Macroscopic redox potential

Flavocytochrome Fumarate Reductase. The flavocytochrome fumarate reductase (Fff) is a soluble periplasmic protein from Shewanella spp. that reduces fumarate but does not oxidize succinate, in contrast to the membrane-bound fumarate reductases that are related to succinate dehydrogenases, and transfer electrons from quinol to fumarate. It is a monomeric protein of 63.8 kDa that is composed of three domains. The N-terminal domain contains four c-type hemes, and the flavin domain contains noncovalently bound FAD and is related to flavoprotein subunits of membrane-bound fumarate reductases and succinate dehydrogenases. There is also a third domain in the flavocytochromes that has considerable flexibility and may be involved in controlling access of substrate to the active site. The macroscopic redox potentials of the fom hemes of Ffr are —102, —146, —196, and -23 8 mV, while that of FAD is —152 mV. The low redox potential of FAD in Ffr compared to that in membrane-bound fumarate reductase (—55 mV) may explain why it is unable to oxidize succinate. 

Table II summarizes the macroscopic redox potentials for the four cytochromes c3. As expected from the strong amino acid sequence homology, the Miyazaki and Hildenborough cytochromes c3 have redox potentials that are very similar and have a redox potential span of 110-120 mV between heme 1 and heme 4. In sharp contrast, Norway cytochrome c3 has a redox potential span of approximately 235 mV. Clearly, the differences in macroscopic redox potentials among the various cytochromes c3 result from the different amino acid sequences and, thus, different heme environments. To date, little can be said about the specific reasons for differences in macroscopic redox potentials among the cytochromes c3, but in view of the large amount of structural information that is accumulating, much progress can be expected in the future.

Recently we carried out kinetic studies with Hildenborough and Miyazaki cytochrome c3 using deazariboflavin semiquinone (dRf ), MV +, and propylene diquat (PDQ +), produced by laser flash photolysis, as reductants (37). Initially, all three reactions were accurately second order, consistent with all hemes being reduced with the same rate constant or with a single site reduced, followed by fast intramolecular electron transfer to reduce the remaining three hemes. However, by measuring reduction kinetics with cytochrome c3 poised at different extents of reduction, the kinetics of reduction of individual hemes could be resolved. Thus, reduction of cytochrome c3 in approximately 5% steps and application of the known macroscopic redox potentials (see previous section) enabled calculation of the concentration of each heme (c.) at each stage of reduction. The plot of kohs versus percent reduction can thereby be fitted to solve for the rate constant for each heme (kt) ... 

Since the relative intensity of any heme-methyl NMR signal belonging to each macroscopic oxidation state is proportional to the mole fraction of its oxidation state, the macroscopic redox potentials of the tetra-heme protein can be determined by the NMR measurement [21, 124, 125]. The equilibrium potential of the redox system in an NMR tube can be correlated with the absorption spectra-equilibrium potential relation obtained from OTTLE measurements. 

Fig. 6 compares the nuclearity effect on the redox potentials [19,31,63] of hydrated Ag+ clusters E°(Ag /Ag )aq together with the effect on ionization potentials IPg (Ag ) of bare silver clusters in the gas phase [67,68]. The asymptotic value of the redox potential is reached at the nuclearity around n = 500 (diameter == 2 nm), which thus represents, for the system, the transition between the mesoscopic and the macroscopic phase of the bulk metal. The density of values available so far is not sufficient to prove the existence of odd-even oscillations as for IPg. However, it is obvious from this figure that the variation of E° and IPg do exhibit opposite trends vs. n, for the solution (Table 5) and the gas phase, respectively. The difference between ionization potentials of bare and solvated clusters decreases with increasing n as which corresponds fairly well to the solvation free energy of the cation deduced from the Born solvation model [45] (for the single atom, the difference of 5 eV represents the solvation energy of the silver cation) [31]. 

Several molecular and supramolecular luminescent systems of varying complexity have been reported, whose emission can be turned on/off at will by means of an external input (either a pH or a redox potential change) and have been therefore considered as molecular level analogues of everyday life light switches [9-13], The Ni(II)-3 system is different in that it controls three levels of illumination high-low-off, as switches operating car headlights do in the macroscopic world. 

The increase of the redox potential of a metal cluster in a solvent with its nuclearity is now well established 1-4). The difference between the single atom and the bulk metal potentials is large (more than 2 V, for example, in the case of silver (3)). The size dependence of the redox potential for metal clusters of intermediate nuclearity plays an important role in numerous processes, particularly electron transfer catalysis. Although some values are available for silver clusters (5, 6), the transition of the properties from clusters (mesoscopic phase) to bulk metal (macroscopic phase) is unknown except for the gas phase (7-9). 

Figure 3. Electron distribution for cytochrome c3. S0 S4 define the five macroscopic states (see text). The 16 microscopic states are shown. Open circles represent oxidized heme, and solid circles represent reduced heme. The microscopic redox potentials of heme i are given by eJkl where j, k, 1 represent hemes that remain oxidized. For clarity only, 12 of the 32 microscopic redox...

Description of redox equilibria of tetra-heme protein Macroscopic formal potential versus microscopic formal potential... 

In the case of a molecule with multiredox centres, such as cytochrome C3, two types of formal potential (mid-point potential) can be defined the macroscopic formal potential (formal potential of the molecule), where each redox site is indistinguishable by using the given technique and the microscopic formal potential (formal potential of each redox site), where each redox site is distinguishable. A tetra-heme protein, such as cytochrome C3, has five macroscopic oxidation states the fully oxidized (Sq), the one-electron reduced (SJ, two-electron reduced (S2), three-electron reduced (S3), and four-electron or fully reduced (S4) states [123], as shown in Fig. 10. 

There are 32 microscopic formal potentials for the redox process of the tetra-heme protein, cytochrome C3, and the deconvolution of these microscopic states distributed over 110 mV is impossible using electrochemical techniques. Many heme-methyl signals are observed separately, e.g. each heme of cytochrome C3 has four methyl groups so that 80 three-proton intensity signals originating from the 16 heme-methyl groups would be expected in the course of a four-electron reduction process. The microscopic formal potentials of the 32 redox processes can be calculated from the chemical shifts of each heme-methyl group at the five macroscopic oxidation states [125, 127]. The results are shown in Table 6 and the macroscopic formal potential can be deduced from these results. 

Figure 5a schematizes LAJs based on two Hg electrodes. By bringing in contact the two Hg-drops inside an electrolyte solution and connecting them to a macroscopic reference electrode by a potentiostat (Figs. 15a and 16a), an electrochemical junction is created this junction allows for independent control of the potentials applied to the two Hg electrodes, so that the cathode can act as electron donor (source) and the anode as electron acceptor (drain) with respect to the redox centre. 

It must be emphasized again that the mid-peak potential is equal to E° for a simple, reversible redox reaction when neither any experimental artifact nor kinetic effect (ohmic drop effect, capacitive current, adsorption side reactions, etc.) occurs, and macroscopic inlaid disc electrodes are used, that is, the thickness of the diffusion layer is much higher than that of the diameter of the electrode. 

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