Mycobacterium tuberculosis Rv2074 is a coenzyme F420-dependent mycobilin reductase

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, scavenges iron from its human host to support survival and pathogenesis. While siderophore-mediated iron acquisition in Mtb is well characterized, the mechanisms of heme‑iron acquisition are less understood. In the Mtb cytosol, the IsdG-type heme-degrading enzyme MhuD converts heme into iron and mycobilin isomers; however, the fate of MhuD-generated mycobilin is unknown. Recent studies have identified four Mtb flavin/deazaflavin oxidoreductases (FDORs), which are predicted to be non-canonical coenzyme F420-dependent biliverdin reductases. One such enzyme, Rv2074, was previously shown to reduce biliverdin IXα (αBV) to bilirubin (BR). Here, we evaluate the activity of four Mtb FDORs toward mycobilin and find that only Rv2074 catalyzes the reduction of mycobilin, yielding a previously uncharacterized tetrapyrrole product as detected by UV/visible spectroscopy. Mass spectrometry fragmentation analysis reveals reduction of mycobilin at the γ-meso carbon, analogous to the reduction of αBV to BR. We have named this product mycorubin. Furthermore, we show that, of the four Mtb FDORs, only Rv2074 can convert MhuD-generated mycobilin to mycorubin in a one-pot reaction containing MhuD and heme as the starting material. These findings provide the first evidence that the product of heme degradation by an IsdG-family enzyme undergoes further enzymatic reduction. This work reveals a previously unrecognized step in mycobacterial heme catabolism.