Cryo-EM Structures of Respiratory bc1-cbb3 type CIII2CIV Super-complex and Electronic Communication Between the Complexes

Abstract

The respiratory electron transport complexes convey electrons from nutrients to oxygen and generate a proton-motive force used for energy (ATP) production in cells. These enzymes are conserved among organisms, and organized as individual complexes or combined forming large super-complexes (SC). Bacterial electron transport pathways are more branched than those of mitochondria and contain multiple variants of such complexes depending on their growth modes. The Gram-negative species deploy a mitochondrial-like cytochrome bc1 (Complex III, CIII2), and may have bacteria-specific cbb3-type cytochrome c oxidases (Complex IV, CIV) in addition to, or instead of, the canonical aa3-type CIV. Electron transfer between these complexes is mediated by two different carriers: the soluble cytochrome c2 which is similar to mitochondrial cytochrome c and the membrane-anchored cytochrome cy which is unique to bacteria. Here, we report the first cryo-EM structure of a respiratory bc1-cbb3 type SC (CIII2CIV, 5.2Å resolution) and several conformers of native CIII2 (3.3Å resolution) from the Gram-negative bacterium Rhodobacter capsulatus. The SC contains all catalytic subunits and cofactors of CIII2 and CIV, as well as two extra transmembrane helices attributed to cytochrome cy and the assembly factor CcoH. Remarkably, some of the native CIII2 are structural heterodimers with different conformations of their [2Fe-2S] cluster-bearing domains. The unresolved cytochrome c domain of cy suggests that it is mobile, and it interacts with CIII2CIV differently than cytochrome c2. Distance requirements for electron transfer suggest that cytochrome cy and cytochrome c2 donate electrons to heme cp1 and heme cp2 of CIV, respectively. For the first time, the CIII2CIV architecture and its electronic connections establish the structural features of two separate respiratory electron transport pathways (membrane-confined and membrane-external) between its partners in Gram-negative bacteria.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsQquinoneQH2Quinol or hydroquinoneComplex IIICIII2 or cytochromebc1ubiquinolcytochrome c oxidoreductaseComplex IV or CIVcbb3-type cytochrome c oxidasecytcytochromecyt c2cytochrome c2, soluble cytochrome ccyt cycytochrome cy, membrane-anchored cytochrome ccyt S-cysoluble part of cytochrome cy without its membrane anchorSCsuper-complexMSmass spectrometryXL-MScross-linking mass spectrometryXLcross-linksTMBZ3,3’,5,5’-tetramethyl-benzidineDBH22,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinoneFeSRieske iron-sulfur proteinFeS-EDmembrane-extrinsic domain of FeS proteinb positionlocation of the [2Fe-2S] cluster near heme bLc positionlocation of the [2Fe-2S] cluster near heme c1cryo-EMcryogenic electron microscopyBN-PAGEblue native polyacrylamide gel electrophoresisSDS-PAGEsodium dodecylsulfate polyacrylamide gel electrophoresisC-terC-terminusN-terN-terminusHis-tag8-histidine tagFLAG-tagDYKDDDDK-tagSECsize exclusion chromatographyTMHtransmembrane helixDSSdisuccinimidyl suberateDSBUdisuccinimidyl dibutyric urea; DMTMM, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chlorideheme-Feheme-ironEmredox midpoint potentialheme cp1N-ter located c-type heme 1 of CcoPheme cp2C-ter located c-type heme 2 of CcoPheme coc-type heme of CcoOSO32-sulfiteSO42-sulfateRMSDroot-mean-square deviationDDMn-dodecyl β-D-maltoside.View Full Text