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

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.

Structural Basis of the Divergent Oxygenation Reactions Catalyzed by the Rieske Nonheme Iron Oxygenase Carbazole 1,9a-Dioxygenase

“…3C). This indicates that the oxygen species binds side on, as it does in other ROs, including WT Oxy (26,44,45). As with I262V-CAR without oxygen, the shift of Ile184 by substrate binding is not observed in the crystal structure of I262V-CAR with oxygen.…”

“…The crystal structure of I262V-CAR with oxygen species close to the mononuclear iron was obtained after soaking the crystals in sodium dithionite and CAR and then exposing them to oxygen for 10 min as described by Ashikawa et al (26). (Fig.…”

“…The position of the dioxygen was calculated on the basis of the assumption that the dioxygen binds to the mononuclear iron at the active site of each Oxy derivative in a manner similar to that observed in the Oxy-Fd-CAR-O 2 complex (PDB accession no. 3VMI) (26). The coordinates of the Oxy-Fd-CAR-O 2 complex were translated and rotated to minimize the root mean square deviation (RMSD) value between the residues coordinating the active-site iron.…”

“…5C), which also gives a relatively high yield of the lateral dioxygenation product. To gain insight into the oxygenation mechanism, we modeled Q282Y-CAR chain C with oxygen on the basis of the crystal structure of WT-CAR with oxygen species (26). C-1 and C-2 were the closest carbon atoms in CAR to modeled oxygen atoms O-1 and O-2, respectively, in chain C of Q282Y-CAR, whereas the closest carbon atoms were C-9a and C-1, respectively, in WT-CAR (Fig.…”

“…strain J3, Novosphingobium sp. strain KA1, and Nocardioides aromaticivorans IC177 have been structurally characterized in detail (T. Umeda et al, unpublished data) (22)(23)(24)(25)(26). CARDO catalyzes dioxygenation at angular (C-9a) and adjacent (C-1) carbon atoms to produce an unstable hemiaminal (1-hydro-1,9a-dihydroxycarbazole) that is spontaneously cleaved to form 2=-aminobiphenyl-2,3-diol ( Fig.…”