Flavoprotein reduction potentials

Reduction potentials can also be quite sensitive to molecular environment. The influence of environment is especially important for flavins, such as FAD/FADHg and FMN/FMNHg. These species are normally bound to their respective flavoproteins the reduction potential of bound FAD, for example, can be very different from the value shown in Table 21.1 for the free FAD-FADHg couple of —0.219 V. A problem at the end of the chapter addresses this... 

The heme cofactors of a and b cytochromes are tightly, but not covalently, bound to their associated proteins the hemes of c-type cytochromes are covalently attached through Cys residues (Fig. 19-3). As with the flavoproteins, the standard reduction potential of the heme iron atom of a cytochrome depends on its interaction with protein side chains and is therefore different for each cytochrome. The cytochromes of type a and b and some of type c are integral proteins of the inner mitochondrial membrane. One striking exception is the cytochrome c of mitochondria, a soluble protein that associates through electrostatic interactions with the outer surface of the inner membrane. We encountered cytochrome c in earlier discussions of protein structure (see Fig. 4-18). 

However, these experiments may not have established a mechanism for natural flavoprotein catalysis because the properties of 5-deazaflavins resemble those of NAD+ more than of flavins.239 Their oxidation-reduction potentials are low, they do not form stable free radicals, and their reduced forms don t react readily with 02. Nevertheless, for an acyl-CoA dehydrogenase the rate of reaction of the deazaflavin is almost as fast as that of natural FAD.238 For these enzymes a hydride ion transfer from the (3 CH (reaction type D of Table 15-1) is made easy by removal of the a-H of the acyl-CoA to form an enolate anion intermediate. 

The one-electron reduction potential of flavin in flavoproteins varies over a wide (>500 mV) potential range, corresponding to a free energy difference of AAG > 10 kcal/mol. Since the redox-active isoalloxazme nucleus is the same in all systems, these extensive variations must arise from apoenzyme-cofactor non-... 

A final distinction from nicotinamides is that the flavin coenzymes generally form tight non-dissociable non-covalent complexes with the apoenzyme. Nicotinamides are released at the end of each catalytic cycle and so are consumed as substrate as part of the redox stoichiometry. Because flavins are tightly bound to the apoprotein (/irD= 10 -10 " M) the coenzyme must be oxidised/reduced at the end each turnover before the enzyme complex again becomes catalytically active. Differential binding of flavin and dihydroflavin is responsible for the wide range of redox potentials for flavoproteins so that oxidation or reduction can be thermodynamically favourable. For example, D-amino acid oxidase binds FAD with a dissociation constant of 10 M but FADHj with one of 10 M which changes the reduction potential from —200 for the FAD/FADHj couple free in solution to 0 mV when bound to the enzyme. 

Searls and Sanadi (1960a) determined the reduction potential of the pig heart dihydrolipoic dehydrogenase from the extent of its reduction at different DPNH DPN ratios. The value is between —0.332 and —0.320 volt at pH 7.0 and 25°C. Thus the reduction potential of the flavoprotein is close to that of the DPNH-DPN system, —0.320 volt at pH 7.0 and 25°C (Burton and Wilson, 1953), and the Lip(SH)2-LipS2 system, —0.325 volt at pH 7.0 and 25°C (see Section II, A). This is consistent with the ready reversibility of reaction (26) as well as the high initial reaction rates in both directions. It would appear that DPN is the physiological electron acceptor for the a-keto acid dehydrogenation complexes in vivo and that the DPNH formed is reoxidized by way of the electron transport chain. However, the... 

A more detailed description of the electron transport chain was obtained by using three other methods selective inhibition, spectrophotometric kinetics, and determination of the respective oxidation-reduction potentials. The first two methods are based on the spectral properties of the various components of the electron transport chain. It is indeed fortunate that the pyridine nucleotides, the cytochromes, and the flavoproteins have specific absorption spectra, and that the spectra undergo considerable changes when they pass from the reduced to the oxidized form. 

Cytochrome b is known to transfer electrons from the substrate to cytochrome c because (1) narcotics allow the oxidation of cytochrome b but leave cytochrome c reduced (2) the oxidation-reduction potential of cytochrome b is below that of cytochrome c and (3) kinetic studies have shown that cytochrome c is oxidized before cytochrome b, and cytochrome b is oxidized before the flavoprotein. 

Both the oxidation-reduction potential and the fluorescence of flavin nucleotides are modified profoundly by attachment of the nucleotide to various proteins. Flavin enzymes have been reported to have oxidation-reduction potentials at pH 7 ranging from —0.4 to 0.187. The combination to proteins also results in shifts of the absorption maxima. The 450 m u band is found at 451 mju in Straub s diaphorase and at 455 m/t in Haas yellow enzyme, while the 375 m/t band appears at 359 m/t and 377 m/t in these preparations. Most flavin enzymes do not fluoresce, and it is assumed that the quenching of fluorescence implies binding of the flavin to the enzyme through N-3. Straub s diaphorase, unlike most other flavoproteins, does fluoresce. This may be evidence that this diaphorase is a partially degraded cytochrome reductase. 

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