Comparative studies on the cumene hydroperoxide- and NADPH-supported <i>N</i>-oxidation of 4-chloroaniline by cytochrome <i>P</i>-450

Hlavica, Peter; Golly, Ines; Mietaschk, J

doi:10.1042/bj2120539

Cited by 22 publications

(12 citation statements)

References 26 publications

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“…In short, introduction of the second electron to pro-and eukaryotic P450s is widely accepted to be limiting in at least some drug oxidations [4,227], though circumstantial analyses with a set of members of the mammalian CYP1, CYP2 and CYP3 families disclosed these enzymes to differ considerably in terms of the mechanisms limiting reaction rates. Thus, overall velocities of turnover were found to be dictated by steps ranging from late oxygen activation [228] to hydrogen abstraction [229], C-H bond cleavage [120,146,[230][231][232] and product release [233][234][235] in dependence on the substrate to be oxidized. Clearly, in these cases selective stimulation of electron flow between donor and terminal acceptor is of minor catalytic importance.…”

Section: Conclusion and Future Prospectsmentioning

confidence: 96%

Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

Hlavica¹

2007

CDM

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Based on initial studies with bacterial CYP101A1, a popular concept emerged predicting that substrate-induced low-to-high spin conversion of P450s is universally associated with shifts of the midpoint potential to a more positive value to maximize rates of electron transfer and metabolic turnover. However, evaluation of the plethora of observations with pro- and eukaryotic hemoproteins suggests a caveat as to generalization of this principle. Thus, some P450s are inherently high-spin, so that there is no need for a supportive substrate-triggered impulse to electron flow. With other enzymes, high-spin content is not consonant with reductive activity, and spin transition as such is not essential to sustaining substrate oxidation. Also, with certain proteins the low-spin conformer is reduced as swift as the high-spin entity. Moreover, there is not regularly a linear relationship between high-spin level and anodic shift of the reduction potential. Similarly, in given cases turnover may proceed despite insignificant or even lacking substrate-provoked alterations in the redox behaviour. Thus, folding of the disparate and sometimes conflicting data into a harmonized overall picture is a lingering problem. Apart from direct perturbation of the electrochemical properties, substrate docking may entail changes in enzyme conformation such as to favour productive complexation with redox partners or modulate electron transfer conduits within preformed donor/acceptor adducts, resulting in elevated ease of flow of reducing equivalents. Substrate-steered ordering of the oligomeric aggregation state of P450s is likely to impose steric constraints on heterodimers, causing one component to more readily align with electron carriers. Careful uncovering of electrochemical mechanisms in these systems will be fruitful to tailoring of novel bioenergetic machines and redox chains via redox-inspired protein engineering or molecular Lego, capable of generating products of interest or degrading toxic pollutants. Finally, availability of P450 nanobiochips for high-throughput screening of substrate libraries might expedite drug development.

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Section: Conclusion and Future Prospectsmentioning

confidence: 96%

Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

Hlavica¹

2007

CDM

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“…After a series of these reactions in the heme crevice a hydroxylated molecule will be released from the enzyme. This mechanism, which may be called an intramolecular free-radical mechanism, will explain various aspects of liver microsomal cytochrome P-450 reactions (97,98,115). It seems possible that electron donors and spin-trapping reagents are released from the enzyme as free radicals just after reacting with free-radical species formed by the homolytic cleavage.…”

Section: O--+ H202mentioning

confidence: 99%

Physiological Aspects of Free-Radical Reactions

Yamazaki¹,

Tamura²,

Nakajima³

et al. 1985

Environmental Health Perspectives

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Enzymes which catalyze the formation of free radicals in vitro will catalyze similar reactions in vivo. We believe that the formation of some kinds of free radicals has definite physiological meanings in metabolism. In this sense, the enzymes forming such free radicals are concluded to be in evolutionally advanced states. Elaborated structure and function of enzymes such as horseradish peroxidase and microsomal flavoproteins support the idea. Deleterious and side reactions caused by free radicals are assumed to be minimized in vivo by localizing the reactions, but this assumption should be verified by future studies.

