5.1.1. Electron Transfer in Metabolic Energy Chains

Most cellular functions, such as cell motility, key molecule biosynthesis or transport across membranes, require energy. Life energy is generated by redox transformations of nutrients present in the environment or by sunlight to create the proton gradient across the membrane. This gradient is later used to produce ATP, the universal molecular "coin" driving metabolic reactions. In these processes, complex but highly specific molecular chains are involved that couple electron transfer (ET) to the diffusion of protons across cell membranes. Focusing on bacteria, a wide range of energy substrates can be metabolized: lactate, glucose, H2, H2S, O2, sulfate, fumarate, etc. thanks to the presence of a large variety of redox enzymes interacting with membrane lipids or with redox proteins acting as electron shuttles. To enable metabolism achievement, ET must proceed fast through highly specific protein–protein interactions. According to Marcus theory, the distance between the donor and the acceptor will drive the rate of ET [193]. It is admitted that electrostatic interactions serve for pre-orientation of interacting partners; then, hydrophobic interactions drive a rearrangement of the transitory complex allowing ET [194]. Pre-orientation is guided by the heterogeneity of charge distribution on the protein surface which induces dipole moments that can be as high as thousands of debyes [112]. As such, one or more amino acids defining a charged patch on the surface of one protein partner are often involved for the specific interaction with the other partner.

## 5.1.2. Electron Transfer Mechanisms Involving Redox Enzymes at Electrochemical Interfaces

Redox enzymes can operate very efficiently in bioelectrocatalytic processes for the redox transformation of a variety of substrates. As stated in the introduction of this review, various applicative domains are concerned from bioelectrosensing, bioenergy to bioelectrosynthesis. One prerequisite is the use of a conductive support, the electrode, to provide or accept the electrons for the redox transformation to take place. The question directly arises whether the electrode would gently replace the physiological redox partner? Actually, many materials used as electrodes (carbon, gold, nano- or porous structures, etc.) can be chemically functionalized to provide an enzyme environment mimicking the physiological one. It was clearly demonstrated that the oriented approach of the enzyme to the electrode surface was guided by electrostatic interactions driven by the enzyme dipole moment. The example of multicopper oxidases (MCO), the key enzymes for O<sup>2</sup> reduction into water, is highly relevant in that sense. Four copper sites are involved in the catalytic process, CuT1 being the first electron acceptor, and electrons generated travelling intramolecularly to the trinuclear Cu site where O<sup>2</sup> binds and is reduced. Bilirubin oxidase (BOD) from the fungus *Myrothecium verrucaria* has a dipole moment of 910 D at pH 5 pointing positive towards the CuT1. At the same pH, BOD from the bacterium *B. pumilus* and laccase (Lac) from *Thermus thermophilus* have dipoles moments of 1830 and 860 D, respectively, but pointing positive opposite to the CuT1. In accordance with this structural feature, ET was obtained on negatively or positively charged electrodes in the case of *M. verrucaria* BOD or *B. pumilus* BOD and *T. thermophilus* LAC, respectively [116,195,196]. One specificity of bioelectrocatalysis stems from the substrate and product coming in and out to/from the active site that may be hampered by the required enzyme orientation for ET. Not only activity but also stability in case of substrate or product accumulation may be affected [197]. This point is however understudied in bioelectrochemistry. When direct ET (DET) is not possible, mainly because there is no surface electron relay identified, either because the enzyme 3D structure is not known or because the active site is electrically isolated in the protein moiety, mediated ET (MET) can be used instead. Typically, MCOs, enzymes with hemic cofactor such as cellulose dehydrogenase, and enzymes with surface FeS clusters such as HASEs are reported to undergo DET, while GOx is only electrochemically addressed through MET. In this latter case, a small molecule acting as a fast and reversible electrochemical system will make the electron shuttle between the electrode and the FAD cofactor in the enzyme. Redox potential of the redox mediator as well as its affinity for the enzyme will drive the electrocatalysis. One important question is whether the enzyme will be more stable under DET or MET processes (Figure 10).

**Figure 10.** Scheme of a typical bioelectrochemical experiment showing the set up and the adsorption of a protein on the electrode surface either in a MET or a DET mode. The active site of the enzyme, the electronic relays and the redox mediator are represented as red squares, dark blue squares, and light blue spheres, respectively. CE, W, and ref represent counter, working, and reference electrodes, respectively.
