Structural insight into the substrate- and dioxygen-binding manner in the catalytic cycle of rieske nonheme iron oxygenase system, carbazole 1,9a-dioxygenase

Yuji Ashikawa,Haruko Noguchi,Hisakazu Yamane,Kengo Inoue,Yusuke Usami,Hideaki Nojiri,Zui Fujimoto

doi:10.1186/1472-6807-12-15

Abstract

BackgroundDihydroxylation of tandemly linked aromatic carbons in a cis-configuration, catalyzed by multicomponent oxygenase systems known as Rieske nonheme iron oxygenase systems (ROs), often constitute the initial step of aerobic degradation pathways for various aromatic compounds. Because such RO reactions inherently govern whether downstream degradation processes occur, novel oxygenation mechanisms involving oxygenase components of ROs (RO-Os) is of great interest. Despite substantial progress in structural and physicochemical analyses, no consensus exists on the chemical steps in the catalytic cycles of ROs. Thus, determining whether conformational changes at the active site of RO-O occur by substrate and/or oxygen binding is important. Carbazole 1,9a-dioxygenase (CARDO), a RO member consists of catalytic terminal oxygenase (CARDO-O), ferredoxin (CARDO-F), and ferredoxin reductase. We have succeeded in determining the crystal structures of oxidized CARDO-O, oxidized CARDO-F, and both oxidized and reduced forms of the CARDO-O: CARDO-F binary complex.ResultsIn the present study, we determined the crystal structures of the reduced carbazole (CAR)-bound, dioxygen-bound, and both CAR- and dioxygen-bound CARDO-O: CARDO-F binary complex structures at 1.95, 1.85, and 2.00 Å resolution. These structures revealed the conformational changes that occur in the catalytic cycle. Structural comparison between complex structures in each step of the catalytic mechanism provides several implications, such as the order of substrate and dioxygen bindings, the iron-dioxygen species likely being Fe(III)-(hydro)peroxo, and the creation of room for dioxygen binding and the promotion of dioxygen binding in desirable fashion by preceding substrate binding.ConclusionsThe RO catalytic mechanism is proposed as follows: When the Rieske cluster is reduced, substrate binding induces several conformational changes (e.g., movements of the nonheme iron and the ligand residue) that create room for oxygen binding. Dioxygen bound in a side-on fashion onto nonheme iron is activated by reduction to the peroxo state [Fe(III)-(hydro)peroxo]. This state may react directly with the bound substrate, or O–O bond cleavage may occur to generate Fe(V)-oxo-hydroxo species prior to the reaction. After producing a cis-dihydrodiol, the product is released by reducing the nonheme iron. This proposed scheme describes the catalytic cycle of ROs and provides important information for a better understanding of the mechanism.

Highlights

Dihydroxylation of tandemly linked aromatic carbons in a cis-configuration, catalyzed by multicomponent oxygenase systems known as Rieske nonheme iron oxygenase systems (ROs), often constitute the initial step of aerobic degradation pathways for various aromatic compounds
Overview Oxy:Fd complex (Oxy):CARDO-F of Pseudomonas resinovorans CA10 (Fd) binary complexes in non-reduced, reduced, and non-reduced CAR-bound forms were called Binary Complex, Complexred, and Complexsubs, respectively, in a previous paper (PDB code 2DE5, 2DE6, and 2DE7) [16], they were respectively renamed Oxy: Fdrest [(1) in Figure 1], Oxy: Fdred (2), and Oxy: FdCAR (2’) in this paper, because the non-reduced complex can be considered to be in the resting state in the catalytic cycle and because the CAR-bound complex was obtained using resting state crystals soaked in a CARcontaining crystallization solution under aerobic conditions
UV-visible absorption spectra of the crystals of Oxy: Fdrest and Oxy: Fdred were previously measured by single-crystal microspectrophotometer [39], suggesting that Rieske clusters in Oxy: Fdrest and Oxy: Fdred were oxidized and reduced, respectively (Figure 2) [16]

Summary

Introduction

Dihydroxylation of tandemly linked aromatic carbons in a cis-configuration, catalyzed by multicomponent oxygenase systems known as Rieske nonheme iron oxygenase systems (ROs), often constitute the initial step of aerobic degradation pathways for various aromatic compounds Because such RO reactions inherently govern whether downstream degradation processes occur, novel oxygenation mechanisms involving oxygenase components of ROs (RO-Os) is of great interest. Many studies have demonstrated that aromatic ring dihydroxylation plays a primary role in the initial step of aerobic bacterial degradation pathways for various natural and synthetic aromatic compounds, including dioxins, polychlorinated biphenyls, and crude oil components such as polycyclic aromatic hydrocarbons and heterocyclic aromatic compounds [1,2,3,4,5,6,7] Such ring dihydroxylation is catalyzed by multicomponent oxygenase systems known as Rieske nonheme iron oxygenase systems (ROs). Further knowledge of the mechanism can lead us to improved application of this important class of enzymes for the bioremediation of various environmentally relevant aromatic compounds, and the synthesis of the chiral precursor compounds [9]

Methods

Results

Conclusion