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 [1–7]. Such ring dihydroxylation is catalyzed by multicomponent oxygenase systems known as Rieske nonheme iron oxygenase systems (ROs). A member of ROs, called as aromatic ring-hydroxylating dioxygenase, catalyze the incorporation of both oxygen atoms of molecular dioxygen as two hydroxyl groups to tandemly linked carbon atoms of an aromatic ring in a cis-configuration. The introduction of both oxygen atoms into aromatic substrates is a key reaction to initiate the transformation of relatively unreactive aromatic compounds, and novel oxygen activation and the addition mechanism of ROs is a subject of great interest. Despite substantial progress in understanding the structure, regulation, and kinetics of ROs in recent studies , no consensus exists on the chemical steps in the catalytic cycle. Further knowledge of the mechanism can lead us to improved application of this important class of enzymes for not only the bioremediation of various environmentally relevant aromatic compounds, but also the synthesis of the chiral precursor compounds .
The multicomponent systems of ROs typically comprise the terminal oxygenase component and the electron transport chain, which consists of either one or two separate proteins: reductase alone or ferredoxin and reductase in combination . Biochemical and/or structural properties of several terminal oxygenase components of RO (RO-Os) have demonstrated that RO-Os have a mononuclear iron site and a Rieske-type [2Fe-2 S] cluster (Rieske cluster) in common . The mononuclear iron site activates dioxygen for reaction with the substrate, and the Rieske cluster transfers electrons to the mononuclear iron site during the catalytic cycle. The structures of RO-Os reveal that each α-subunit contains a mononuclear iron site and a Rieske cluster separated by a distance of approximately 45 Å. However, the functional pair appears to be constituted by the mononuclear iron and the Rieske cluster in neighboring subunits located within ~12 Å distance. The nonheme iron site is coordinated by two histidine residues and one carboxylate residue called the 2-His-1-carboxylate facial triad, which is a versatile platform of nonheme iron-containing oxygenases including ROs . In most RO-O structures, the carboxylate residue is coordinated to the iron in a bidentate manner [12–17], although monodentate structures have also been reported [18–22]. Additional water molecules are found to be coordinated to the nonheme iron, forming a five- or six-coordinate catalytic ferrous site, depending on the number of water molecules. The Rieske cluster has two iron and two sulfides; one iron is coordinated by two histidine residues, and the other is coordinated by two cysteine residues. The nonheme iron site and the Rieske cluster can be bridged by a conserved aspartate residue located at the subunit interface, which may be important for electron transfer  and regulation  during the catalytic cycle.
The metal site composition of RO-Os generally suggests that three oxidation states can stably exist: both metal sites are oxidized, the Rieske cluster is oxidized and mononuclear iron is reduced, and two metal sites are reduced. Another potential oxidation state with a reduced Rieske cluster and oxidized mononuclear iron is not stable because of the relative redox potentials of the metal sites. Spectroscopic studies report that substrate binding and dioxygen activation occur at the mononuclear iron center for naphthalene 1,2-dioxygenase (NDO) and benzoate 1,2-dixoygenase (BZDO) [25–28]. Wolf et al. demonstrated that the oxygenase component of BZDO (BZDO-O) alone in the fully reduced state could activate dioxygen and generate the cis-dihydrodiol product in a single turnover reaction . Following the single turnover of BZDO-O, this protein was found to be in the fully oxidized state with most of the products retained in the active site, suggesting that the mononuclear iron and Rieske cluster each provide one of two electrons required by the reaction stoichiometry. The same was found to be true for the oxygenase component of NDO (NDO-O), which produced an essentially stoichiometric yield of product in a single turnover based on the number of populated mononuclear irons present . The binding of the dioxygen to the ferrous catalytic site was regulated by both substrate binding and Rieske cluster reduction, implying that a structural change occurs in the vicinity of the ferrous iron site during the catalytic cycle. Indeed, this was demonstrated from crystallography for an allosteric effect of the Rieske cluster in 2-oxoquinoline 8-monooxygenase (OMO) . Martins et al. showed that reduction of the Rieske cluster modulated the ferrous nonheme iron through a chain of structural changes across the subunit interface, resulting in the movement of the nonheme iron and its ligand histidine away from a substrate-binding site . The ferrous nonheme iron changes from five- to six-coordinate, which was also found for NDO , BZDO , and phthalate 4,5-dioxygenase (PDO) [24, 29]. However, the crystal structures of the ferrous nonheme iron in NDO  and carbazole (CAR) 1,9a-dioxygenase (CARDO)  with a reduced Rieske cluster are five-coordinate, which differs from OMO-O. In addition, binding of the substrate to the active site of RO-Os is a key step in regulating the reactivity toward dioxygen. Until now, spectroscopic studies on NDO-O and the oxygenase component of PDO (PDO-O) found that the NDO-O (Rieske cluster is either oxidized or reduced) and PDO-O with the oxidized Rieske cluster showed conversion of ferrous iron from six- to five-coordinate upon binding of the substrate to the active site [30–32]. However, the crystal structures of the nonheme iron site in the native and substrate-bound forms of NDO-O did not indicate a significant change in the coordination environment, maintaining five-coordinate structures . The five-coordinate nonheme iron in the oxygenase component of OMO (OMO-O) with the oxidized Rieske cluster also did not show a change in coordination number even when a substrate was bound to the active site . On the other hand, Daughtry et al. have reported that five-coordinate geometry leaves one site open for oxygen binding and activation or the single solvent ligand could be displaced by oxygen binding, compared between the unliganded and the substrate binding structures of stachydrine demethylase . As noted above, spectroscopy and current crystal structures have provided different perspectives of the catalytic mechanism involved in ROs.
