MhuD from Mycobacterium tuberculosis: Probing a Dual Role in Heme Storage and Degradation

Abstract

The Mycobacterium tuberculosis (Mtb) heme oxygenase MhuD liberates free iron by degrading heme to the linear tetrapyrrole mycobilin. The MhuD dimer binds up to two hemes within the active site of each monomer. Binding the first solvent-exposed heme allows heme degradation and releases free iron. Binding a second heme renders MhuD inactive, allowing heme storage. Native-mass spectrometry revealed little difference in binding affinity between solvent-exposed and solvent-protected hemes. Hence, diheme-MhuD is formed even when a large proportion of the MhuD population is in the apo form. Apomyoglobin heme transfer assays showed MhuD-diheme dissociation is far slower than monoheme dissociation at ∼0.12 min–1 and ∼0.25 s–1, respectively, indicating that MhuD has a strong affinity for diheme. MhuD has not evolved to preferentially occupy the monoheme form and, through formation of a diheme complex, it functions as part of a larger network to tightly regulate both heme and iron levels in Mtb.

Structure–function characterization of the mono- and diheme forms of MhuD, a noncanonical heme oxygenase from Mycobacterium tuberculosis

“…When the diheme samples in our study were applied to a Sephadex G-75 column (GE Healthcare), no such separation of oligomeric states was observed. Similarly, analytical ultracentrifugation studies recently showed that diheme MhuD with the His-tag removed by TEV protease exists only as a dimeric protein (19). The aggregation of tagged diheme MhuD would likely impede access of electrons to heme that are required in the catalytic cycle and explain its apparent inactivity as reported previously.…”

“…Reconstituting apo-MhuD protein with heme is also not a trivial endeavor. Since MhuD can bind two hemes in the same active site, special measures must be taken to ensure reliable and consistent heme:protein stoichiometry during reconstitution of monoheme samples to prevent formation of diheme, which was shown to occur even when incubated with heme in a 1:1 molar ratio (19). Therefore, MhuD samples in this study were reconstituted using a CN-CO replacement method that has provided an apparent solution to this challenging problem.…”

“…Functional implications. Since its discovery, the evolutionary significance of the capability for MhuD to bind either one or two hemes in the same active site has been theorized (10,19,39). It was proposed that diheme MhuD functions primarily as a heme storage protein during periods of high intracellular heme concentration to prevent iron-mediated toxicity, a function comparable to that of the iron storage role of Mtb ferritin proteins, BfrA and BfrB (10).…”

“…It was proposed that MhuD has much higher affinity for the first heme than the second, implying that monoheme functions as a heme degrader in low intracellular heme conditions and when heme concentrations rise, MhuD binds the second heme and its diheme form acts as a heme storage or regulatory protein (18). However, more recent studies showed that MhuD did not evolve to preferentially bind one heme molecule and that the diheme form is comparably favored, implicating a more complex functional role of diheme MhuD in Mtb physiology (19).…”

HutW from Vibrio cholerae Is an Anaerobic Heme-Degrading Enzyme with Unique Functional Properties

“…These K d values are in a range that is similar to what has been reported for other bacterial heme-degrading enzymes. 12,30,31 The binding also appears to saturate at 10 μM, which is a 1:1 HutW:heme ratio. Turnover of HutW Using Metalated and Nonmetalated Porphyrins.…”