Theoretical Studies of the Mechanism of N-Hydroxylation of Primary Aromatic Amines by Cytochrome P450 1A2: Radicaloid or Anionic?

Abstract

Primary aromatic and heteroaromatic amines are notoriously known as potential mutagens and carcinogens. The major event of the mechanism of their mutagenicity is N-hydroxylation by P450 enzymes, primarily P450 1A2 (CYP1A2), which leads to the formation of nitrenium ions that covalently modify nucleobases of DNA. Energy profiles of the NH bond activation steps of two possible mechanisms of N-hydroxylation of a number of aromatic amines by CYP1A2, radicaloid and anionic, are studied by dispersion-corrected DFT calculations. The classical radicaloid mechanism is mediated by H-atom transfer to the electrophilic ferryl-oxo intermediate of the P450 catalytic cycle (called Compound I or Cpd I), whereas the alternative anionic mechanism involves proton transfer to the preceding nucleophilic ferrous-peroxo species. The key structural features of the catalytic site of human CYP1A2 revealed by X-ray crystallography are maintained in calculations. The obtained DFT reaction profiles and additional calculations that account for nondynamical electron correlation suggest that Cpd I has higher thermodynamic drive to activate aromatic amines than the ferrous-peroxo species. Nevertheless, the anionic mechanism is demonstrated to be consistent with a variety of experimental observations. Thus, energy of the proton transfer from aromatic amines to the ferrous-peroxo dianion splits aromatic amines into two classes with different mutagenicity mechanisms. Favorable or slightly unfavorable barrier-free proton transfer is inherent in compounds that undergo nitrenium ion mediated mutagenicity. Monocyclic electron-rich aromatic amines that do not follow this mutagenicity mechanism show significantly unfavorable proton transfer. Feasibility of the entire anionic mechanism is demonstrated by favorable Gibbs energy profiles of both chemical steps, NH bond activation, and NO bond formation. Taken together, results suggest that the N-hydroxylation of aromatic amines in CYP1A2 undergoes the anionic mechanism. Possible reasons for the apparent inability of Cpd I to activate aromatic amines in CYP1A2 are discussed.

An attempt to evaluate the effect of proton-coupled electron transfer on the H-abstraction step of the reaction between 1,1-dimethylhydrazine and cytochrome P450 compound I

“…The SNO is also seen to have only a small distribution at the oxo site. An analogous significant spin accumulation at the amine nitrogen was observed recently by Ripa et al for the reaction of an aromatic amine [33]. The distribution of the SNO for UDMH suggests that an electron is transferred from the lone pair orbital to the a 2u orbital, while a proton is transferred from the UDMH nitrogen to the oxo unit.…”

Heteroatom–Heteroatom Bond Formation in Natural Product Biosynthesis

“…82 Nucleophilic attack of the distal hydroperoxo oxygen of 23 by the nitrogen atom of this anionic intermediate affords the corresponding hydroxylamine. This alternative mechanism has also been previously proposed for N–O bond formation by NOS based on inconsistent experimental evidence for the involvement of 24 as the active oxygenating species 99 and ArNH 2 hydroxylase modeling studies by Shamovsky et al 82 As mentioned above, whether N- oxygenation proceeds via radical or anionic pathway is currently unresolved for P450-dependent ArNH 2 hydroxylases. Nonetheless, these potential mechanisms should provide adequate background knowledge for discussing heme-based N -hydroxylation and oxime installation (Section 2.2) in natural product biosynthesis.…”

Nitro-Group-Containing Drugs

“…It was concluded that structural alterations in ArNH 2 moieties enabling the disruption of the geometric compatibility with CYP1A2 either hinder the proton abstraction or strongly destabilize the nitrenium ion, thus preventing genotoxicity. The authors continued their investigations on the mechanistic aspects of N-hydroxylation by P450 enzymes leading to mutagenicity and deduced logical strategies to avoid genotoxicity issues by hindering N-hydroxylation . It is well-known that hydroxylamino, nitro, and nitroso groups are able to generate amine groups by metabolic conversion.…”