*4.1. Electrochemical Methods*

Protein film electrochemistry (PFE) is a well-established and important technique to study protein modified electrodes and has proven powerful to probe the thermodynamic, kinetic and catalytic properties of redox proteins, typically with voltammetry [97–99]. PFE is also compatible with membrane-modified electrode and allows to probe the membrane proteins within detergent solutions, polymer matrices or model-membrane environment [100,101]. We refer to the following reviews for a detailed description of PFE methods [102–106].

Besides voltammetry, impedance spectroscopy has been used to study the electrode-membrane protein interface. The quality and the structure of electrode surfaces modified with planar lipid membranes (Figure 2e–h) are particularly suitable for investigation by electrochemical impedance spectroscopy (EIS). EIS can monitor the resistance and the capacitance of a planar membrane covering the electrode. For ideal planar lipid bilayers on the electrode, the capacitance value should be in the range of ~0.5 µF·cm−<sup>2</sup> with a resistance of >MΩ cm<sup>2</sup> [107]. Disorders and defects of these bilayers will result in lower resistance and/or higher capacitance values. For instance, we monitored the formation of multilayer membrane stacks (Figure 2j) by EIS [86] and observed only small reductions in capacitance upon the formation of each additional bilayer, indicating that the additional lipid bilayers permeable to ions and thus contain large or many defects. EIS can also provide information on whether protein incorporation in the membranes affects the electrode structure. For instance, incorporation of cytochrome *bo*<sup>3</sup> into tethered membranes (tBLM, Figure 2g) had almost no effect on the capacitance, indicating that cytochrome *bo*<sup>3</sup> did not induce large defects in the tBLM [75]. Similarly, only small changes in capacitance were observed of a hBLM (Figure 2e) after integrating human enzyme cytochrome P450, indicating that the hBLM retains its integrity upon protein adsorption [108]. Besides the membrane, the quality of a SAM on metal electrodes is also often evaluated with EIS. Compact, well-formed SAMs act as ideal insulating layers with high resistance values. The compactness of the SAM will influence subsequent protein immobilisation steps and can determine the thickness of the SAM, impacting on interfacial electron transfer kinetics.

Protein-film photoelectrochemistry (PF-PEC) is a PFE technique which has been developed for photosynthetic proteins [22,109–111]. PF-PEC combines illumination with PFE to investigate photoactive proteins. Voltammetry and chronoamperometry can be used to illustrate the charge transfer processes and the kinetics of the light driven reactions. Recently, other electrochemical setups have been used to investigate photosynthetic proteins, including rotating ring disk electrode (RRDE) and scanning electrochemical microscopy (SECM). The set-up of RRDE includes a central rotating disc electrode and a ring electrode surrounding it. The potential on these two electrodes can be controlled independently and products generated by a protein film at the central disk electrode are transferred to the ring electrode for electrochemical analysis. For instance, oxygenic photoreactivity of PSII was studied with RRDE [112] and products generated by PSII at the central disc, e.g., oxygen and radical species, were detected by the ring electrode. This study revealed ET pathways that generate reactive oxygen species and O<sup>2</sup> by PSII. SECM employs a microelectrode as a tip above the protein modified electrode to detect the products generated locally. It was used to monitor H<sup>2</sup> evolution of a PSI-Pt complex within redox polymer under illumination [113]. SECM has also been used to quantify the reduction of charge carriers (methyl viologen) by PSI and compared this to the photocurrent [114]. Methyl viologen is often used as a charge carrier to collect electron from PSI in biophotovoltaic systems, but reoxidation on the electrode leads to charge recombination. The authors compared PSI on gold and silicon surfaces, both with Os-based redox polymers. SECM showed that gold and silicon exhibits different photocurrents due to different charge recombination kinetics, i.e., electron transfer kinetics for the reoxidation of the methyl viologen radical cation. Finally, by monitoring the photocurrent and H2O<sup>2</sup> generation of a PSI photocathode with SECM, it was shown that light-induced formation of reactive oxygen species caused degradation of PSI modified electrodes [115].
