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

Milo R van Moort; Michael R Jones; Raoul N Frese; Vincent M Friebe

doi:10.1021/acssuschemeng.2c06769

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

ACS Sustain Chem Eng. 2023 Feb 7;11(7):3044-3051. doi: 10.1021/acssuschemeng.2c06769. eCollection 2023 Feb 20.

Authors

Milo R van Moort^{1

2}, Michael R Jones³, Raoul N Frese^{1

2}, Vincent M Friebe^{1

2

4}

Affiliations

¹ Biophysics of Photosynthesis, Department of Physics and Astronomy, Faculty of Science, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands.
² LaserLaB Amsterdam, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands.
³ School of Biochemistry, Biomedical Sciences Building, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.
⁴ Campus Straubing for Biotechnology and Sustainability, Technical University of Munich, Uferstraße 53, 94315 Straubing, Germany.

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.