Respirasomes are macromolecular assemblies of the respiratory chain complexes I, 
III and IV in the inner mitochondrial membrane. We determined the structure of 
supercomplex I1III2IV1 from bovine heart mitochondria by cryo-EM at 9 Å 
resolution. Most protein-protein contacts between complex I, III and IV in the 
membrane are mediated by supernumerary subunits. Of the two Rieske iron-sulfur 
cluster domains in the complex III dimer, one is resolved, indicating that this 
domain is immobile and unable to transfer electrons. The central position of the 
active complex III monomer between complex I and IV in the respirasome is 
optimal for accepting reduced quinone from complex I over a short diffusion 
distance of 11 nm, and delivering reduced cytochrome c to complex IV. The 
functional asymmetry of complex III provides strong evidence for directed 
electron flow from complex I to complex IV through the active complex III 
monomer in the mammalian supercomplex.