Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Matthew D. Liptak

Second Advisor

Christopher S. Francklyn


MhuD is a heme degrading enzyme found across mycobacteria. In the heme uptake machinery of Mycobacterium Tuberculosis, MhuD is a heme oxygenase (HO) involved in catalyzing the degradation of heme. Canonical HOs, such as human HO, have been shown to degrade heme to non-heme iron, carbon monoxide (CO) and biliverdin. However, MhuD has been demonstrated to degrade heme to non-heme iron and mycobilin without the release of CO. Formation of a novel chromophore by MhuD revealed that it follows a unique heme degradation mechanism compared to that of canonical HOs. Binding of heme 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 would mean that MhuD may be an inefficient HO 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 uM. These data strongly suggest that MhuD is a competent heme oxygenase in vivo.

Next, heme degradation by MhuD was investigated, which is shown to proceed via formation of a meso-hydroxyheme intermediate. Time-resolved and tandem mass spectrometry (MS) identified formation of both mycobilin (major product) and biliverdin (minor product) by wild type (WT) MhuD. This interesting discovery warranted further investigation. Heme bound to the MhuD active site undergoes a dynamic distortion between two substrate conformations. Thus, we hypothesized that the conformation of heme may be responsible for the product identity and that both heme conformations may be required for an efficient enzymatic activity of MhuD. To test these hypothesis, different variants of MhuD were investigated. F23W MhuD has been shown to stabilize more ruffled heme and W66F MhuD has been shown to stabilize less ruffled heme than WT. MS studies of these variants identified mycobilin and biliverdin as the major products for F23W and W66F MhuD, respectively. Thus, suggesting that the heme confirmation plays a role in product identity. Also, the biliverdin formation by WT and F23W MhuD lacked regioselectivity, which suggests that its formation proceeds via a non-enzymatic pathway. In addition, UV/Vis study identified meso-hydroxyheme intermediate for the heme degradation by F23W MhuD. Kinetic analysis of the reaction catalyzed by F23W MhuD revealed that the meso-hydroxyheme dioxygenation rate is considerably slower for this variant. In summary, these data strongly suggest that heme degradation product of MhuD depends upon the degree of heme ruffling induced by the active site and that dynamic heme is essential for MhuD.



Number of Pages

247 p.