UC Irvine

Iron is important for pathogen survival and pathogens such as Mycobacterium tuberculosis (Mtb) have evolved pathways to acquire host iron. One such mechanism Mtb has evolved is the acquisition of heme, where Mtb imports host heme for degradation and iron release. In the cytosol, the heme degrading enzyme MhuD is responsible for the cleavage of heme and the liberation iron for use by Mtb. In this work we document our ongoing efforts to better understand MhuD and its larger role in Mtb.

Although the structure of the inactive diheme form of MhuD was previously determined, the structure of its active monoheme form was unresolved. We were able to determine the crystal structure of the cyano-derivative of MhuD-monoheme to obtain insights into MhuD heme degradation and product uniqueness. Heme ruffling, proposed to be responsible for heme’s reactivity within the active site of MhuD homologs, was observed in the MhuD-monoheme structure. Mutations to apparent heme ruffling residues Phe23 and Trp66 appear to abolish MhuD’s heme degradation activity suggesting the importance of heme ruffling in MhuD activity. Residues were also identified potentially responsible for MhuD’s unique degradation products. We found that mutation of one such residue, Arg26 into a serine, alters MhuD’s heme degradation into biliverdin and formaldehyde. Efforts to biophysically and structurally characterize the MhuD R26S mutant suggests that there is a reduction in heme ruffling in the mutant which may lead to the altered degradation product.

We also explored the role MhuD plays in the pharmacological inhibition of Mtb growth of mice infected with Mtb by the heme analog tin-protoporphrin IX (SnPPIX). Despite its ability to inhibit the human heme degrader, heme oxygenase, SnPPIX did not appear to inhibit MhuD’s ability to degrade heme. Lastly, we also carried out preliminary research towards the identification of a MhuD electron donor as well as of a potential protein partner for MhuD product release. To date, neither proposed MhuD interaction partners have been identified, however more candidate proteins remain to be purified and tested.