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“…The functional importance of hydroperoxo‐iron or iron‐coordinated hydrogen peroxide as the putative second oxidant in P450 catalysis is also corroborated by studies on heteroatom oxidation. Thus, comparative investigations on the NADPH/O 2 ‐ and cumene hydroperoxide‐driven N‐hydroxylation of 4‐chloroaniline by CYP2B4 indicated discrepancies in the positions of the Soret maxima in the absolute spectra of the individual oxy complexes [116]. Noteworthy, transformation of P450 to the denatured P420 form through treatment with either p ‐chloromercuribenzoate or deoxycholate rendered the hemoprotein a more powerful peroxygenase [116], but disrupted NADPH‐linked monooxygenase activity [117].…”

Section: Evidence From Kinetic Analysis Of P450 Functionmentioning

confidence: 99%

“…Thus, comparative investigations on the NADPH/O 2 ‐ and cumene hydroperoxide‐driven N‐hydroxylation of 4‐chloroaniline by CYP2B4 indicated discrepancies in the positions of the Soret maxima in the absolute spectra of the individual oxy complexes [116]. Noteworthy, transformation of P450 to the denatured P420 form through treatment with either p ‐chloromercuribenzoate or deoxycholate rendered the hemoprotein a more powerful peroxygenase [116], but disrupted NADPH‐linked monooxygenase activity [117]. Hence, resonanace stabilization via the thiolate ‘push effect’ (see above) did not appear to be obligatory when peroxide substituted for reduced cofactor and dioxygen.…”

Section: Evidence From Kinetic Analysis Of P450 Functionmentioning

confidence: 99%

“…Hence, resonanace stabilization via the thiolate ‘push effect’ (see above) did not appear to be obligatory when peroxide substituted for reduced cofactor and dioxygen. While N ‐(4‐chlorophenyl) hydroxylamine was found to be the major metabolic product under mixed‐function conditions, a marked change to the preponderant formation of 1‐chloro‐4‐nitrobenzene was observed when organic hydroperoxide served as the oxygen donor [116]. Involvement in the N‐oxidative process of CmO · () radicals could be safely ruled out owing to insensitivity of the reaction toward radical scavengers, whereas blockage of turnover by cyanide hinted at an iron‐based mechanism [116].…”

Section: Evidence From Kinetic Analysis Of P450 Functionmentioning

confidence: 99%

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Models and mechanisms of O‐O bond activation by cytochrome P450

Hlavica¹

2004

European Journal of Biochemistry

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Cytochrome P450 enzymes promote a number of oxidative biotransformations including the hydroxylation of unactivated hydrocarbons. Whereas the long-standing consensus view of the P450 mechanism implicates a high-valent ironoxene species as the predominant oxidant in the radicalar hydrogen abstraction/oxygen rebound pathway, more recent studies on isotope partitioning, product rearrangements with Ôradical clocksÕ, and the impact of threonine mutagenesis in P450s on hydroxylation rates support the notion of the nucleophilic and/or electrophilic (hydro) peroxo-iron intermediate(s) to be operative in P450 catalysis in addition to the electrophilic oxenoid-iron entity; this may contribute to the remarkable versatility of P450s in substrate modification. Precedent to this mechanistic concept is given by studies with natural and synthetic P450 biomimics. While the concept of an alternative electrophilic oxidant necessitates C-H hydroxylation to be brought about by a cationic insertion process, recent calculations employing density functional theory favour a Ôtwo-state reactivityÕ scenario, implicating the usual ferryl-dependent oxygen rebound pathway to proceed via two spin states (doublet and quartet); state crossing is thought to be associated with either an insertion or a radicalar mechanism. Hence, challenge to future strategies should be to fold the disparate and sometimes contradictory data into a harmonized overall picture.

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Comparative studies on the cumene hydroperoxide- and NADPH-supported N-oxidation of 4-chloroaniline by cytochrome P-450

Cited by 22 publications

References 26 publications

Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

Physiological Aspects of Free-Radical Reactions

Models and mechanisms of O‐O bond activation by cytochrome P450

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