Oxidation-reduction potential cytochrome

Conroy CW, Tyma P, Daum PH et al (1978) Oxidation-reduction potential measurements of cytochrome c peroxidase and pH dependent spectral transitions in the ferrous enzyme. Biochim Biophys Acta 537 62-69... 

It has been generally assumed that iron is transported across biological membranes in the ferrous form and that ferric iron would have to be reduced before it can be used by the organism. Thus, based on nutritional studies it has long been recognized that Fe(II) is1 more effectively absorbed than Fe(III), and this has been attributed to differences in the thermodynamic and kinetic stability of the complexes and chelates formed by these cations (for review, see Ref. 2). The experimental proof of a transport in the ferrous form has, however, not been given until quite recently in studies of iron transport in isolated mitochondria (23) as well as in enterobacteria (33). In rat liver mitochondria we have found that Fe(III) donated from a metabolically inert water soluble complex of sucrose interacts with the respiratory chain at the level of cytochrome c (and possibly cytochrome a) (23, 32) (Figure 1 B), which has a oxidation-reduction potential of around +250 mV (34) and is localized to the outer phase of the mitochondrial inner membrane (35). 

McDonald, T. L., Gutheim, W. G., Martin, R. B., and Guengerich, F. P. (1989) Oxidation of substituted N, N-dimetylanylines by cytochrome P-450 estimation of the effective oxidation-reduction potential of cytochrome P-450, Biochemistry 28, 2071-2077. 

Gray, K. A., Knaff, D. B., Husain, M., and Davidson, V. L., 1986, Measurement of the oxidation-reduction potentials of amicyanin and c-type cytochromes from Paracoccus denitrificans, FEES Lett. 207 39n242. 

Oxidation-reduction potentials are expressed in volts relative to the standard hydrogen electrode at 0 volts, and represent the tendency of a compound to lose electrons (oxidation) or gain electrons (reduction). Various enzyme systems in respiration reactions (e.g., the cytochromes) utilizing ascorbic acid, hemoglobin, as well as reactions in the Krebs cycle (succinic and fumaric acids) involve electron transfers. Drugs can affect all these systems, and the consequences must be taken into account. 

A characteristic of the cytochromes c3 is a very low oxidation-reduction potential. Moreover, it is obvious from the multiheme nature of these cytochromes that the redox properties should be complex. In the simplest situation, four individual redox potentials could be expected, one for each heme. In addition to the axial ligands on the hemes, a number of factors are anticipated to influence the individual heme redox potentials. First and foremost, the environment of each heme can exert an influence on its oxidation-reduction potential. This influence will be manifested in two ways the packing of the specific amino acid side chains about each heme and the extent of solvent exposure of each heme. It is quite apparent from the structural data (Figures 1 and 2) that the four hemes, which are in nonequivalent environments, are expected to have different oxidation-reduction potentials. Moreover, at least with Miyazaki cytochrome c3> one of the hemes (heme II) is substantially more exposed to solvent, which may result in a lower oxidation-reduction potential (13). Finally, it is apparent that in a small molecule that contains four hemes within close proximity (< 18 A), heme-heme interactions, principally as a result of electrostatic interactions, are likely to influence oxidation-reduction potentials (14). Indeed, on electrostatic grounds the redox state of one heme should influence another. This influence results from the fact that addition of electrons changes the formal... 

Table II. Cytochrome c3 Macroscopic Oxidation-Reduction Potentials...

Shen, A.L. and C.B. Kasper (1996). Role of Ser457 of NADPH-cytochrome P450 oxidoreductase in catalysis and control of FAD oxidation-reduction potential. Biochemistry iS, 9451-9459. 

Macdonald TL, Gutheim WG, Martin RB, Guengerich FP. Oxidation of substituted A,A-dimethylanilines by cytochrome P450 estimation of the effective oxidation-reduction potential of cytochrome P450. Biochemistry 1989 28 2071-2077. 

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. 

