Overexpression of recombinant Bacillus cereus TSPO (BcTSPO) in E. coli bacteria leads to its recovery with a bound hemin both in bacterial membrane (MB) and inclusion bodies (IB). Unlike mouse TSPO, BcTSPO purified in SDS detergent from IB is well structured and can bind various ligands such as high-affinity PK 11195, protoporphyrin IX (PPIX) and δ-aminolevulinic acid (ALA). For each of the three ligands, 1H-15N HSQC titration NMR experiments suggest that different amino acids of BcTSPO binding cavity are involved in the interaction. PPIX, an intermediate of heme biosynthesis, binds to the cavity of BcTSPO and its fluorescence can be significantly reduced in the presence of light and oxygen. The light irradiation leads to two products that have been isolated and characterized as photoporphyrins. They result from the addition of singlet oxygen to the two vinyl groups hence leading to the formation of hydroxyaldehydes. The involvement of water molecules, recently observed along with the binding of heme in Rhodobacter sphaeroides (RsTSPO) is highly probable. Altogether, these results raise the question of the role of TSPO in heme biosynthesis regulation as a possible scavenger of reactive intermediates.
Keywords: Aminolevulinic acid; Functional recombinant TSPO; Hemin; Membrane protein; Porphyrin.
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