研究セミナー High resolution cryoEM studies of cytochrome bc1 and Nitric Oxide reductase

Recent advances in CryoEM has allowed us to obtain high-resolution structures of Bovine cytochrome bc1 and a quinol-dependent NO Reductase (qNORs) from Alcaligenes xylosoxidans, an opportunistic pathogen and a denitrifying bacterium of importance in the nitrogen cycle. qNORs are considered members of the respiratory heme-copper oxidase superfamily, are unique to bacteria, and are commonly found in pathogenic bacteria where they play a role in combating the host immune response. NORs are also essential enzymes in the denitrification pathway, catalysing the reduction of nitric oxide to nitrous oxide. We recently determined a 2.2 Å cryoEM structure of the as isolated qNOR in dimeric state and structure of its E494A mutant in both monomeric (~4.5Å resolution) and dimeric (2.56 Å resolution) states. The structures of E494A mutants reveal different levels of movements of TMI helix and dimer-stabilising TMII helix which in addition to destabilising the dimeric functional assembly results in the destabilisation of catalytic Fe. The mutant has less than 10% of wild-type enzyme consistent with no detectable density of Fe in the 2.56Å structure of the mutant dimer. Bovine cytochrome bc1 is a 500 kDa heterodimeric mitochondrial complex containing 12 different subunits. Recently obtained ~2 Å structures of the as-isolated protein and complexes with inhibitors allowed us to see details of positions for 4 lipids; cardiolipin (CDL); phosphatidyl-ethanolamine (LOP); phosphatidylcholine (PSC) and phosphatidylinositol (T7X) within the transmembrane part of the complex and extensive water networks in outer membrane space. High resolution also allowed us to understand the reason behind the non-toxicity of SG-1-14 ligand, bound to Qi site and toxicity of CK-2-67 bound in both Qi and Qo sites.