3.1.1. Cytochrome *c*

As an electrochemical paradigm redox metalloprotein target, cytochrome *c* (cyt *c*) is a soluble heme protein extensively studied also as a model metalloprotein on thiol SAM surfaces [64,82–87]. Comprising 105 amino acid residues, cyt *c* (horse heart, ca. MW: 12.4 kDa) is an electron transport protein largely present in eukaryotic cells [88]. Cyt *c* is an ideal model system enabling an understanding of protein ET mechanisms in electrochemistry and homogeneous solution. The interfacial ET rate constant (*kapp*) can be obtained based on the Laviron equation [89]. Electroreflectance spectroscopy (ER) has been utilized to obtain more accurate *kapp* due to the elimination of the capacitive double-layer charging current [83]. Horse heart cyt *c* is the most studied cytochrome, containing a number of positively charged lysine residues around the heme edge. Cyt *c* docks electrostatically with natural partners including cyt *c* oxidases/peroxidases. To immobilize cyt *c*, SAMs with carboxyl terminal groups are suitable due to favorable electrostatic binding [84]. Collinson and associates demonstrated that horse heart cyt *c* shows similar orientations on the carboxyl terminated SAM-modified electrode for both covalent bonding and electrostatic adsorption, but covalent bonding led to more stable immobilization [85]. It was also noted that the formal redox potential (E◦ ) of electrostatically adsorbed horse heart cyt *c* is shifted negatively due to the electrostatic interactions with the negatively charged SAM surface.

SAMs, consisting of a mixture of long-chain pyridine alkanethiols and short-chain alkanethiols, enhance the interfacial *kapp* because of more favorable electronic coupling between cyt *c* and the electrodes [84,86] than for pure SAMs. The effect of lysine residues on interfacial ET was explored by substituting lysine residues at specific positions. Niki and associates reported that replacement of lysine-13 with alanine in rat cyt *c* (RC9-K13A) showed a more than five-fold ET rate decrease compared with replacing lysine-72 and lysine-79 [90], which suggests that lysine-13 exhibits optimized coupling with the carboxyl SAM-modified electrode. Direct bonding to the heme group with axial pyridine or imidazole ligands onto the gold surfaces is another effective method for narrow orientation distribution of cyt *c* [84,87]. The tunneling distance-dependent ET was also investigated by surface-enhanced resonance Raman (SERR) spectroscopy, showing a declining signal with increasing SAM chain length from 2-mercaptoacetic acid to 16-mercaptohexadecanoic acid [91].

AuNPs enhance the interfacial ET rate of cyt *c* in bioelectrocatalysis. Insertion of 3–4 nm coated AuNPs between cyt *c* and the a SAM-modified Au(111)-electrode surfaces was shown to increase *kapp* by more than an order of magnitude [89] in spite of an ET distance increase exceeding 50 Å. This raises issues relating to the mechanism of the AuNP promotion even of simple ET processes, discussed in detail recently [92,93]. Engelbrekt and associates reported ultra-stable starch-coated AuNPs, enabling a clear redox signal of yeast cyt *c* on AuNP-modified basal plane graphite (BPG) electrodes but no signals on bare BPG and Au(111) electrode [94].

Other cytochromes, such as cyt *b* and cyt *c*4, have also been investigated. Della Pia and associates reported that ET between the heme group in cyt *b*<sup>562</sup> and the Au(111) electrode can be promoted by replacing the original aspartic acid residue with a cysteine residue, which provided specific protein orientation through a Au-S bond [95]. Chi and associates studied the interfacial and intramolecular ET kinetics of di-heme *Pseudomonas stutzeri* cyt *c*<sup>4</sup> compared with horse heart cyt *c* (Figure 3) [64]. *In situ* STM showed directly that the dipolar cyt *c*<sup>4</sup> is vertically oriented on the carboxyl SAM-modified Au(111) electrode (Figure 3c), resulting in intriguing asymmetric CVs. The authors could show that electrons were first transferred to the heme with the higher potential and then to the second, low-potential heme by fast intramolecular ET. Lisdat and coworkers reported extensive studies on a multilayered protein–enzyme system on SAM-modified gold electrodes [96–99]. For example, they described a sulfite oxidase/cyt *c* (SOx/cyt *c*) multilayer system without polyelectrolyte, repeatedly incubating the prepared cyt *c-*modified Au electrode into a mixture of SOx/cyt *c* solution and pure cyt *c* solution [97]. A notable current density was observed even up to eight SOx/cyt *c* layers, which could be explained by the direct electronic interactions between the two proteins.

ω **Figure 3.** (**a**) Schematic illustration of *P. stutzeri* cyt *c*<sup>4</sup> (left) and horse heart cyt *c* (right) on SAM-modified Au(111); in situ STM images of a ω-mercapto-decanoic acid SAM-modified Au(111)-electrode surface (**b**) without protein as a reference, (**c**) with the two-domain *P. stutzeri* cyt *c*<sup>4</sup> vertically oriented (sharp roughly circular spots), and (**d**) horse heart cyt *c* (sharp roughly circular spots) in 5 mM pH 7.0 phosphate buffer under potential control in constant current mode; scan area, 60 × 60 nm<sup>2</sup> Reproduced with permission from [64]. Copyright 2010, American Chemical Society.

Overall, these reports highlight cyt *c* as a core electron carrier enabling efficient ET between redox enzymes and the electrode surface across suitably chosen SAMs and along with the blue ET protein azurin as a case for characterization in unique detail, right down to the level of the single molecule. Many oxidoreductases furthermore rely on cytochrome domains or subunits as "built-in" ET relays between the catalytically active cofactor and the electrode surfaces and will be discussed in the following sub-sections [100].
