*4.2. Spectroscopic Methods*

While electrochemical methods provide information about redox reactions, in situ spectroscopic methods can be applied to characterise the structure of the protein and the electrode assembly. Surface-enhanced Raman spectroscopy (SERS) is a surface-sensitive technique for molecules and proteins immobilised on roughened metallic surfaces, in particular Ag [116]. The SERS effect is due to a large increase in the Raman cross-section of molecules in contact with the metal surface, which is

enhanced at metallic nanostructured surfaces due to local surface plasmon resonance (LSPR) effects. SERS has been used to successfully characterise oxidised and reduced forms of adsorbed cytochromes (cytochrome *c*, haemoglobin and myoglobin) under precise potential control with low laser power [117]. In 2005, Hrabakova et al. showed that no structural changes occurred to the haem sites (e.g., *a* and *a*3) of cytochrome *c* oxidase embedded in a phospholipid bilayer tethered to a functionalised silver electrode via a histidine-tag [118]. In 2011, Weidinger group used SERS to study the electron transfer of MBH from *R. eutropha* H16 [119]. Interestingly, the authors compared the behaviour of the entire enzyme with just the transmembrane "anchor" subunit (*b*-type cytochrome) and determined two independent pathways for the electrons from the active site of the enzyme to the electrode. A slow rate pathway crosses all the three subunits of the enzyme, whilst a faster pathway only crosses two subunits and leaves out the transmembrane anchor subunit which, however, contributes to the stabilisation of the enzyme on the electrode.

A different variation of conventional infrared spectroscopy is represented by surface-enhanced infrared absorption spectroscopy (SEIRAS) in which the signal enhancement is due to plasmon resonance from a nanostructured metal thin film [120]. The Hellwig group used SEIRAS to characterise the deposition of gold NPs on gold electrodes, the modification of gold NPs with thiols and, finally, the absorption of cytochrome *bo*<sup>3</sup> on these modified electrodes [51,54]. Wiebalck et al. characterised the formation of a tBLM and incorporation of functional cytochrome *bo*<sup>3</sup> from *E. coli* by SEIRA spectroscopy [121].
