Characterization of a Heme-Binding Radical SAM Enzyme HemW from Moraxella catarrhalis.

Moraxella catarrhalis is an emerging human respiratory pathogen responsible for various infectious diseases. Recent transposon sequencing analysis identified yggW (renamed hemW) as essential for M. catarrhalis growth under iron-limiting conditions, mimicking host-imposed nutritional immunity. HemW is annotated as a putative radical S-adenosylmethionine (SAM) enzyme and belongs to the HemN-like subfamily, but its biochemical properties remain unclear. Here, we report on the first experimental characterization of M. catarrhalis HemW (McHemW). Our bioinformatic analysis confirmed its evolutionary relationship with the putative heme-binding radical SAM enzyme HemW in Escherichia coli. We experimentally demonstrated that McHemW contains a catalytically active [4Fe-4S] cluster and binds heme through biochemical assays and spectroscopy. Using hydrogen-deuterium exchange mass spectrometry, we demonstrated, for the first time, that the heme-binding site resides within the C-terminal auxiliary domain unique to HemW. These findings provide mechanistic insight into the molecular basis of heme recognition in McHemW and establish a foundation for understanding its role in the pathogenicity of M. catarrhalis under iron-limited conditions.