Cytochrome c Provides an Electron-Funneling Antenna for Efficient Photocurrent Generation in a Reaction Center Biophotocathode

Abstract

The high quantum efficiency of photosynthetic reaction centers (RCs) makes them attractive for bioelectronic and biophotovoltaic applications. However, much of the native RC efficiency is lost in communication between surface-bound RCs and electrode materials. The state-of-the-art biophotoelectrodes utilizing cytochrome c (cyt c) as a biological wiring agent have at best approached 32% retained RC quantum efficiency. However, bottlenecks in cyt c-mediated electron transfer have not yet been fully elucidated. In this work, protein film voltammetry in conjunction with photoelectrochemistry is used to show that cyt c acts as an electron-funneling antennae that shuttle electrons from a functionalized rough silver electrode to surface-immobilized RCs. The arrangement of the two proteins on the electrode surface is characterized, revealing that RCs attached directly to the electrode via hydrophobic interactions and that a film of six cyt c per RC electrostatically bound to the electrode. We show that the additional electrical connectivity within a film of cyt c improves the high turnover demands of surface-bound RCs. This results in larger photocurrent onset potentials, positively shifted half-wave reduction potentials, and higher photocurrent densities reaching 100 μA cm^-2. These findings are fundamental for the optimization of bioelectronics that utilize the ubiquitous cyt c redox proteins as biological wires to exploit electrode-bound enzymes.

Keywords: biophotoelectrochemistry; biophotovoltaics; biosolar cells; cytochrome c; reaction center.

Figure 1

Biophotocathode schematic. (a) Proposed ET mechanism in the Ag^R|mSAM|cyt c|RC working electrode. (b) Photocurrent density response upon illumination beginning at 50 s and ending at 170 s at −50 mV vs Ag/AgCl.

Figure 2

Cyt c-electrode-binding affinity. (a) Baseline-subtracted CVs of a Ag^R|mSAM electrode as a function of solution cyt c concentration. (b) Γ_cytc derived from baseline-subtracted peak integration of plot (a) on electrodes prefunctionalized with RCs (Ag^R|mSAM|cyt c|RC) and without RCs (Ag^R|mSAM|cyt c). The preloaded electrode had a Γ_RC of 53 pmol cm^–2. (c) Γ_cytc as a function of ionic strength. The loading measurements were taken after a 2 min incubation in the presence of KCl at the concentrations indicated. KCl was not present in the cell during CV measurements. Error bars represent 1 standard deviation, with n = 4.

Figure 3

Binding of RCs to the electrode. Peak photocurrents from Ag^R|mSAM|cyt c|RC electrodes, whereby the cyt c was drop-casted first, followed by the RC, and vice versa. The latter electrode was then sequentially treated with 1 M KCl, drop-casted with cyt c (50 μM for 5 min), treated with 1% DDM and again drop-cast with cyt c. This last incubation (cyt c fourth) step ensured the cyt c monolayer was re-established, and that any photocurrent decrease attributed to RC desorption. Each step was interceded with rinsing in milli-Q for 15 min. Error bars represent standard deviation, n = 4.

Figure 4

Effects of cyt c loading on electrode performance. (a) Cyclic voltammetry at low (292 pmol cm^–2), medium (155 pmol cm^–2), and high (55 pmol cm^–2) Γ_cytc. (b) CV E°′_cytc vs E_hw from the pCV in full vs striped bars. (c) pCVs recorded under forced convection at a sweep rate of 10 mV s^–1. (d) Schematic depicting the electrode surface at low Γ_cytc. (e) Schematic depicting the electrode surface at high Γ_cytc.

Figure 5

Improvement of RC turnover at high Γ_cytc. Photocurrents measured as a function of irradiance at high, medium, and low Γ_cytc are plotted in black, red, and blue, respectively. Michaelis–Menten fits are shown as dashed lines, with R² > 0.999. Error bars represent standard deviation with n = 4. The photon absorption rate of the RC as a function of irradiance is shown on the top axis.

Figure 6

Effect of cyt c binding and mobility on photocurrent. (a) Photocurrent density as a function of ionic strength (left axis, black) and Γ_cytc (right axis, red). (b) Effect of cross-linking Ag^R|mSAM|cyt c|RC electrodes with carbodiimide (EDC) and glutaraldehyde (GLUT) on photocurrent (black) and Γ_cytc (red).