Cytochrome cyclic voltammetry

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... 

Figure 3.96 The effect of increasing time of exposure (as indicated) of a gold electrode once-modified with SSBipy to thiophenol on the cyclic voltammetry of horse heart cytochrome t (0.4mM). 20 mM sodium phosphate/0.1 M NaCI04 pH 7.0. Scan rate 20mVs l. From Hill...

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. 

The molecular interaction of cytochrome c and cardiolipin has been extensively studied. A mode of the interaction has been confirmed to be both electrostatic and hydrophobic, by using infrared spectroscopy (Choi and Swanson, 1995), fluorescence resonance energy transfer method (Rytdmaa and Kinnunen, 1994), protease digestion (de Jongh et al., 1995), cyclic voltammetry (Salamon and ToUin, 1997), deuterium and phosphorus NMR measurements (Spooner et al., 1993), and surface plasmon resonance spectroscopy (Salamon and Tollin, 1996). 

Cyclic voltammetry has been also used for estimation of the rate constants for oxidation of water-soluble ferrocenes in the presence of HRP (131). There is a perfect match between the data obtained spectrophotometrically and electrochemically (Table IV), which proves that the cyclic voltammetry reveals information on the oxidation of ferrocenes by Compound II. It is interesting to note that an enzyme similar to HRP, viz. cytochrome c peroxidase, which catalyzes the reduction of H202 to water using two equivalents of ferrocytochrome c (133-136), is ca. 100 times more reactive than HRP (131,137). The second-order rate constant equals 1.4 x 106 M-1 s 1 for HOOCFc at pH 6.5 (131). There is no such rate difference in oxidation of [Fe(CN)e]4- by cytochrome c peroxidase and HRP (8). These comparisons should not however create an impression that the enzymatic oxidation of ferrocenes is always fast. The active-R2 subunit of Escherichia coli ribonucleotide reductase, which has dinuclear nonheme iron center in the active site, oxidizes ferrocene carboxylic acid and other water-soluble ferrocenes with a rate constant of... 

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-... 

One of the first reports on the quasireversible electrochemistry of redox proteins appeared in 1977 when Eddowes and Hill demonstrated (10) cyclic voltammetry of horse heart cytochrome c at a gold electrode in the presence of 4,4 -bipyridyl (Bipy) in solution. In the voltammo-grams (Fig. 1), the peak-to-peak separations were close to 60 mV and the faradaic currents varied linearly with (scan rate), indicating a quasireversible one-electron transfer process controlled by linear diffusion of redox species to the electrode surface. The midpoint potential... 

The reduction potentials of the Dsm.baculatum cytochrome c3 (Mr 26,000) hemes were determined by cyclic voltammetry, and found to be as low as those of cytochrome c3 [72]. Based on the four values obtained (-210, -270, -325, -365 mV at pH 7.6) the authors proposed that the hemes are bis-histidinyl coordinated. The amino-acid sequence of the Dsm.baculatum cytochrome c3 (Mr 26,000) was compared to the sequences of several polyheme cytochromes, giving evidence that this cytochrome is distinct from cytochrome c3 (Mr 13,000) [73]. The sequence of the D. gigas cytochrome has also been determined [71]. The crystal structure of the Dsm. baculatum cytochrome c3 (Mr 26,000) was obtained at 2.16 A resolution [74], Preliminary X-ray studies have also been reported for the D.gigas cytochrome [75]. 

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