*2.2. Membrane Proteins in Photosynthesis*

Photosynthesis is the natural process by which photosynthetic organisms convert light energy into chemical forms. The key steps, which include light harvesting, charge separation and electron transport, occur in the membrane. For oxygenic photosynthesis in algae, higher plants and cyanobacteria, two photosynthetic complexes are involved: photosystem I (PSI) and photosystem II (PSII) [17], each with a peripheral antenna system: light harvesting complex I (LH I) for PSI and light harvesting complex II (LH II) for PSII [18]. Anoxygenic photosynthesis in bacteria, such as purple bacteria, is conducted by just one type of photosystem [19]. There are two types of light-harvesting complexes in most purple bacteria. The light-harvesting complex I and reaction centre forms the RC-LH I complex which is surrounded by multiple light-harvesting complex II [20]. In both oxygenic and anoxygenic photosynthesis, light-harvesting complexes exert the vital function to effectively absorb light and transfer the energy to the reaction centres [21]. Light-harvesting complexes absorb a limited spectral range depending on their natural pigment. The light-induced charge separation occurs at the reaction centres: PSII catalyses the light-driven water oxidation to reduce quinones, while the reaction centre of purple bacteria only catalyses the reduction of quinones (part of the cyclic electron transfer process). PSI is not catalytically active but instead serves as an "electron pump" which can be potentially coupled to the other redox catalysts. Electrodes interfaced with photosynthetic proteins have found broad applications in biosensors, biophotovoltaic cells and solar fuels generation [5,22,23].

## **3. Membrane Protein Electrode Design**

Redox enzymes are molecular electrocatalysts that can function either in solution or while immobilised on an electrode surface. Immobilising enzymes on the electrodes has several advantages for electrocatalysis and this review will consider only immobilised systems. It is important that the enzymes retain their structural integrity and catalytic activity upon immobilisation [24]. Unlike soluble proteins, transmembrane proteins exist within lipid membranes and, consequently, are less stable in an aqueous environment where the amphiphilic properties of membrane proteins can lead to aggregation and denaturation. Therefore, it is challenging to retain the stability and function of membrane proteins in in vitro studies [25]. Suitable detergents or mixed lipid/detergent systems are needed to maintain an amphiphilic environment surrounding the membrane protein and mimic the original membrane conditions [26,27]. Alternatively, for some multisubunit, heteromeric membrane proteins the solubility problem can be circumvented by either purifying only the soluble subunits or engineering the protein to express only the soluble subunits [28]. For instance, the large and small subunits of membrane-bound hydrogenases can be separated from the transmembrane "anchor" subunit (*b*-type cytochrome). More sophisticated approaches have been developed for the mammalian membrane-bound cytochrome P450 that has been bioengineered without the hydrophobic membrane anchor domain [29]. However, similar methods are not always applicable for other proteins, either because the catalytic centre is located in a polytopic membrane subunit or because the water-soluble subunits on their own are not stable. It is worth mentioning that the activity of certain membrane proteins can be dependent on the presence of particular annular lipids [30] and therefore the strategies that improve the solubility by removing the need of the membrane environment can have some potential downsides.

Various immobilisation methods have been developed to achieve efficient electron transfer between a membrane protein and an electrode. There are several aspects to consider when designing and assembling (membrane) protein modified electrodes for bioelectrocatalysis: (1) orientation of the protein on the electrode surface, (2) preservation of the protein structural integrity and functionality, (3) low overpotential to minimise the energy loss, (4) protein loading of the electrode [24,31,32]. Some methods developed for electrocatalysis using soluble proteins can be adapted to detergent solubilised membrane proteins. An alternative strategy to the use of detergent is represented by reconstitution of membrane proteins within a lipid membrane on the electrode surface and several strategies have been developed to achieve this. We will discuss the most commonly used immobilisation methods.
