*3.4. Redox Polymers*

Redox polymers are widely used for wiring redox proteins on electrode surface (Figure 2d) [59]. Redox polymers act simultaneously as immobilisation matrix and as redox mediators. High coverages of proteins can be achieved and the ubiquitous localisation of redox mediators bound to the polymer matrix negates the need to control protein orientation. These polymer-bound redox mediators overcome mass transport limitations, typically observed with freely diffusing redox mediators. The chemical and physical properties of the redox polymers can be tailored by tuning the polymer backbone and the redox mediators. The Schuhmann group has extensively explored Os redox polymers to "wire" DDM-solubilised PSI [60] and PSII [61] on electrode surfaces for photocurrent generation. A two-compartment cell with a PSII photoanode and a PSI photocathode was constructed to mimic the Z-scheme of natural photosynthesis and an open-circuit voltage (OCV) of 90 mV was achieved [62]. By tuning the redox potential of Os complexes in the redox polymers to match the redox sites of the proteins (PSII and PSI), the OCV could be increased from 90 mV to 372 mV [63]. Interestingly, by exploiting the pH-dependent properties of Os-modified polymer, the group of Plumeré improved the interfacial electron transfer rates for the PSI photocathode, even exceeding rates observed in natural photosynthesis [64].

#### *3.5. Membrane Modified Electrode*

Because of the nature and functions of the membrane proteins, it is important that their assembly on electrodes preserves their structural integrity and functionality. In the examples provided so far, detergents are used to retain stability of the immobilised membrane enzymes. Although detergents are required to accommodate the amphiphilic nature of membrane proteins, they also have adverse effects to membrane protein stability and are likely to influence the electrode-protein interaction. To mimic the native environment of membrane proteins, electrodes have been modified with model membranes, as reviewed previously [65–67]. These membrane-modified electrodes can be categorised into hybrid bilayer lipid membrane (hBLM) system (Figure 2e), solid supported bilayer lipid membrane (sBLM) system (Figure 2f), tethered bilayer lipid membrane (tBLM) system (Figure 2g), and protein tethered bilayer lipid membrane (ptBLM) system (Figure 2h).

In hBLM systems a phospholipid layer is absorbed onto a self-assembled monolayer (SAM) of alkylthiols (Figure 2e). This strategy was used by the Hawkridge group to study cytochrome *c* oxidase immobilised on gold electrode [68,69]. In sBLMs, a lipid bilayer is non-covalently bound to the electrode surface (Figure 2f). Photosynthetic reaction centres including *Rhodobacter sphaeroides* RC [70,71], spinach photosystem I [72] and photosystem II [73] have been integrated into sBLMs on pyrolytic graphite. Electron transfer between electrode and the aforementioned proteins was achieved and showed well-defined peaks in voltammetry which corresponded to the redox sites of the proteins. Noji et al. incorporated *Rhodopseudomonas palustris* RC-LH I into sBLMs on an ITO electrode. Anionic phospholipid like phosphatidylglycerol (PG) were shown to stabilise the charge-separated state of RC-LH I and enhance the photocurrent [74]. In tBLM systems, the membrane is 'tethered' to the lipid-modified electrode surface via a linker (Figure 2g). Cytochrome *bo*<sup>3</sup> has been incorporated into tBLMs and was shown to retain its catalytic activity [75]. We previously included the membrane-bound [NiFe]-hydrogenases (MBH) from *R. eutropha* into a tBLM approach and used electrochemistry to study the activity of the entire heterotrimeric membrane-bound form of the enzyme [76]. In 2016, Pelster and Minteer isolated mitochondrial electron transport chain (ECT) enzymes, reconstituted them into liposomes and immobilised them onto a gold electrode in a tBLM. The authors used this reconstructed mitochondrial inner membrane biomimic to show the interdependence of the different complexes on bioelectrocatalytic activity [77]. In ptBLMs, the membrane protein is first anchored to an electrode via a His-tag/NTA interaction and subsequently reconstituted into a lipid membrane (Figure 2h). Ataka et al. have immobilised the *Rhodobacter sphaeroides* cytochrome *aa*<sup>3</sup> (a cytochrome *c* oxidase) by using a ptBLM system [78–80]. Two opposite protein orientations were compared, by varying the position of the His-tag. Cytochrome *c* bound and exchanged electrons with cytochrome *c* oxidase only when

the latter was orientated with its subunit II (the binding domain for cytochrome *c*) facing the bulk aqueous medium.
