Dearomatizing benzoyl-coenzyme A reductases (BCR) from facultatively anaerobic bacteria are key enzymes in the anaerobic degradation of aromatic compounds. They catalyze the ATP-dependent reduction of benzoyl-CoA (BCoA) to cyclohexa-1,5-diene-1-carboxyl-CoA (dienoyl-CoA). A Birch reduction mechanism involving alternate electron transfer and protonation steps has been proposed for BCR. In this work we reacted BCoA in H2O and D2O, and d5-BCoA in H2O with BCR and the second enzyme of the pathway, dienoyl-CoA hydratase (DCH). The 1,4 hydration product formed from the dienoyl-CoA, 6-hydroxycyclohex-1-ene-1-carbonyl-CoA, was analyzed by several NMR techniques. The results obtained indicate that BCR stereoselectively forms the trans-dienoyl-CoA product, and DCH stereoselectively catalyzes a trans-1,4 water addition. Moreover, unexpected proton exchanges at C-2 and C-6 were observed. They indicate that a free radical intermediate with an unusual low pKa is formed during BCR catalysis. This finding provides evidence for the proposed Birch reduction mechanism of BCR and is in agreement with the established radical mechanism of homologous alpha-hydroxyacyl-CoA dehydratases.

Benzoyl-CoA reductases (BCRs) are key enzymes of anaerobic aromatic metabolism in facultatively anaerobic bacteria. The highly oxygen-sensitive enzymes catalyze the ATP-dependent reductive dearomatization of the substrate, yielding cyclohexa-1,5-diene-1-carbonyl-CoA (1,5-dienoyl-CoA). In extracts from anaerobically grown denitrifying Thauera aromatica, we detected a benzoate-induced, benzoyl-CoA-forming, 1,5-dienoyl-CoA:acceptor oxidoreductase activity. This activity co-purified with BCR but could be partially separated from it by hydroxyapatite chromatography. After activity staining on native gels, a monomeric protein with a subunit molecular weight of M r 76,000 was identified. Mass spectrometric analysis of tryptic digests identified peptides from NADH oxidases/2,4-dienoyl-CoA reductases/"old yellow" enzymes. The UV-visible spectrum of the enriched enzyme suggested the presence of flavin and Fe/S-cofactors, and it was bleached upon the addition of 1,5-dienoyl-CoA. The enzyme had a high affinity for dioxygen as electron acceptor (K m ‫؍‬ 10 M) and therefore is referred to as 1,5-dienoyl-CoA oxidase (DCO). The likely product formed from dioxygen reduction was H 2 O. DCO was highly specific for 1,5-dienoyl-CoA (K m ‫؍‬ 27 M). The initial rate of DCO followed a Nernst curve with half-maximal activity at ؉10 mV. We propose that DCO provides protection for the extremely oxygensensitive BCR enzyme when the bacterium degrades aromatic compounds at the edge of steep oxygen gradients. The redox-dependent switch in DCO guarantees that DCO is only active during oxidative stress and circumvents futile dearomatization/ rearomatization reactions catalyzed by BCR and DCO.The central intermediate generated by most anaerobic bacteria capable of using aromatic compounds as growth substrates is benzoyl-CoA, which then serves as the substrate for dearomatizing benzoyl-CoA reductases (BCRs) 2 (1-5). With the exception of the enzyme from Rhodopseudomonas palustris, all BCRs appear to catalyze the two-electron reduction of benzoyl-CoA, yielding cyclohexa-1,5-diene-1-carbonyl-CoA (1,5-dienoyl-CoA; Fig. 1). The catalytic mechanism of BCR is considered to proceed in analogy to the Birch reduction in chemical synthesis and involves electron transfer steps at extremely low redox potentials (6). Two completely different classes of dearomatizing benzoyl-CoA reductases have been described. In BCR from facultative anaerobes, the electron transfer from reduced ferredoxin to benzoyl-CoA is coupled to a stoichiometric ATP-hydrolysis (1 ATP/electron transferred) (7,8). The biochemistry of ATP-dependent BCR enzymes has so far only been studied with the enzyme from the denitrifying bacterium Thauera aromatica. It has an ␣␤␥␦ subunit composition with two functionally different modules: (i) the electron activation module is composed of the ␣␦ subunits (49 and 29 kDa, respectively) and harbors two ATP-binding sites and the so-called electron entry 1ϩ/2ϩ cluster; (ii) the benzoylCoA reduction module is formed by the ␤␥ subunits (48 and 44 kDa, respe...

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