MhuD is a protein found across mycobacteria that can catalyze the degradation of heme to mycobilin. MhuD can also bind two heme molecules per protein monomer, however, the degradation function is inhibited after binding the second heme. Heme binding to MhuD is the first step in the degradation process and previous studies have reported the binding affinity of heme to MhuD to be micromolar. This suggests that MhuD may be an inefficient heme oxygenase in vivo, especially at the recently measured cytosolic labile heme concentration of 20-40 nM. Therefore, heme binding to MhuD was re-investigated using fluorescence spectroscopy. Based on a recently developed fluorescence-based assay, the Kd1 for heme-bound MhuD (MhuD—heme) was determined to be 7.6 ± 0.8 nM. In addition, a UV/Vis absorption-based assay was developed to measure the Kd2 for heme dissociation from diheme-bound MhuD (MhuD—diheme), which was determined to be 3.3 ± 1.1 mM. These data strongly suggest that MhuD is a competent heme oxygenase in vivo. The binding of heme is followed by its degradation to mycobilin, which is shown to proceed via formation of a meso-hydroxyheme intermediate. WT MhuD—heme has been shown to stabilize a ruffled ground state with a thermally-accessible planar excited state. Recent data suggests that both conformations may play important roles in heme degradation by MhuD.

Insights into binding and degradation of heme by Mycobacterium tuberculosis MhuD

2019 Spring National Meeting

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