The Role of Electrostatic Binding Interfaces in the Performance of Bacterial Reaction Center Biophotoelectrodes

Abstract

Photosynthetic reaction centers (RCs) efficiently capture and convert solar radiation into electrochemical energy. Accordingly, RCs have the potential as components in biophotovoltaics, biofuel cells, and biosensors. Recent biophotoelectrodes containing the RC from the bacterium Rhodobacter sphaeroides utilize a natural electron donor, horse heart cytochrome c (cyt c), as an electron transfer mediator with the electrode. In this system, electrostatic interfaces largely control the protein-electrode and protein-protein interactions necessary for electron transfer. However, recent studies have revealed kinetic bottlenecks in cyt-mediated electron transfer that limit biohybrid photoelectrode efficiency. Here, we seek to understand how changing protein-protein and protein-electrode interactions influence RC turnover and biophotoelectrode efficiency. The RC-cyt c binding interaction was modified by substituting interfacial RC amino acids. Substitutions Asn-M188 to Asp and Gln-L264 to Glu, which are known to produce a higher cyt-binding affinity, led to a decrease in the RC turnover frequency (TOF) at the electrode, suggesting that a decrease in cyt c dissociation was rate-limiting in these RC variants. Conversely, an Asp-M88 to Lys substitution producing a lower binding affinity had little effect on the RC TOF, suggesting that a decrease in the cyt c association rate was not a rate-limiting factor. Modulating the electrode surface with a self-assembled monolayer that oriented the cyt c to face the electrode did not affect the RC TOF, suggesting that the orientation of cyt c was also not a rate-limiting factor. Changing the ionic strength of the electrolyte solution had the most potent impact on the RC TOF, indicating that cyt c mobility was important for effective electron donation to the photo-oxidized RC. An ultimate limitation for the RC TOF was that cyt c desorbed from the electrode at ionic strengths above 120 mM, diluting its local concentration near the electrode-adsorbed RCs and resulting in poor biophotoelectrode performance. These findings will guide further tuning of these interfaces for improved performance.

Figure 1

Reaction center structure and mutations. (Left) overview of the interaction complex between the Rba. sphaeroides RC and cyt c₂, taken from an X-ray crystal structure of the cocomplex (Protein Data Bank ID: 1L9B) and rendered using ChimeraX. The RC L-subunit (green), M-subunit (tan), and H-subunit (purple) are labeled. (Right) a zoomed-in view of the RC/cyt c binding interface depicting the location of the substituted residues Gln-L264 (blue carbons: changed to Glu), Asp-M88 (magenta carbons: changed to Lys), and Asn-M188 (cyan carbons: changed to Asp).

Figure 2

Biophotoelectrode configuration and mechanism. (a) Schematic depicting the composition and arrangement of the RC, cyt c, Q₀, and the mesoporous silver electrode (Ag^R). The mesoporous structure of the Ag^R is omitted for clarity. The electron transfer pathway is indicated by the black arrows. (b) Plot of the midpoint potentials of all components involved in the electron transfer pathway, including the bacteriochlorophyll pair (P870), sequential monomeric bacteriochlorophyll (BChl), bacteriopheophytin (BPhe), and ubiquinone (Q_A and Q_B) electron carriers. The added water-soluble Q₀ carries electrons to the Pt counter electrode (not shown).

Figure 3

Dependence of electrode performance on cyt c concentration. (a) Averaged photocurrents from WT RCs at an increasing cyt c concentration. Error bars are omitted for clarity. The period of illumination is indicated by the yellow bar. (b) Peak cathodic photocurrents as a function of cyt c concentration for four bioelectrodes with different RCs. (c) RC TOF as a function of cyt c concentration (symbols) overlaid with a Hill equation fit (lines), which converged with an R² over 0.99. All shown error bars represent standard deviations (n = 4).

Figure 4

Peak photocurrents at different ionic strengths as a function of cyt c concentration. In addition to KCl, the electrolyte buffer also contained 20 mM Tris-Cl, which was included in the ionic strength calculation. Lines show Hill equation fits of the data, all of which converged with an R² > 0.99.

Figure 5

Electrode functionalization. (a) Peak photocurrents are shown as a function of solution cyt c concentration from WT RCs adsorbed onto a bare or SAM-functionalized Ag^R electrode. The SAM consisted of a 3:1 ratio of mercaptoundecanol and mercaptoundecanoic acid. (b) RC TOF as a function of cyt c concentration. Error bars represent standard deviations (n = 3). Lines show Hill equation fits, all of which converged with an R² over 0.99.

Figure 6

Electrostatic interfaces and the proposed mechanism of electron transfer. This schematic depicts the adsorption and desorption of cyt c (mauve dashed arrows) onto an electrode until a binding equilibrated concentration (K_E) is reached. The cyt c heme is depicted in black. Following electrode reduction of oxidized cyt (k_et2/black solid arrow), cyt c diffuses (blue dashed arrow) and binds to the RC (green arrow) at a rate k_ON. An electron is transferred from cyt c to the photo-oxidized P870⁺ (yellow solid arrow) at a rate k_et. Finally, oxidized cyt c dissociates from the RC (red dashed arrow) at a rate k_OFF. Dashed arrows indicate diffusional processes, and solid arrows represent electron transfers.