A Dynamic Substrate is Required for MhuD-catalysed Degradation of Haem to Mycobilin

The non-canoncial haem oxygenase MhuD from Mycobacterium tuberculosis binds a haem substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between haem substrate dynamics and MhuD-catalysed haem degradation resulting in a revised enzymatic mechanism. UV/Vis absorption (Abs) and electrospray ionisation mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favouring population of the ruffled haem conformation changed the rate-limiting step of the reaction resulting in a measurable build-up of the monooxygenated meso-hydroxyhaem intermediate. In addition, UV/Vis Abs and ESI-MS data for a second-sphere variant that favoured the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an alpha-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of haem monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyhaem dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled haem conformation supports rapid haem monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered haem monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyhaem. Altogether, these data yielded a revised enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into haem yielding mycobilin.