Investigating the mechanism and consequences of heme binding to the Bach1 transcription factor

The majority of iron in the body is in the form of heme. Heme usually acts as the cofactor of proteins, which carry out biological functions including oxygen transport, election transport, enzyme reaction and regulation of various signalling pathways. The central iron atom of the heme molecule can cause excess reactive oxygen species (ROS) production which can cause inflammation. Heme oxygenase-1 (HO-1) is an antioxidant defence enzyme, which degrades heme to protect against free heme-induced toxicity. ROS accumulation can promote development and progression of tumours.

Bach1 (BTB and CNC homology 1) is a transcription repressor that belong to the Cap ‘n’Collar and basic region leucine zipper family. Bach1 forms a heterodimer with another bZIP family member, MafK. Once formed, the heterodimer binds to the multiple Maf recognition elements (MAREs) resulting in the transcriptional repression of some oxidative stress-response genes such as HO-1. Bach1 contains 4 heme binding CP motifs. A proposal is that heme interacting with Bach1 leads to inhibit the binding of Bach1/MafK to DNA and exclude Bach1 from the nucleus. To date, there is not a clear understanding of the structural mechanism for heme to interact with Bach1 and how heme inhibits DNA binding.

Bach1 protein has been successfully expressed and purified from E. coli. I have determined a novel method to prepare a heterodimer of Bach1 and MafK. Mutations of potential heme interacting residues have been generated and investigated using UV–vis absorption spectroscopy and NMR. The results show that Bach1 has various heme binding types and a potential heme binding pocket. Electrophoretic Mobility Shift assays (EMSA) shows that heme binding to CP motifs mediates DNA dissociation. Both EMSA and Fluorescence correlation spectroscopy show that high heme concentration increases the dissociation constant of DNA-protein complex, suggesting heme changes the folding properties of Bach1.