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 (Q_n and Q_p). Typically, this ET occurs from the low- to high-potential heme b, but in photosynthetic cytochrome b₆f, the redox midpoint potentials (E_ms) 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 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.