Cytochrome protein—electrode complex

A more detailed kinetic investigation of the Au/Bipy/cytochrome c system was carried out using the rotating ring-disk technique (12). It was found that rate constants for adsorption and desorption of the protein were 3 x cm sec" and 50 sec", respectively. The limiting first-order rate constant within the protein-electrode complex was determined as 50 sec", a reasonable value as compared to that of long-range electron transfer between or within proteins. 

These results support the electrode reaction mechanism originally proposed by Hill et al. (17), i.e., hydrogen bonding between the lysine residues surrounding the exposed heme edge of cytochrome c and the pyridyl nitrogens at the electrode surface stabilizes a transient protein-electrode complex oriented so as to allow rapid electron transfer to and from the heme group. 

The involvement of lysine amino acid residues on cytochrome c in the heterogeneous reactions with functionalized electrodes seems to have been established. Importantly, it is now thought that the proposed protein-promoter complex is more likely to be dynamic, as revealed by the results of a recent investigation (28) of site-specific 4-chloro-3,5-dinitrophenyl (CDNP)-substituted cytochrome c. It was found that monosubstitution of either Lys 13 or Lys 72 did not result in any significant change in its electrochemical response, whereas two modifications greatly decreased the heterogeneous rate constant, and complete loss of electrochemical activity was observed upon modification of more lysines. It was proposed that the electrode reaction occurred in numerous rotational conformations. Therefore, for the mono-... 

Since mitochondrial cytochrome c was available commercially (horse heart muscle being the most common source) and could readily be purified to a high level, it formed the basic subject for most of the pioneering studies. Many ideas concerning the electrochemical mechanism, in particular, the mode of interaction with the electrode, have developed around the considerable wealth of information that is available [14, 18] on the structure and properties of the protein molecule. The extent to which the metal centre is buried is illustrated well in Fig. 1 which shows the 3D structure [19] of yeast (iso-1) cytochrome c and a view of the exposed active site. The major function of cytochrome c is as electron donor to cytochrome c oxidase (Complex IV), the membrane-bound enzyme that is the terminus of the aerobic respiratory chain and a site for proton translocation. Another physiological oxidant of cytochrome c (in yeasts) is cytochrome c peroxidase, a soluble enzyme whose crystal structure is known (see Sect. 7). The most important reduc-tant of cytochrome c is the cytochrome Cj component of the membrane-bound hcj complex (Complex III), but others (see Sect. 6, Scheme 5) include cytochrome b, sulfite oxidase, and flavocytochrome (lactate dehydrogenase, found in yeasts). 

Redox catalysis is the catalysis of redox reactions and constitutes a broad area of chemistry embracing biochemistry (cytochromes, iron-sulfur proteins, copper proteins, flavodoxins and quinones), photochemical processes (energy conversion), electrochemistry (modified electrodes, organic synthesis) and chemical processes (Wacker-type reactions). It has been reviewed altogether relatively recently [2]. We will essentially review here the redox catalysis by electron reservoir complexes and give a few examples of the use of ferrocenium derivatives. 

Recently, de novo-synthesized four-helix polypeptides were applied to mimic functions of cytochrome b and to tailor layered cross-linked electrocatalytic electrodes. A four-helix bundle de novo protein (14728 Da) that includes four histidine units in the respective A -helices was assembled on Au electrodes (Figure 22A). Two units of Fe(III)-protoporphyrin IX were reconstituted into the assembly to yield a vectorial electron-transfer cascade [157]. The de novo-synthesized protein assembly forms affinity complexes with the cytochrome-dependent nitrate reductase (NR) and with Co(II) protoporphyrin IX-reconstituted myoglobin [158]. The resulting layered complex of Fe(III) de novo protein-NR or Fe(lll)-de novo protein-Co(II)-reconstituted myoglobin was cross-linked with glutaric dialdehyde to yield electrically contacted electrocatalytic electrodes. The Fe(lll)-de novo protein-NR electrode assembly was applied for the electrocatalyzed reduction NO3 to NOt" and acted as an amperometric sensor (Figure 22B). The Fe(III)-de novo... 

