ATP-Dependent Electron Activation Module of Benzoyl-Coenzyme A Reductase from the Hyperthermophilic Archaeon Ferroglobus placidus.

ABSTRACT

The class I benzoyl-coenzyme A (BzCoA) reductases (BCRs) are key enzymes in the anaerobic degradation of aromatic compounds that catalyze the ATP-dependent dearomatization of their substrate to a cyclic dienoyl-CoA. The phylogenetically distinct Thauera- and Azoarcus-type BCR subclasses are iron-sulfur enzymes and consist of an ATP-hydrolyzing electron activation module and a BzCoA reduction module. More than 20 years after their initial identification, all biochemical information about class I BCRs derives from studies of the wild-type enzyme from the denitrifying bacterium Thauera aromatica (BCRTaro). Here, we describe the first heterologous production and purification of the ATP-hydrolyzing, electron-activating module of an Azoarcus-type BCR from the hyperthermophilic archaeon Ferroglobus placidus, BzdPQFpla. The Fe content, UV/vis spectroscopic, and Mössbauer spectroscopic properties of the 57Fe-enriched enzyme clearly identified a [4Fe-4S]+/2+ cluster with a redox potential (E°') of -376 mV as a cofactor. ATP hydrolysis is required to overcome a redox barrier of ∼250 mV for stoichiometric electron transfer from the [4Fe-4S]+ cluster to the substrate benzene ring (E°'BzCoA/dienoyl-CoA = -622 mV). BzdPQFpla exhibited ATPase activity (15 nmol min-1 mg-1; Km = 270 µM) at 75 °C, which was relatively stable in air in contrast to BCRTaro. The results obtained revealed high levels of functional and molecular similarity between Azoarcus-type BCRs and the homologous ATP-dependent activator components of 2-hydroxyacyl-CoA dehydratases involved in amino acid fermentations. Insights into the diversity and evolution of ATP-dependent electron-activating modules for catalytic or stoichiometric low-potential electron transfer processes are presented.