The binding of photosystem I (PS I) from Thermosynechococcus elongatus to the native cytochrome (cyt) c₆ and cyt c from horse heart (cyt c_HH) was analyzed by oxygen consumption measurements, isothermal titration calorimetry (ITC), and rigid body docking combined with electrostatic computations of binding energies. Although PS I has a higher affinity for cyt c_HH than for cyt c₆, the influence of ionic strength and pH on binding is different in the two cases. ITC and theoretical computations revealed the existence of unspecific binding sites for cyt c_HH besides one specific binding site close to P₇₀₀ Binding to PS I was found to be the same for reduced and oxidized cyt c_HH Based on this information, suitable conditions for cocrystallization of cyt c_HH with PS I were found, resulting in crystals with a PS I:cyt c_HH ratio of 1:1. A crystal structure at 3.4-Å resolution was obtained, but cyt c_HH cannot be identified in the electron density map because of unspecific binding sites and/or high flexibility at the specific binding site. Modeling the binding of cyt c₆ to PS I revealed a specific binding site where the distance and orientation of cyt c₆ relative to P₇₀₀ are comparable with cyt c₂ from purple bacteria relative to P₈₇₀ This work provides new insights into the binding modes of different cytochromes to PS I, thus facilitating steps toward solving the PS I-cyt c costructure and a more detailed understanding of natural electron transport processes.

Photosystem I (PS I) has a symmetric structure with two highly similar branches of pigments at the center that are involved in electron transfer, but shows very different efficiency along the two branches. We have determined the structure of cyanobacterial PS I at room temperature (RT) using femtosecond X-ray pulses from an X-ray free electron laser (XFEL) that shows a clear expansion of the entire protein complex in the direction of the membrane plane, when compared to previous cryogenic structures. This trend was observed by complementary datasets taken at multiple XFEL beamlines. In the RT structure of PS I, we also observe conformational differences between the two branches in the reaction center around the secondary electron acceptors A_1A and A_1B. The π-stacked Phe residues are rotated with a more parallel orientation in the A-branch and an almost perpendicular confirmation in the B-branch, and the symmetry breaking PsaB-Trp673 is tilted and further away from A_1A. These changes increase the asymmetry between the branches and may provide insights into the preferential directionality of electron transfer.