Figure 22. (A) The assembly of a nitrate-sensing electrode by the cross-linking of an affinity complex formed between nitrate reductase (cytochrome-dependent, EC 1.9.6.1), NR and an Fe(III)-protoporphyrin reconstituted de novo four-helix-bundle protein. (B) Cyclic voltammograms of the NR-two heme-reconstituted de novo protein-layered Au electrode at nitrate concentrations of (a) 0, (b) 12, (c) 24, (d) 46 and (e) 68 mM. Inset calibration curve for the amperometric response of the electrode at different nitrate concentrations (at E = —0.48 V vs. SCE). Potential scan rate, 5 mV s" 0.1 M phosphate buffer, pH 7.0, under argon electrode roughness factor, ca. 20.

Reduction of horse cytochrome C with [Colsepll ", [Co(diAMsar)]2+, and [Co(NOcapten)]2+ cations was reported in Refs. 316-320. The intrinsic reactivity of these complexes with proteins make it possible the use of clathrochelates as potential protein redox titrants, electrochemical mediators, and electrode modifiers. 

Figure 3-32. (B) The assembly of an electroswitchable, electrically-contacted electrode for the bioelectrocatalyzed reduction of O2 consisting of a crosslinked affinity complex of cytochrome c/cytochrome oxidase (cyt c/COx) on a polyacrylic acid-polyethylene imine film that includes incorporated Cu -ions. The conductivity of the film and the electrical contacting of the redox-proteins is accomphshed by applying a potential of -0.5 V vs. SCE and the generation of Cu-clusters in the film. Reproduced with permission from ref. 89. Copyright 2003 American Chemical Society.

As discussed before in the case of nucleic acids the authors have also considered the incidence of the interfacial conformation of the hemoproteins on the appearance of the SERRS signals from the chromophores. Although under their Raman conditions no protein vibration can be observed, the possibility of heme loss or protein denatura-tion are envisaged to explain a direct interaction of the heme chromophores with the electrode surface in the case of the adsorl Mb. extensive denaturation of Cytc at the electrode appears unlikely to the authors on the basis of the close correspondence of the surface and solution spectra. Furthermore, the sluggish electron transfer kinetics measured by cyclic voltammetry in the case of Cytc is also an argument in favour of some structural hindrance for the accessibility to the heme chromophore in the adsorbed state of Cytc. This electrochemical aspect of the behaviour of Cytc has very recently incited Cotton et al. and Tanigushi et al. to modify the silver and gold electrode surface in order to accelerate the electron transfer. The authors show that in the presence of 4,4-bipyridine bis (4-pyridyl)disulfide and purine an enhancement of the quasi-reversible redox process is possible. The SERRS spectroscopy has also permitted the characterization of the surface of the modified silver electrode. It has teen thus shown, that in presence of both pyridine derivates the direct adsorption of the heme chromophore is not detected while in presence of purine a coadsorption of Cytc and purine occurs In the case of the Ag-bipyridyl modified electrode the cyclicvoltammetric and SERRS data indicate that the bipyridyl forms an Ag(I) complex on Ag electrodes with the appropriate redox potential to mediate electron transfer between the electrode and cytochrome c. 

Although the whole-cell membrane is non-conductive, there are several redox proteins anchored on/in the membrane that confer nano-scale conductivity to the membrane and directly enable electron transfer across the cell membrane. These proteins usually assemble together in the periplasm and/or on/across the outer-surface membrane and act as an electron transfer chain to relay the electron across the membrane. For example, the membrane-bound electron transfer chain of Shewanella oneidensis is a trans icosa-heme complex, MtrCAB, that can move electrons across the membrane. The MtrC is a decaheme cytochrome located on the outside of the outer cell membrane that mediates the electron transfer to the extracellular substrate e.g, solid electrode). MtrAB is the transmembrane electron transfer module that is responsible for electron transport from the periplasm to MtrC. More interestingly, recent findings indicates that this electron conduit is capable of reverse electron transfer, ie., electron up-take from extracellular electrodes. ... 

---