Inserm U955-IMRB, UPEC, Ecole Nationale Vétérinaire d'Alfort, F-94010, Créteil, France.
University of Reims Champagne-Ardenne, CNRS, ICMR UMR 7312, 51687, Reims, France.
Biochimie. 2024 Sep;224:16-28. doi: 10.1016/j.biochi.2024.01.011. Epub 2024 Jan 25.
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, H-N 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.
重组蜡样芽胞杆菌 TSPO（BcTSPO）在大肠杆菌中的过表达导致其在细菌膜（MB）和包涵体（IB）中与结合的血红素一起回收。与小鼠 TSPO 不同，从 IB 中的 SDS 洗涤剂中纯化的 BcTSPO 结构良好，并且可以结合各种配体，如高亲和力 PK 11195、原卟啉 IX（PPIX）和 δ-氨基酮戊酸（ALA）。对于这三种配体中的每一种，H-N HSQC 滴定 NMR 实验表明，BcTSPO 结合腔的不同氨基酸参与相互作用。PPIX 是血红素生物合成的中间体，与 BcTSPO 的腔结合，并且在光和氧存在下其荧光可以显著降低。光照射导致两种产物被分离和表征为光卟啉。它们是由于单线态氧加成到两个乙烯基基团上而形成的，因此导致形成羟基醛。水分子的参与，最近在 Rhodobacter sphaeroides（RsTSPO）结合血红素时观察到，是高度可能的。总之，这些结果提出了 TSPO 在血红素生物合成调节中作为活性中间体清除剂的作用的问题。
