Unexpected Heme Redox Potential Values Implicate an Uphill Step in Cytochrome b 6 f .

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 (Q n and Q p ). Typically, this ET occurs from the low- to high-potential heme b , but in photosynthetic cytochrome b 6 f , the redox midpoint potentials ( E m s) 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 b 6 f and revealed that E m of heme b n 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 Q p and Q n sites and modulate the efficiency of cyclic and linear ET in photosynthesis.