The ATP-independent dihydro-2-phenanthroyl-CoA reductase AprC catalyzes two consecutive two-electron reduction steps of dihydro-2-phenanthroyl-CoA to hexahydro-2-phenanthroyl-CoA in anaerobic phenanthrene degradation

Polycyclic aromatic hydrocarbons (PAHs) are widespread pollutants and especially difficult to degrade under anoxic conditions. The non-substituted three-ring PAH phenanthrene is activated through carboxylation to 2-phenanthroic acid which is then ligated to Co-enzyme A to produce 2-phenanthroyl-CoA, followed by step-wise reduction of the aromatic rings. The first reduction reaction is catalyzed by 2-phenanthroyl-CoA reductase producing dihydro-2-phenanthroyl-CoA. In this study, we elucidated the second reductase enzyme in the pathway, catalyzing the reduction of dihydro-2-phenanthroyl-CoA to hexahydro-2-phenanthroyl-CoA. Dihydro-2-phenanthroyl-CoA reductase (AprC) was heterologously overproduced in <i>Escherichia coli</i> and purified and characterized. AprC has a specific activity of 15.8 ± 0.3 nmol min^-1 mg^-1 and an apparent <i>K</i>_<i>m</i> value of 59.9 ± 3.8 nM at pH 7.5. Dithionite-reduced methyl viologen is the preferred <i>in vitro</i> electron donor for the four electron-reduction reaction, but it also functions with NADH but not with NADPH. The intermediate appearance of tetrahydro-2-phenanthroyl-CoA during the reduction to hexahydro-2-phenanthroyl-CoA indicates that AprC catalyzes two consecutive two-electron reduction steps. AprC is a monomeric enzyme with a molecular mass of ≈73 kDa and contains one FMN, one FAD, and one 4Fe-4S cluster, indicating that the reductase belongs to the type III aryl-CoA reductases of the old-yellow enzyme family. The high amino acid sequence similarity between dihydro-2-phenanthroyl-CoA reductase and 2-naphthoyl-CoA reductase, as well as the similar cofactor coordination in both enzymes, suggested that dihydro-2-phenanthroyl-CoA reductase follows the same biochemical principle as naphthalene ring reduction by 2-naphthoyl-CoA reductase. Quantum chemical calculation indicated that 5,6,7,8,9,10-hexahydro-2-phenanthroyl-CoA (isomer <b>6a</b>) is the most stable product of AprC out of eight possible isomers, although the real isomer is not known. The reductase also performed a two-electron reduction of chemically synthesized 9,10-dihydro-2-phenanthroyl-CoA to tetrahydro-2-phenanthroyl-CoA but at much lower reaction rates indicating that the 9,10-dihydro-2-phenanthroyl-CoA isomer is not the natural substrate of the enzyme and, thus, also not the real product of 2-phenanthroyl-CoA reduction by 2-phenanthroyl-CoA reductase AprB. Since 9,10-dihydro-2-phenanthroyl-CoA has two non-conjugated benzene rings, this also indicates that, surprisingly, type III aryl-CoA reductases can reduce benzene ring structures without additional ATP requirement.IMPORTANCEPolycyclic aromatic hydrocarbons (PAHs) are highly toxic, persistent pollutants found in the environment. The anaerobic degradation of larger three-ring PAHs like phenanthrene is still poorly understood. Here, we show that after activation to a CoA-ester, the resonance energy of the aromatic ring system of phenanthrene is overcome by consecutive two-electron reduction steps catalyzed by ATP-independent type III aryl-CoA reductases belonging to the old-yellow enzyme family. This finding contributes to our understanding of the anaerobic degradation of PAHs with three or more rings.