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