Evidence for two distinct loci catalyzing substrate monooxygenation and hydrogenation

2-Aminobenzoyl-CoA monooxygenaselreductase catalyzes both monooxygenation and hydrogenation of anthraniloyl-CoA. Its reactivity with 11 substrate analogs has been investigated. Only 2-aminobenzoylCoA (anthraniloyl-CoA) in its normal and deuterated (5-'H) form is a full substrate, and only traces of 2hydroxybenzoyl-CoA (salicyloyl-CoA) are probably monooxygenated but not hydrogenated. The purified enzyme is a homodimer and has been resolved preparatively into three major species by anion-exchange chromatography on Mono Q. All three species have the same specific activity when reconstituted to full content of FAD, they differ, however, substantially in their mode of binding FAD. The oxidized or fully reduced enzyme forms bind tightly 0.5 mol/mol of the substrate 2-aminobenzoyl-CoA (Kd = 1-2 pM). The enzyme can be depleted of ~ 5 0 % of its FAD, which corresponds to essentially complete removal from one of the two binding sites, reflecting a large difference in the affinity for FAD. From this it is deduced that the two sites are not equivalent. Removal of FAD from one binding site leads to loss of the hydrogenation capacity of the enzyme, while monooxygenation catalysis is retained. The FAD cofactors of the two binding sites differ drastically in their reactivities towards NADH, oxygen and N-ethylmaleimide. Exchange of reducing equivalents between the two FAD cofactors at the respective binding sites is very slow and irrelevant compared to the rates of catalysis. It is concluded that the enzyme, which has been proposed to consist of two identical polypeptide chains [Altenschmidt, U., Bokranz, M. & Fuchs, G. (1992) Eur: J. Biochem. 207, 715-7221, contains two active centers which differ substantially in their catalytic activity. One center belongs to the class of monooxygenases, the other one to the (de)hydrogenases. This must result from substantially different interaction of the same flavin cofactors with protein functional groups and is, to our knowledge, unprecedented in flavoprotein enzymology.