Abstract
The cytochromes P450 are heme-dependent enzymes that catalyze many vital reaction processes in the human body related to biodegradation and biosynthesis. They typically act as mono-oxygenases; however, the recently discovered P450 subfamily TxtE utilizes O2 and NO to nitrate aromatic substrates such as L-tryptophan. A direct and selective aromatic nitration reaction may be useful in biotechnology for the synthesis of drugs or small molecules. Details of the catalytic mechanism are unknown, and it has been suggested that the reaction should proceed through either an iron(III)-superoxo or an iron(II)-nitrosyl intermediate. To resolve this controversy, we used stopped-flow kinetics to provide evidence for a catalytic cycle where dioxygen binds prior to NO to generate an active iron(III)-peroxynitrite species that is able to nitrate l-Trp efficiently. We show that the rate of binding of O2 is faster than that of NO and also leads to l-Trp nitration, while little evidence of product formation is observed from the iron(II)-nitrosyl complex. To support the experimental studies, we performed density functional theory studies on large active site cluster models. The studies suggest a mechanism involving an iron(III)-peroxynitrite that splits homolytically to form an iron(IV)-oxo heme (Compound II) and a free NO2 radical via a small free energy of activation. The latter activates the substrate on the aromatic ring, while compound II picks up the ipso-hydrogen to form the product. The calculations give small reaction barriers for most steps in the catalytic cycle and, therefore, predict fast product formation from the iron(III)-peroxynitrite complex. These findings provide the first detailed insight into the mechanism of nitration by a member of the TxtE subfamily and highlight how the enzyme facilitates this novel reaction chemistry.
Highlights
The cytochromes P450 (P450s or CYPs) are vital enzymes for human health with, e.g., key functions in the liver related to the metabolism of drugs and xenobiotics; and as such their activity and reactivity is of interest to drug delivery and development.[1−5] In addition, they catalyze key steps in the biosynthesis of natural products and hormones, including estrogen.[6−10] Their diverse reactivities and ability to activate inert C−H bonds have resulted in the P450s gaining significant interest due to their potential as biocatalysts in the synthesis of high value chemicals.[11−13] factors including requirement for expensive redox partners, as well as a lack of knowledge of many facets of their structure and activity, has limited their applications
The P450 catalytic cycle for a mono-oxygenation reaction starts with substrate binding in the active site close to the iron(III)-heme in its resting state, followed by release of water from the sixth ligand position of the heme and resulting in conversion of iron from low spin (LS) to high spin (HS)
This is followed by a single electron reduction of the heme from HS iron(III) to iron(II) using a redox partner, enabling molecular oxygen binding to form the iron(III)-superoxo complex.[1−3,17−19,67,68] A further reduction and two protonation steps result in the formation of Compound I (CpdI), an iron(IV)-oxo heme cation radical species that is the active oxidant in oxygen atom transfer reactions
Summary
The cytochromes P450 (P450s or CYPs) are vital enzymes for human health with, e.g., key functions in the liver related to the metabolism of drugs and xenobiotics; and as such their activity and reactivity is of interest to drug delivery and development.[1−5] In addition, they catalyze key steps in the biosynthesis of natural products and hormones, including estrogen.[6−10] Their diverse reactivities and ability to activate inert C−H bonds have resulted in the P450s gaining significant interest due to their potential as biocatalysts in the synthesis of high value chemicals.[11−13] factors including requirement for expensive redox partners, as well as a lack of knowledge of many facets of their structure and activity, has limited their applications. O2 is reduced to catalyze diverse reactions, including aliphatic or aromatic hydroxylation, epoxidation and sulfoxidation, in addition to pathways leading to desaturation, C−C bond cleavage and O-dealkylation.[20,21]. Two novel P450 subfamilies were discovered that are able to regiospecifically nitrate aromatic amino acids including L-tryptophan and L-tyrosine.[22−31] The P450 TxtE isozyme activates L-tryptophan at the 4-position, while the P450 RufO isozyme binds L-tyrosine and adds an NO2 group to the 3-position. TxtE catalyzed nitration is part of the biosynthetic pathway of the phytotoxin thaxtomin in Streptomyces scabies, while tyrosine nitration catalyzed by RufO is a key step in the biosynthesis of rufomycin, a natural product peptide antibiotic with activity against Mycobacterium tuberculosis. The L-Phe residue in the N,Ndimethyl-diketopiperazine intermediate in thaxtomin biosynthesis is hydroxylated at the Cα and aromatic ring positions by the bifunctional P450 TxtC.[22−24,32−34] The substrate nitration
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