Aromatic-ring-hydroxylating dioxygenases (ARHD) incorporate two atoms of dioxygen (O<sub>2</sub>) into their substrates in the dihydroxylation reaction. The product is (substituted) cis-1,2-dihydroxycyclohexadiene, which is subsequently converted to (substituted) benzene glycol by a cis-diol dehydrogenase.
A large family of multicomponent mononuclear (non-heme) iron oxygenases has been identified. Components of bacterial aromatic-ring dioxygenases constitute two different functional classes: hydroxylase components and electron transfer components. Hydroxylase components are either (ñò)<sub>n</sub> or (ñ)<sub>n</sub> oligomers. Two prosthetic groups, a Rieske-type [Fe<sub>2</sub>S<sub>2</sub>] center and a mononuclear iron, are associated with the ñ-subunit in the (ñò)<sub>n</sub>-type enzymes. Electron transfer components are composed of flavoprotein (NADH:ferredoxin oxidoreductase) and Rieske-type [Fe<sub>2</sub>S<sub>2</sub>] ferredoxin. In benzoate and toluate 1,2-dioxygenase systems, a single protein containing reductase and Rieske-type ferredoxin domains transfers the electrons from NADH to the hydroxylase component. In the phthalate 4,5-dioxygenase system, phthalate dioxygenase reductase (PDR) has the same function. PDR is a single protein comprising FMN-binding reductase and plant-type ferredoxin domains. Thus, the electron transfer in ARHD systems can be summarised as:
benzene 1,2-dioxygenase
phthalate 4,5-dioxygenase
4-sulfobenzoate 3,4-dioxygenase
4-chlorophenylacetate 3,4-dioxygenase
benzoate 1,2-dioxygenase
toluene dioxygenase
naphthalene 1,2-dioxygenase
terephthalate 1,2-dioxygenase
biphenyl 2,3-dioxygenase
The crystal structure of the hydroxylase component of naphthalene 1,2-dioxygenase from Pseudomonas has been determined. The protein is an (ñò)<sub>3</sub> hexamer. The ò-subunit belongs to the ñ+ò class. It has no prosthetic groups and its role in catalysis is unknown. The ñ-subunit can be divided into two domains: a Rieske domain that contains the [Fe<sub>2</sub>S<sub>2</sub>] center and the catalytic domain that contains the active site mononuclear iron. The Rieske domain (residues 38-158) consists of four ò-sheets. The overall fold is very similar to that of the soluble fragment of the Rieske protein from bovine heart mitochondrial cytochrome bc<sub>1</sub> complex. In the [Fe<sub>2</sub>S<sub>2</sub>] center, Fe1 is coordinated by two cysteine residues (Cys-81 and Cys-101) while Fe2 is coordinated by N<sup>ô</sup> atoms of two histidine residues (His-83 and His-104). The catalytic domain belongs to the ñ+ò class and is dominated by a nine-stranded antiparallel ò-sheet. The iron of the active site is located at the bottom of a narrow channel, approximately 15 àfrom the protein surface. The mononuclear iron is coordinated by His-208, His-213, Asp-362 (bidentate) and a water molecule. The geometry can be described as a distorted octahedral with one ligand missing. The structure of the hexamer suggests cooperativity between adjacent ñ-subunits, where electrons from the [Fe<sub>2</sub>S<sub>2</sub>] center in one ñ-subunit (A) are transferred to the mononuclear iron in the adjacent ñ-subunit (B) through Asp<sub>B</sub>-205, which is hydrogen-bonded to His<sub>A</sub>-104 of the Rieske center and His<sub>B</sub>-208 of the active site.