Cyclic voltammetry, proteins

Fig. 13. Midpoint potential (E°) as a function of pH. Data points ( 3 mV) were obtained with cyclic voltammetry. Protein concentration was 0.10-0.15 roM in 50 mAf cacodylate/NaOH or HEPES/NaOH buffer. The solid circles and the left y axis refer to wild-t3T3e cyt C550, whereas the open circles and the right y axis are for the MIOOK cytochrome C550 variant.

The use of direct electrochemical methods (cyclic voltammetry Pig. 17) has enabled us to measure the thermodynamic parameters of isolated water-soluble fragments of the Rieske proteins of various bci complexes (Table XII)). (55, 92). The values determined for the standard reaction entropy, AS°, for both the mitochondrial and the bacterial Rieske fragments are similar to values obtained for water-soluble cytochromes they are more negative than values measured for other electron transfer proteins (93). Large negative values of AS° have been correlated with a less exposed metal site (93). However, this is opposite to what is observed in Rieske proteins, since the cluster appears to be less exposed in Rieske-type ferredoxins that show less negative values of AS° (see Section V,B). 

Cyclic voltammetry and other electrochemical methods offer important and sometimes unique approaches to the electroactive species. Protein organization and kinetic approaches (Correia dos Santos et al. 1999, Schlereth 1999) can also be studied by electrochemical survey. The electron transfer reaction between cytochrome P450scc is an important system for... 

The first reports on direct electrochemistry of a redox active protein were published in 1977 by Hill [49] and Kuwana [50], They independently reported that cytochrome c (cyt c) exhibited virtually reversible electrochemistry on gold and tin doped indium oxide (ITO) electrodes as revealed by cyclic voltammetry, respectively. Unlike using specific promoters to realize direct electrochemistry of protein in the earlier studies, recently a novel approach that only employed specific modifications of the electrode surface without promoters was developed. From then on, achieving reversible, direct electron transfer between redox proteins and electrodes without using any mediators and promoters had made great accomplishments. 

A variety of physical methods has been used to ascertain whether or not surface ruthenation alters the structure of a protein. UV-vis, CD, EPR, and resonance Raman spectroscopies have demonstrated that myoglobin [14, 18], cytochrome c [5, 16, 19, 21], and azurin [13] are not perturbed structurally by the attachment of a ruthenium complex to a surface histidine. The reduction potential of the metal redox center of a protein and its temperature dependence are indicators of protein structure as well. Cyclic voltammetry [5, 13], differential pulse polarography [14,21], and spectroelectrochemistry [12,14,22] are commonly used for the determination of the ruthenium and protein redox center potentials in modified proteins. 

Reduction potentials of the S. obliqms His59 Ru(NH3)5-modified protein have been determined by cyclic voltammetry using as electrode the oxidized surface obtained by polishing the edge plane of pyrolytic graphite [137]. The modified protein responds well at the electrode, whereas the native protein requires multi-eharged cations, e.g. Mg or [Cr(NH3)g] as mediators to give satisfactory reversibility. Separate reduction potentials at 1=0.10 M(NaCl) for native S. obliquus plastocyanin (389 mV) and [Ru(NH3)5 (imidazole)]... 

The Fe2S2 complex derived from the 20-peptide exhibits a visible spectrum typical of plant-type 2-Fe proteins (10). (See Figure 4.) But at least two isomeric 2-Fe species are detect by the cyclic voltammetry (CV) and also by the differential-pulse polarography in DMF. One isomer has an E1/2 at -0.64 V (SCE), which is very similar to that of the native protein, and this complex presumably adopts a structure... 

Fd I, cyclic voltammetry, 38 130-131 Vfe proteins, 36 90-91 Azotobacter Fd 1, redox properties, 38 HSUS... 

Cyclic silylphosphanes, see Silylphosphanes, phosphorus-rich, cyclic Cyclic sulfur-nitrogen compounds, see Sulfur-nitrogen compounds, cyclic Cyclic trithiolate ligand, 38 8-9 Cyclic voltammetry A. chroococcum Fd 1, 38 130-131 fullerene adducts, 44 19 nickel(ll) macrocyclic complexes, 44 112 Rieske proteins, 47 138, 139 Cyclidenes, as cobalt complex ligands, 44 282-284... 

Apart from the above techniques, the electromodulated reflectance spectroscopy combined with cyclic voltammetry has been utilized by Gaigalas et al. [14] in the investigations of electron transfer between the 2Fe-2S protein putidaredoxin and either bare or bekanamycin-modified Ag electrode. Of the two models considered, the free diffusion model, as compared to the adsorbed layer model, exhibited better concordance with the experimental data. After modification of the Ag electrode with bekanamycin, it exhibited only a small increase in the observed redox reaction... 

Two different protein phosphatases were used the one from Upstate Biotechnology (New York, USA), from human red blood cells, and the one from GTP Technology (Toulouse, France), isolated from SF9 insect cells infected by baculovirus. The enzymatic activity of these two enzymes towards several substrates was investigated by cyclic voltammetry and steady-state chronoamperometry (see experimental details in Refs. [86,87]). First, commercial substrates were tested. Ascorbic acid 2-phosphate and phenyl phosphate were not recognised by the protein... 

In the last 20 years, a big effort has been made to characterize different reaction schemes taking place at modified electrodes, with special focus on the case of biomolecules in what has been called Protein Film Voltammetry [79-83]. Among the different situations analyzed with multipulse techniques (including Cyclic Voltammetry), it can be cited the surface ECE process [89, 90] and surface reactions preceded by homogeneous chemical reactions [91]. For a more detailed revision of the different mechanisms analyzed in the case of SWV, see [19]. 

Aprotic solvents mimic the hydrophobic protein interior. However, a functional artificial receptor for flavin binding under physiological conditions must be able to interact with the guest even in competitive solvents. As found by spectroscopic measurements with phenothiazene-labeled cyclene, the coordinative bond between flavin and Lewis-acidic macrocyclic zinc in methanol was strong enough for this function. Stiochiometry of the complex was proved by Job s plot analysis. Redox properties of the assemblies in methanol were studied by cyclic voltammetry which showed that the binding motif allowed interception of the ECE reduction mechanism and stabilisation of a flavosemiquinone radical anion in a polar solvent. As a consequence, the flavin chromophore switched from a two-electron-one-step process to a two-step-one-electron-each by coordination. 

Cytochrome c is a single-heme protein, MW = 12 kDa, fairly universal in respiratory ET chains [77]. The reduction potential is 265 mV (SHE, horse heart). ET reactions in homogeneous solution and promoter-induced reversible cyclic voltammetry are well characterized. Horse heart cyt c has been in recent focus in some of the first STM investigations of metalloprot-... 

CV = cyclic voltammetry Fd = ferredoxin HP = high potential iron-sulfur protein IRP = iron regulatory protein LS3 = l,3,5-tris((4,6-dimethyl-3-mercaptophenyl)thio)-2,4,6-tris(/ -tolylthio)benzene (3-) Rd = rubredoxin SCE = standard calomel electrode tibt = 2,4,6-triisopropylphenyl Tp = tris(pyrazolyl)hydroborate (1-). 

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