High-resolution cryo-EM structures of plant cytochrome b6f at work.

Plants use solar energy to power cellular metabolism. The oxidation of plastoquinol and reduction of plastocyanin by cytochrome b6f (Cyt b6f) is known as one of the key steps of photosynthesis, but the catalytic mechanism in the plastoquinone oxidation site (Qp) remains elusive. Here, we describe two high-resolution cryo-EM structures of the spinach Cyt b6f homodimer with endogenous plastoquinones and in complex with plastocyanin. Three plastoquinones are visible and line up one after another head to tail near Qp in both monomers, indicating the existence of a channel in each monomer. Therefore, quinones appear to flow through Cyt b6f in one direction, transiently exposing the redox-active ring of quinone during catalysis. Our work proposes an unprecedented one-way traffic model that explains efficient quinol oxidation during photosynthesis and respiration.

Unexpected Heme Redox Potential Values Implicate an Uphill Step in Cytochrome b6f.

Cytochromes bc, key enzymes of respiration and photosynthesis, contain a highly conserved two-heme motif supporting cross-membrane electron transport (ET) that connects the two catalytic quinone-binding sites (Qn and Qp). Typically, this ET occurs from the low- to high-potential heme b, but in photosynthetic cytochrome b6f, the redox midpoint potentials (Ems) of these hemes remain uncertain. Our systematic redox titration analysis based on three independent and comprehensive low-temperature spectroscopies (continuous wave and pulse electron paramagnetic resonance (EPR) and optical spectroscopies) allowed for unambiguous assignment of spectral components of hemes in cytochrome b6f and revealed that Em of heme bn is unexpectedly low. Consequently, the cross-membrane ET occurs from the high- to low-potential heme introducing an uphill step in the energy landscape for the catalytic reaction. This slows down the ET through a low-potential chain, which can influence the mechanisms of reactions taking place at both Qp and Qn sites and modulate the efficiency of cyclic and linear ET in photosynthesis.