Online

Graves, Amanda

Chemistry

2016

PhD

MhuD is a heme degrading enzyme from Mycobacterium tuberculosis that catalyzes both monooxygenation and dioxygenation leading to novel chromophores, mycobilins. The X-ray crystal structure of heme-bound, cyanide-inhibited MhuD displays a ruffled heme substrate where pyrrole rings are rotated around the Fe-N bonds, distorting the heme. The electronic structure of the heme in heme-bound, cyanide-inhibited MhuD (MhuD-heme-CN) is characterized here to provide insight into the mechanism of heme degradation by MhuD. Analysis of nuclear magnetic resonance (NMR) and magnetic circular dichroism (MCD) data for MhuD-heme-CN leads to the conclusion that the ground, (dxz,yz)4(dxy)1 electron configuration (2B2g state) and excited (dxy)2(dxz,yz)3 electron configuration (2Eg state) are close in energy allowing thermal population of the excited state at cryogenic and physiologically-relevant temperatures. The accessibility of two electronic states is studied with a combination of microbiology, spectroscopy, and computational analysis. The computational model developed describes a ground state that is close in energy to the excited state at ruffled geometries. Additionally, the heme is shown to be dynamic in the MhuD active site as the ground state hosts a double well potential in the ruffling coordinate allowing interconversion between a planar and a ruffled heme. To investigate the role of a ruffled, 2B2g heme and a planar, 2Eg heme in the MhuD reaction, substitutions are made to alter the active site residues Trp66 and Phe23. The Trp66 variants reduce heme ruffling resulting in decreased activity and a more planar heme as determined by optical spectroscopy and density functional theory calculations. F23W MhuD is shown to increase heme ruffling stabilizing the 2B2g state as determined by MCD. The F23W mutation stabilizes the meso hydroxyheme intermediate suggesting the more planar heme substrate conformation is needed to convert meso hydroxyheme into mycobilins. In summary, the data presented here strongly suggests that MhuD promotes formation of meso hydroxyheme with a ruffled substrate conformation and the formation of mycobilins with a more planar substrate conformation.