Aromatic-ring-hydroxylating dioxygenases

Aromatic ring hydroxylating dioxygenases (ARHD) incorporate two atoms of dioxygen (O2) 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 (αβ)n or (α)n oligomers. Two prosthetic groups, a Rieske type [Fe2S2] center and a mononuclear iron, are associated with the α subunit in the (αβ)n type enzymes. Electron transfer components are composed of flavoprotein (NADH:ferredoxin oxidoreductase) and Rieske type [Fe2S2] 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:

NADH →reductase FAD or FMN → ferredoxin [Fe2S2] → hydroxylase α subunit
[Fe2S2], Fe

The crystal structure of the hydroxylase component of naphthalene 1,2 dioxygenase from Pseudomonas has been determined. The protein is an (αβ)3 hexamer. The β subunit belongs to the
α+β class. It has no prosthetic groups and its role in catalysis is unknown. The α subunit can be divided into two domains: a Rieske domain that contains the [Fe2S2] center and the catalytic domain that contains the active site mononuclear iron. The Rieske domain (residues 38 158) consists of four β sheets. The overall fold is very similar to that of the soluble fragment of the Rieske protein from bovine heart mitochondrial cytochrome bc1 complex. In the [Fe2S2] center, Fe1 is coordinated by two cysteine residues (Cys 81 and Cys 101) while Fe2 is coordinated by Nδ atoms of two histidine residues (His 83 and His 104). The catalytic domain belongs to the α+β class and is dominated by a nine stranded antiparallel β sheet. The iron of the active site is located at the bottom of a narrow channel, approximately 15 Å from 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 α subunits, where electrons from the [Fe2S2] center in one α subunit (A) are transferred to the mononuclear iron in the adjacent α subunit (B) through AspB 205, which is hydrogen bonded to HisA 104 of the Rieske center and HisB 208 of the active site.