FMN binding to the PNPOx-like scaffold of PM0042 from Pasteurella multocida facilitates electron transfer in heme degradation

The HugZ/HutZ family has been shown to degrade heme in the presence of ascorbic acid. However, its inability to utilize canonical cytochrome P450 reductase has raised questions regarding its physiological role as a true heme oxygenase. PM0042 from Pasteurella multocida, a member of the HugZ/HutZ family, belongs to the pyridoxamine 5'-phosphate oxidase (PNPOx) family, which utilizes FMN as a cofactor. In this study, we examined the role of FMN in heme-degradation activity. Fluorescence analysis revealed that PM0042 binds FMN with a dissociation constant in the micromolar range. PM0042 degrades heme in the presence of FMN and NADH at an efficient rate comparable to the fastest previously described condition using ascorbic acid and ferrous ion. Based on AlphaFold3 predictions, PM0042 binds to FMN near the heme, which enables efficient electron transfer from NADH. The LC-MS product analysis supports the proposal that FMN binds in close proximity to the heme. Furthermore, similar FMN-dependent activity was observed for HutZ from Vibrio cholerae (VcHutZ). These results reveal that the PNPOx-like domain of PM0042 serves as an integrated redox module, relaying electrons from NADH via FMN to the heme. This mechanism likely represents a conserved strategy among the HutZ family, distinguishing it from canonical heme oxygenase systems.