Dissecting the cytochrome c₂-reaction centre interaction in bacterial photosynthesis using single molecule force...

The reversible docking of small, diffusible redox proteins onto a membrane protein complex is a common feature of bacterial, mitochondrial and photosynthetic electron transfer (ET) chains. Spectroscopic studies of ensembles of such redox partners have been used to determine ET rates and dissociation constants. Here, we report a single-molecule analysis of the forces that stabilise transient ET complexes. We examined the interaction of two components of bacterial photosynthesis, cytochrome c₂ and the reaction centre (RC) complex, using dynamic force spectroscopy and PeakForce quantitative nanomechanical imaging. RC-LH1-PufX complexes, attached to silicon nitride AFM probes and maintained in a photo-oxidised state, were lowered onto a silicon oxide substrate bearing dispersed, immobilised and reduced cytochrome c₂ molecules. Microscale patterns of cytochrome c₂ and the cyan fluorescent protein were used to validate the specificity of recognition between tip-attached RCs and surface-tethered cytochrome c₂ Following the transient association of photo-oxidised RC and reduced cytochrome c₂ molecules, retraction of the RC-functionalised probe met with resistance, and forces between 112 and 887 pN were required to disrupt the post-ET RC-c₂ complex, depending on the retraction velocities used. If tip-attached RCs were reduced instead, the probability of interaction with reduced cytochrome c₂ molecules decreased 5-fold. Thus, the redox states of the cytochrome c₂ haem cofactor and RC 'special pair' bacteriochlorophyll dimer are important for establishing a productive ET complex. The millisecond persistence of the post-ET cytochrome c₂[oxidised]-RC[reduced] 'product' state is compatible with rates of cyclic photosynthetic ET, at physiologically relevant light intensities.