From Sensor to Catalyst: Heme-Dependent Oxidative N -Demethylase Redefines Per-ARNT-Sim-Domain Chemistry via Amine Oxidase Mechanisms for Dimethylamine Demethylation, Vital N–C Bond Cleavage in Global N -Cycle

Microbial N–C bond cleavage plays a vital role in the nitrogen biogeochemical cycle. Heme-dependent oxidative N-demethylase (HODM), an enzyme identified within the Per-ARNT-Sim (PAS) domain family, challenges the traditional view of PAS domains as merely sensory modules by catalyzing the dimethylamine (DMA) demethylation to methylamine (MA) and formaldehyde via N–C bond dissociation. In this study, we employed density functional calculations to investigate the mechanistic landscape of the HODM reaction across multiple spin states. Among nine mechanistic pathways examined, two (mechanisms A1 and A3) proceeding on the doublet-state surface were found to be viable, mainly involving (1) addition of an electron to the enzyme-substrate complex generating Fe(III)–O22– anion, (2) proton transfer from Arg224 forming Fe(III)––OOH (Cpd 0), (3) homolytic O─O bond cleavage producing heme high-valent O═Fe(IV)(Por) (Cpd II) and •OH radical, (4) hydrogen-atom transfer (HAT) from DMA to •OH yielding water, and (5) second HAT from DMA to Cpd II producing imine that hydrolyzes nonenzymatically to MA and formaldehyde. The DMA hydrogen abstraction by •OH is rate-limiting. Our findings establish HODM as an amine oxidase with •OH as the initial active species to abstract substrate hydrogens, instead of a monooxygenase using high-valent iron(IV)–oxo. Cpd 0 could not work as the H-abstraction species. Without an electron gained, the Fe(III)–O2•– superoxide radical is unable to initiate the HODM reaction. The revelation of HODM nature expands the PAS chemistry and enriches the understanding of enzymatic N–C bond dissociation and heme chemistry.