Dissecting the cytochrome c2–reaction centre interaction in bacterial photosynthesis using single molecule force spectroscopy

Abstract

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.

Capacity and kinetics of light-induced cytochrome oxidation in intact cells of photosynthetic bacteria

“…Several other studies showed preferential binding of oxidized cytochrome c 2 to RCs 33 , to the extent that the oxidized cytochrome impeded the access of reduced cytochrome c 2 to its docking site on the RC. Using single molecule force spectroscopy, surprisingly large forces between 112 and 887 pN were required to pull apart the P–oxidized cytochrome c 2 complex 34 . They found a duration of milliseconds for the persistence of the post-ET oxidized cytochrome c 2 –P “product” state compatible with rates of cyclic photosynthetic ET.…”

Capacity and kinetics of light-induced cytochrome oxidation in intact cells of photosynthetic bacteria

“…Several other studies showed preferential binding of oxidized cytochrome c 2 to RCs 29 , to the extent that the oxidised cytochrome impeded the access of reduced cytochrome c 2 to its docking site on the RC. Using single molecule force spectroscopy, surprisingly large forces between 112 and 887 pN were required to pull apart the P-oxidized cytochrome c 2 complex 30 . They found a duration of milliseconds for the persistence of the post-ET oxidized cytochrome c 2 -P "product" state compatible with rates of cyclic photosynthetic ET.…”

Light- and Redox-Dependent Force Spectroscopy Reveals that the Interaction between Plastocyanin and Plant Photosystem I Is Favored when One Partner Is Ready for Electron Transfer

“…Additionally, the authors indicated the importance of the redox states of both C c 2 and RC redox prosthetic groups ( haem cofactor for C c 2 and special pair bacteriochlorophyll dimer for RC, respectively). In particular, they observed higher binding frequency when the proteins were in a redox state ready for physiological ET (reduced C c 2 and oxidized RC). , …”

“…They observed an occurrence of Cyt b6f -Pc binding interactions (binding frequency) that was 5-fold higher when the redox states of the proteins were opposite (e.g., oxidized/reduced), compared to the same redox states (e.g., oxidized/oxidized or reduced/reduced). However, they did not observe significant differences in force. , Vasilev et al studied the interaction between cytochrome c 2 (C c 2 ) and the photo-oxidized reaction center (RC) from purple photosynthetic bacteria. , In those studies, RC photo-oxidation was induced using nonspecific illumination (white light) with low intensity (between 1 and 5% of the intensity of sunlight). They found that the dissociation of the RC-C c 2 complex was not spontaneous after the ET process and required the application of an external force.…”

“…11,12 Vasilev et al studied the interaction between cytochrome c 2 (Cc 2 ) and the photo-oxidized reaction center (RC) from purple photosynthetic bacteria. 12,13 In those studies, RC photo-oxidation was induced using nonspecific illumination (white light) with low intensity (between 1 and 5% of the intensity of sunlight). They found that the dissociation of the RC-Cc 2 complex was not spontaneous after the ET process and required the application of an external force.…”