Cytochrome Ci acts before cytochrome c because (1) cytochrome c is oxidized more rapidly than cytochrome Cl (2) the oxidation-reduction potential of cytochrome Ci falls between the oxidation-reduction potential of cytochrome c and that of cytochrome b (3) antimycin prevents the oxidation of cytochrome Cj and (4) NADH or succinate cytochrome c reductase reduces cytochrome Ci in the absence of cytochrome c. 

That cytochromes a and a3 must act after cytochrome c is indicated by the fact that purified cytochrome a oxidizes cytochrome c. Cytochrome a3 is by definition at the end of the chain since it has been demonstrated to be identical with cytochrome oxidase. Cytochrome a is located between cytochromes c and a3. In fact, the two cytochromes are practically inseparable, and since a3 is terminal, a must be between a3 and c. Furthermore, the oxidation-reduction potential of cytochrome a is somewhat higher than that of cytochrome cbut slightly below that of cytochrome a3. 

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. 

The sequence of reactions in which the cytochromes participate is a mechanism for transferring electrons to molecular oxygen via iron complexes that are alternately in ferric and ferrous states. The order of the transfer has been deduced from studies with inhibitors, in which the electron-transport chain is broken so that components below the break are reduced, those above are oxidized from studies with poised potentials, in which the relative degrees of oxidation and reduction define the oxidation-reduction potentials of the various components and from rapid kinetic measurements, in which the order of reduction or oxidation can be seen. These methods agree on the following sequence ... 

As discussed previously, cytochrome c is reduced by several flavo-proteins. On enzymatic reduction or reaction with hydrosulfite, ascorbic acid, or any of several other reducing reagents, the typical spectrum of reduced cytochrome c is produced. The band at 550 m/ is usually used to assay reduced cytochrome c. The reduction causes a change in the iron from the ferric to the ferrous state. The oxidation-reduction potential of the couple, ferric3rtochrome Jerrocytochrome at neutral pH values (5-8) is -H0.256 volts. ... 

Cytochrome Oxidase. Reduced cytochrome c is oxidized in particles by cytochrome a. This is consistent with the difference in oxidation-reduction potentials measured by Ball of - -0.25 volts for cytochrome c and -fO.29 volts for cytochrome a. Despite years of intensive investigation, both the chemical nature and physiological functions of cytochrome a remain uncertain. One of the major questions concerns the relation of cytochrome a to the terminal enzyme of the series, cytochrome oxidase, which reacts with molecular oxygen. 

The kinetics of reduction [K4Fe(CN)s, SO2", Sa04 , sodium ascorbate, and cytochrome C2] and oxidation [K3Fe(CN)6 and cytochrome Cg] of HIPIP from Chro-matium vinosum have been reported, and it is concluded that HIPIP undergoes rapid outer-sphere electron transfer (there being no indication of complex formation with the various reactants). Moreover, steric restrictions and differences in oxidation-reduction potential are less important than electrostatic attraction and/or repulsion in determining the absolute rate constants (Table 2). Rawlings et al. have deter-... 

C. W. Conroy, P. Tyma, P. H. Daum, J. E. Erman, Oxidation-Reduction Potential Measurements of Cytochrome c Peroxidase and pH Dependent Spectral Transitions in the Ferrous Enzyme. Biochim. Biophys. Acta, 537 (1978) 62-69. 

NADH and FADHg are produced as a result of substrate level dehydrogenations. Oxidation of these reduced coenzymes by oxygen is accomplished by the intervention of a series of electron carriers between the primary reductant and the terminal oxidant (Fig. 2). The electron-transport components represent redox couples of increasing redox potential and are therefore favored thermodynamically. The respiratory chain can be separated into four multienzyme complexes NADH-Q reductase (complex I), succinate-Q reductase (complex II), QH2"Cytochrome c reductase (complex III), and cytochrome c oxidase (complex IV). At each of these successive oxidation-reduction steps, a certain amount of free energy is available, the amount being determined by the difference in the oxidation-reduction potential of the two sequential components. The difference in the redox potential between... 

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