*4.3. Spectroelectrochemistry*

Spectroelectrochemistry combines electrochemistry and spectroscopic approaches to monitor a specific molecular response while varying the electrode potential [122,123]. This can be done with proteins in solution [124] or immobilised on the electrode surface [125,126]. Insights into reaction mechanisms of membrane enzymes could be gained by methods such as UV-vis spectroelectrochemistry and infrared spectroelectrochemistry. UV-vis spectra of a protein are often specific for the redox state of a cofactor, e.g., haems, FeS clusters or flavins. A common application is a spectroelectrochemical titration to determine the reduction potential of a cofactor in an enzyme in solution, where results are usually evaluated by fitting the titration data to the Nernst equation. An example is a study of the cytochrome *bc<sup>1</sup>* complex where the midpoint potentials of the cofactors were determined by UV-vis and IR spectroelectrochemical titrations [127,128]. The redox potentials of the primary electron acceptor pheophytin *a* in photosystem II [129] and the primary electron donor P700 in photosystem I [130] were also revealed by UV-vis spectroelectrochemistry with proteins in solution. UV-vis spectroelectrochemistry can also be used to study proteins on electrode surfaces. For instance, Haas et al. used UV-vis spectroelectrochemical titrations to determine the midpoint potentials for cytochrome *c* oxidase either in solution or in a Langmuir-Blodgett monolayer film [131]. A multi-protein system was studied with UV-vis spectroelectrochemistry in which an outer-membrane cytochrome, MtrC from *Shewanella oneidensis* MR-1 (a peripheral membrane protein), was used as an electron conduit between an electrode and other redox enzymes. MtrC was shown to be an effectively transfer-electron conduit by monitoring the absorbance of reduced FeII-haems in MtrC [132].

Infrared (IR) spectroscopy is a powerful technique to detect structural changes of proteins during or in response to an electrochemical reaction although few studies have been reported for membrane proteins. Reactions can be triggered either by light or redox potential and infrared difference spectroscopy of proteins is typically measured to investigate the reaction mechanisms. This approach has been developed for membrane proteins involved in photosynthesis, respiration and metabolic pathways [100]. Electrochemical SEIRA was used to study the conformational changes of the *R. sphaeroides* cytochrome *c* oxidase induced by direct electron transfer in ptBLM system [133]. The cytochrome *c* oxidase was shown to change from a non-activated to an activated state after it involving enzymatic reaction. By applying periodic potential pulses switching between −800 mV and open circuit potential to control the state of cytochrome *c* oxidase, the kinetics of the conformational

changes was monitored by time-resolved SEIRA spectroelectrochemistry [134]. The bacterial respiratory ubiquinol/cytochrome *bo*<sup>3</sup> couple was incorporated into a tethered bilayer lipid membrane (tBLM) on SAM modified electrode. The transmembrane proton gradient was successfully monitored by spectroelectrochemical SEIRA [121].
