FRET measurement of cytochrome bc1 and reaction centre complex proximity in live Rhodobacter sphaeroides cells.

Abstract In the model purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides, solar energy is converted via coupled electron and proton transfer reactions within the intracytoplasmic membranes (ICMs), infoldings of the cytoplasmic membrane that form spherical ‘chromatophore’ vesicles. These bacterial ‘organelles’ are ideal model systems for studying how the organisation of the photosynthetic complexes therein shape membrane architecture. In Rba. sphaeroides, light-harvesting 2 (LH2) complexes transfer absorbed excitation energy to dimeric reaction centre (RC)-LH1-PufX complexes. The PufX polypeptide creates a channel that allows the lipid soluble electron carrier quinol, produced by RC photochemistry, to diffuse to the cytochrome bc1 complex, where quinols are oxidised to quinones, with the liberated protons used to generate a transmembrane proton gradient and the electrons returned to the RC via cytochrome c2. Proximity between cytochrome bc1 and RC-LH1-PufX minimises quinone/quinol/cytochrome c2 diffusion distances within this protein-crowded membrane, however this distance has not yet been measured. Here, we tag the RC and cytochrome bc1 with yellow or cyan fluorescent proteins (Y/CFP) and record the lifetimes of YFP/CFP Forster resonance energy transfer (FRET) pairs in whole cells. FRET analysis shows that that these complexes lie on average within 6 nm of each other. Complementary high-resolution AFM of intact, purified chromatophores verifies the close association of cytochrome bc1 complexes with RC-LH1-PufX dimers. Our results provide a structural basis for the close kinetic coupling observed by spectroscopy between RC-LH1-PufX and cytochrome bc1, and explain how quinols/quinones and cytochrome c2 shuttle on a millisecond timescale between these complexes, sustaining efficient photosynthetic electron flow.