Single-molecule study of redox control involved in establishing the spinach plastocyanin-cytochrome b₆f electron...

Small diffusible redox proteins play a ubiquitous role in bioenergetic systems, facilitating electron transfer (ET) between membrane bound complexes. Sustaining high ET turnover rates requires that the association between extrinsic and membrane-bound partners is highly specific, yet also sufficiently weak to promote rapid post-ET separation. In oxygenic photosynthesis the small soluble electron carrier protein plastocyanin (Pc) shuttles electrons between the membrane integral cytochrome b₆f (cytb₆f) and photosystem I (PSI) complexes. Here we use peak-force quantitative nanomechanical mapping (PF-QNM) atomic force microscopy (AFM) to quantify the dynamic forces involved in transient interactions between cognate ET partners. An AFM probe functionalised with Pc molecules is brought into contact with cytb₆f complexes, immobilised on a planar silicon surface. PF-QNM interrogates the unbinding force of the cytb₆f-Pc interactions at the single molecule level with picoNewton force resolution and on a time scale comparable to the ET time in vivo (ca. 120 micros). Using this approach, we show that although the unbinding force remains unchanged the interaction frequency increases over five-fold when Pc and cytb₆f are in opposite redox states, so complementary charges on the cytb₆f and Pc cofactors likely contribute to the electrostatic forces that initiate formation of the ET complex. These results suggest that formation of the docking interface is under redox state control, which lowers the probability of unproductive encounters between Pc and cytb₆f molecules in the same redox state, ensuring the efficiency and directionality of this central reaction in the 'Z-scheme' of photosynthetic ET.