Single-Molecule Detection of the Encounter and Productive Electron Transfer Complexes of a Photosynthetic Reaction Center

Abstract

Small, diffusible redox proteins play an essential role in electron transfer (ET) in respiration and photosynthesis, sustaining life on Earth by shuttling electrons between membrane-bound complexes via finely tuned and reversible interactions. Ensemble kinetic studies show transient ET complexes form in two distinct stages: an "encounter" complex largely mediated by electrostatic interactions, which subsequently, through subtle reorganization of the binding interface, forms a "productive" ET complex stabilized by additional hydrophobic interactions around the redox-active cofactors. Here, using single-molecule force spectroscopy (SMFS) we dissected the transient ET complexes formed between the photosynthetic reaction center-light harvesting complex 1 (RC-LH1) of Rhodobacter sphaeroides and its native electron donor cytochrome c₂ (cyt c₂). Importantly, SMFS resolves the distribution of interaction forces into low (∼150 pN) and high (∼330 pN) components, with the former more susceptible to salt concentration and to alteration of key charged residues on the RC. Thus, the low force component is suggested to reflect the contribution of electrostatic interactions in forming the initial encounter complex, whereas the high force component reflects the additional stabilization provided by hydrophobic interactions to the productive ET complex. Employing molecular dynamics simulations, we resolve five intermediate states that comprise the encounter, productive ET and leaving complexes, predicting a weak interaction between cyt c₂ and the LH1 ring near the RC-L subunit that could lie along the exit path for oxidized cyt c₂. The multimodal nature of the interactions of ET complexes captured here may have wider implications for ET in all domains of life.

Figure 1

Affinity mapping of surface-attached wild-type (WT) RC-LH1 complexes using a cyt c_2-modified AFM probe. (A) The lumenal surface of the RC-LH1 complex of Rba. sphaeroides, showing acidic residues (red) and uncharged residues (yellow) involved in the binding interface with cyt c₂ (pale blue, with basic residues in blue). The RC is in pink and the surrounding LH1 complex is in gray. The binding interfaces for RC-LH1 and cyt c₂ are displayed in an open book form (PDB: RC-LH1 dimer–7PIL; RC-cyt c₂–1L9J). (B) The ET complex between RC-LH1 and cyt c₂ with color coding of residues as in panel A. The heme (red) and bacteriochlorophyll (BChl) (green) ET cofactors are also highlighted. (C) Schematic representation of cyt c₂ molecules attached to the AFM probe and RC-LH1 complexes attached to the functionalized SiOx substrate. The blue circled insets show protein attachment points, each of which is distal to the cyt c₂–RC binding interface. In the case of cyt c₂ a His₆-tag follows the C-terminal residue Pro124, and then a PEG linker is used to attach the His-tag to the AFM probe. For RC-LH1, the C-terminal Ala260 of the RC H-subunit is followed by a 14-residue linker including a thrombin cleavage sequence and then a His₁₀-tag. (D) AFM topography image (in liquid buffer) of individual WT RC-LH1 complexes on the SiOx substrate. The surface density is ∼300–500 molecules per μm². (E) Typical force–distance curves that exhibit the specific interaction between RC-LH1 complexes and cyt c₂ with a separation distance ∼10 nm.

Figure 2

Distribution of the interaction force between oxidized WT RC-LH1 complexes and reduced cyt c₂ versus the interaction probability. The histograms represent the distribution of the interaction forces measured by SMFS as oxidized WT RC-LH1 complexes and reduced cyt c₂ are brought into contact and then separated. (A–D) The salt concentration in the imaging buffer was increased from 10 to 200 mM, as indicated next to each histogram. The solid curves represent the best fit for each of the histogram peaks, with the low force component in red, the high force component in green, and the cumulative fit in dark blue. The respective forces, in pN, are shown above each component.

Figure 3

Force spectroscopy parameters in the WT versus D(M184)K, Q(L264)E and N(M188)D mutants of the RC-LH1 complex. The data were acquired upon the separation of complexes, initially brought into contact as oxidized RC-LH1 and reduced cyt c₂, at different salt concentrations in the imaging buffer. (A) Cumulative interaction probability. (B) Probability of the low force interaction event. (C) Interaction force for the low-force peak. (D) Interaction force for the high-force peak. The WT and mutant RC-LH1 complexes are color coded in panel A.

Figure 4

Force distributions for interactions between oxidized WT and mutant RC-LH1 complexes and oxidized or reduced cyt c₂. (A–D) Histograms representing the distribution of the interaction forces measured at 10 mM NaCl using either reduced (green bars) or oxidized (red bars) cyt c₂ as a partner for oxidized WT (A), N(M188)D (B), Q(L264)E (C) and D(M184)K (D) RC-LH1 complexes. The darker areas in each panel show the areas where the histograms for reduced cyt c₂ and oxidized cyt c₂ overlap. The solid curves in represent the best fit for each of the histogram peaks, with the low force component in red (reduced cyt c₂) and orange (oxidized cyt c₂), the high force component in green (reduced cyt c₂) and olive (oxidized cyt c₂), and the cumulative fit in dark blue (reduced cyt c₂) and light blue (oxidized cyt c₂).

Figure 5

BD simulations of cyt c₂ binding poses on the lumenal surface of the dimeric RC-LH1 complex. (A,B) BD simulation snapshots of a top-down (A) and side view (B) of the proximal (blue) and distal (green) cyt c₂ interaction with the RC core (red) and LH1 periphery (gray). Panel (B) includes a zoomed-in image of the RC/cyt c₂ interaction viewed from the side. (C) The distribution of the calculated K_D values for each binding pose on the proximal and distal sites.

Figure 6

Surface distribution and SMFS interaction force distribution of oxidized RC-only complexes with reduced cyt c₂. Surface topography of immobilized RC complexes (A) and SMFS results (B) showing distribution of the interaction force between the oxidized RC and reduced cyt c₂ complexes versus the interaction probability. The solid curves in (B) represent the best fit for each of the histogram peaks, with the low force component in red, the high force component in green, and the cumulative fit in dark blue. The respective forces, in pN, are shown above each component.

Figure 7

RC-only SMD simulations of cyt c₂ entering along the M-subunit and exiting along the L-subunit. (A) SMD simulation snapshots of a top-down view of cyt c₂ (blue) being pulled horizontally across both the M and L-subunit of the RC (pink). Snapshots correspond with the peak interaction energies on the outsets in (B). Cyt c₂ is in its reduced state when pulling along the M-subunit and oxidized when pulling along the L-subunit. The RC is kept in a neutral state. (B) Distribution of interaction energies between cyt c₂ and RC as the cyt c₂ travels along the entrance pathway (M-side, maroon color) and exit pathway (L-side, blue color). The x-axis represents the distance between donor and acceptor. There are two separate energy graphs, one for the entry pathway and the other for the exit pathway, with a central gap corresponding to the S0 state. The two histograms on the outsets show the distribution of interaction energies between (left) reduced cyt c₂ and the RC for the M-side entry pathway, and (right) oxidized cyt c₂ and the RC for the L-side exit pathway.