-limitingaddition of Cpd I to substrate mainly through a side-on approach and a subsequentproton shuttle to form the phenol product. A comparison of the energy barriers forboth routes indicates that the N-demethylation reaction is thermodynamically morefavorable than the aromatic hydroxylation process. This is in good agreement with theexperimental product distribution, viz., the N-demethylation product PTP is more than the aromatic hydroxylation product MPTP-OH. Taken together, these observationsnot only enrich our knowledge on the mechanistic details of the N-dealkylation andthe aromatic hydroxylation by P450s, but also provide certain insights into themetabolism of other analogous toxin.