In the aerobic metabolism of aromatic substrates, oxygenases use molecular oxygen to hydroxylate and finally cleave the aromatic ring. In the case of the common intermediate benzoate, the ring cleavage substrates are either catechol (in bacteria) or 3,4-dihydroxybenzoate (protocatechuate, mainly in fungi). We have shown before that many bacteria, e.g. Azoarcus evansii, the organism studied here, use a completely different mechanism. This elaborate pathway requires formation of benzoyl-CoA

The coenzyme A (CoA)-dependent aerobic benzoate metabolic pathway uses an unprecedented chemical strategy to overcome the high aromatic resonance energy by forming the nonaromatic 2,3-epoxybenzoyl-CoA. The crucial dearomatizing reaction is catalyzed by three enzymes, BoxABC, where BoxA is an NADPH-dependent reductase, BoxB is a benzoyl-CoA 2,3-epoxidase, and BoxC is an epoxide ring hydrolase. We characterized the key enzyme BoxB from Azoarcus evansii by structural and Mössbauer spectroscopic methods as a new member of class I diiron enzymes. Several family members were structurally studied with respect to the diiron center architecture, but no structure of an intact diiron enzyme with its natural substrate has been reported. X-ray structures between 1.9 and 2.5 Å resolution were determined for BoxB in the diferric state and with bound substrate benzoyl-CoA in the reduced state. The substrate-bound reduced state is distinguished from the diferric state by increased iron-ligand distances and the absence of directly bridging groups between them. The position of benzoylCoA inside a 20 Å long channel and the position of the phenyl ring relative to the diiron center are accurately defined. The C2 and C3 atoms of the phenyl ring are closer to one of the irons. Therefore, one oxygen of activated O 2 must be ligated predominantly to this proximate iron to be in a geometrically suitable position to attack the phenyl ring. Consistent with the observed iron/phenyl geometry, BoxB stereoselectively should form the 2S,3R-epoxide. We postulate a reaction cycle that allows a charge delocalization because of the phenyl ring and the electron-withdrawing CoA thioester.The metabolism of aromatic compounds has attracted broad attention due to its importance in the biogeochemical carbon cycle (includes 10 -20% of the biomass) and because of the chemistry necessary to overcome the high resonance energy of the conjugated cyclic ring. A central intermediate of the aromatic metabolism is benzoate, which can be degraded by microorganisms using three different strategies. The first strategy in the presence of oxygen uses mono-and dioxygenases to hydroxylate benzoate to catechol or protocatechuate. Central ring-cleaving dioxygenases with mononuclear iron centers subsequently cleave the aromatic ring either between the two hydroxyl groups (ortho cleavage, ␤-ketoadipate pathway) or next to one of the hydroxyl groups (meta cleavage) (1). The second strategy under anoxic conditions involves benzoylCoA, which is reduced by two electrons to a non-aromatic cyclic diene followed by hydrolytic ring opening. Benzoyl-CoA reduction is accomplished by two completely different enzyme systems that may catalyze a Birch-like reduction (2).The third, semi-aerobic strategy to metabolize benzoate shares characteristic features of both of these options (3). Oxygen is still required for attacking the ring, as in the aerobic metabolism, whereas all metabolites are activated to CoA thioesters and ring cleavage is performed hydrolytically, as in the anaerobic pathwa...

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