Cryo-EM Structures of Respiratory bc₁-cbb₃ type CIII₂CIV Supercomplex 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 c_y 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 c_y 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 c_y suggests that it is mobile, and it interacts with CIII₂CIV differently than cytochrome c₂. Distance requirements for electron transfer suggest that cytochrome c_y and cytochrome c₂ donate electrons to heme c_p1 and heme c_p2 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.