Abstract Electron transfer pathways in photosynthesis involve interactions between membrane-bound complexes such as reaction centres with an extrinsic partner. In this study, the biological specificity of electron transfer between the reaction centre-light-harvesting 1-PufX complex and its extrinsic electron donor, cytochrome c 2, formed the basis for mapping the location of surface-attached RC-LH1-PufX complexes using atomic force microscopy (AFM). This nano-mechanical mapping method used an AFM probe functionalised with cyt c 2 molecules to quantify the interaction forces involved, at the single-molecule level under native conditions. With surface-bound RC-His12-LH1-PufX complexes in the photo-oxidised state, the mean interaction force with cyt c 2 is approximately 480 pN with an interaction frequency of around 66 %. The latter value lowered 5.5-fold when chemically reduced RC-His12-LH1-PufX complexes are imaged in the dark to abolish electron transfer from cyt c 2 to the RC. The correspondence between topographic and adhesion images recorded over the same area of the sample shows that affinity-based AFM methods are a useful tool when topology alone is insufficient for spatially locating proteins at the surface of photosynthetic membranes.