The observation that two electrons present in the Rieske cluster and the mononuclear iron of NDO-O and BZDO-O are used during a single turnover supports the mechanism by which dioxygen is activated by reduction to the peroxo state after binding to the active site iron [Fe(III)-(hydro)peroxo]. Following dioxygen activation, this state is considered to react directly with bound substrate or to result in O–O bond cleavage generating Fe(V)-oxo-hydroxo species prior to reaction [8, 34, 35]. An alternative mechanism involves an additional step in which one electron is donated to the initially formed Fe(III)-(hydro)peroxo to produce the Fe(II)-(hydro)peroxo intermediate, which could react either directly or as Fe(IV)-oxo-hydroxo species after O–O bond cleavage. The latter mechanism is supported by studies that used PDO-O in which the mononuclear iron was left in Fe(II) state after yielding product [29, 36], while single turnover occurred as observed for NDO-O and BZDO-O. These studies suggest that an additional electron was transferred to the nonheme iron at some stage of the catalytic cycle from a Rieske cluster in a neighboring α-subunit.
To understand the molecular basis of the catalytic cycle, Karlsson et al. formed complex crystals of NDO-O with substrate, dioxygen, substrate plus dioxygen, or product and determined their structures by X-ray crystallography . The complex structure with substrate and dioxygen showed that dioxygen was bound to the nonheme iron in a side-on fashion, which allowed each oxygen atom to attack the neighboring aromatic carbon atom from the same face of a planar aromatic ring, producing a cis-dihydrodiol. In addition, based on the Fe–O bond lengths in the crystal structure, the complex is likely to be an Fe(III)-(hydro)peroxo species. However, conformational changes by the binding of substrate and/or oxygen in the above-mentioned crystal structures were hardly observed. In these situations, determining whether conformational changes including coordination conversion at the active site occur by binding of substrate and/or oxygen is very important for a better understanding of the catalytic mechanisms in ROs.
We have investigated the enzymatic function of CARDOs, members of ROs, from various bacteria: Pseudomonas resinovorans CA10, Janthinobacterium sp. J3, Novosphingobium sp. KA1, and Nocardioides aromaticivorans IC177 [7, 37]. All CARDOs commonly consist of three components: terminal oxygenase (CARDO-O), ferredoxin (CARDO-F), and ferredoxin reductase (CARDO-R). We determined the structures of CARDO-Os of J3 and IC177 [15, 17] and CARDO-Fs of CA10 and IC177 [17, 38]. In addition, the structures of the CARDO-O: CARDO-F binary complex were determined in non-reduced, reduced, and CAR-bound forms using CARDO-O of J3 and CARDO-F of CA10 . These structures provide a structure-based interpretation of inter-component electron transfer between two Rieske clusters of ferredoxin and oxygenase in ROs as well as, conformational changes upon CAR binding, which result in the closure of a lid over the substrate-binding pocket .
In the present study, we used CARDO-O of J3 and CARDO-F of CA10, hereafter simply termed Oxy and Fd, respectively, and aimed to clarify the holistic catalytic mechanism including conformational changes in ROs by determining various structures of the Oxy:Fd binary complex at different steps.