Structural bases of electron transfer and reaction cycle of ringhydroxylating dioxygenase systems
Hideaki NOJIRIBiotechnology Research Center, The University of Tokyo
Report: Using carbazole (CAR) 1,9a-dioxygenase (CARDO), crystal structures of reduced
ferredoxin component (Fdred), CAR-bound reduced oxygenase component (Oxyred/red; see Fig.
1), both CAR- and O2-bound oxidized Oxy (Oxyox/ox), and product-bound Oxyox/ox weresolved. These are found in the catalytic cycle of CARDO and clearly show the molecularaction of the enzyme from the reduction of Oxy by Fdred to product formation. In addition,
mutant analyses show that the Fd-binding site of α3β3-type Oxy is located on the side of Oxy
mushroom structure, distinct from that in α3-type CARDO Oxy.
Research aims
Our previous studies clarified the crystal structures of oxidized Oxy (Oxyox/red, I inFig. 1)1), oxidized Fd (Fdox, II)2), and the complex of Oxyox/red:Fdox (III)4). Although thesestructures provided basic information on the structures and functions of CARDO, structuralbases of Oxy reduction and product formation were still limited. In this study, we solved thecrystal structures of reduced Fd (Fdred, IV) and the substrate-, oxygen-, and product-bindingstructures (VII, VIII, IX). Together with the structures of the complex of Oxyred/red:Fdox andOxyred/red (Matsuzawa et al., in preparation), we clarified the molecular mechanism ofOxyox/red reduction and product formation. On the other hand, we accessed whether theFd-binding site of α3β3-type Oxy is the same as that in α3-type CARDO Oxy.
Methods
Fdred was crystalized under anaerobic conditions, and its crystal structure wassolved. Using the V crystals soaked with CAR, or both CAR and O2, the crystal structures ofVII, VIII, and IX were solved. As the α3β3-type Oxy, it was used in a cumene
2,3-dioxygenase (CDO) system. Ala replacements were introduced into putative bindingsites between Oxy and Fd in the CDO system, and Oxy-reduction activity in thereconstituted CDO system was assessed.

Fig. 1. Catalytic cycle of CARDO OxyLowercase “ox” and “red” indicate the “oxidized” and “reduced” states,
respectively, of Oxy ([2Fe:2S] cluster/active site iron) and Fd ([2Fe:2S] cluster).
Results
The crystal structure of Fdred (VI) was solved at 1.8 Å resolution. Because rapid
electron transfer could occur after Fdred-binding to I, it could be difficult to solve the crystal
structure of the state shown in parentheses in Fig. 1. Therefore, we instead used the Oxystructure of III, because the redox states of Oxy in both crystals are the same, and thestructural changes from I to V were analyzed. Among the structural changes, the movementof Oxy Arg118 side chain was important (Fig. 2A). The side chain of Arg118 constituted salt
bridges with Fd Glu43 upon Fd binding (Fig. 2A Left). After electron transfer, the salt bridge
broke and the Arg118 side chain formed a hydrogen bond with the Oxy amino acid residue(Fig. 2A Center and Right). Similar movement was observed in Oxy Arg210, and these twoare considered to be an important switch for Fd association/dissociation. The CAR-binding
structure of Oxyred/red (VII) and its O2-exposed structure was also solved (both at 2.2 Åresolution). In the latter structure, both CAR and O2 were bound at one active site (VIII),
while the products were bound at other two sites (IX). In the product-bound structure (IX),
two phenyl moieties are twisted (Fig. 2B). As shown in Fig. 2C, a water molecule bound at
the active site iron was removed upon CAR-binding, and O2 bound at the resultant vacant
position (Fig. 2C).
Two possible Fd-binding sites were predicted for the α3β3-type Oxy-specificmushroom structure; the upper part corresponds to that in CARDO Oxy (α subunit) and theside part is constituted by both α and β subunits. We introduced Ala to Asp158, Trp159,Glu180, and Arg407 of α subunit (upper); Lys117 and Lys141 of α subunit; and Arg65,Leu98, and Trp100 of β subunit (side). As a result, the Oxy-reducing activities were reduced
in Lys117Ala (to less than 20%) and Arg65Ala (to 40~60%), suggesting that Fd could bindat the side part of the α3β3-type mushroom-like Oxy.

Fig. 2. Structure comparisons reveal the structural changes specific forassociation/dissociation (A) and CAR-binding followed by product formation (B, C)

FdConclusion
We succeeded in clarifying the crystal structures of VI, VII, VIII, and IX in thecatalytic cycle of CARDO Oxy (Fig. 1). The next targets are X and XI, which would allow
us to clarify a complete picture of the catalytic cycle. Based on the results on the putative
Fd-binding state of α3β3-type Oxy, we plan to evaluate the binding affinities of aminoacid-replaced enzymes and to determine the complex structure of α3β3-type Oxy and Fd.
References
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