Cytochrome c reduction

J.M. McCord and I. Fridovich, Utility of superoxide dismutase in studying free radical reactions. II. Mechanism of the mediation of cytochrome c reduction by a variety of electron carriers. J. Biol. Chem. 245,1374-1377 (1970). 

It is extremely important that the interaction of quinones with XO (Reaction (3)) is reversible that can lead to receiving erroneous results at the measurement of superoxide production by SOD-inhibitable cytochrome c reduction [28,29] (see also Chapter 27). Lusthof et al. [30] demonstrated that 2,5-bis(l-aziridinyl)-l,4-benzoquinones are directly reduced by XO. Interestingly at quinone concentrations greater than 25pmol I 1, quinones entirely suppressed one-electron reduction of dioxygen, and cytochrome c was completely reduced by the semiquinones formed. It is well known that cytochrome c and lucigenin are effective superoxide scavengers and due to that, these compounds are widely used in the quantitative assays of superoxide detection. Nonetheless, under certain experimental conditions they can be directly reduced by XO [31]. 

Controversial results of oxygen radical detection in mitochondria have been described in the literature. Owing to experimental difficulties many authors were obliged to work with sub-mitochondrial particles instead of the whole mitochondria. However, it is quite possible that oxygen radical production by submitochondrial particles may be artificially enhanced due to exposure to oxygen. On the other hand, some analytical methods of superoxide detection such as cytochrome c reduction cannot be used due to the direct reduction of cytochrome by mitochondrial components. 

Superoxide-dismuting activity of copper rutin complex was confirmed by comparison of the inhibitory effects of this complex and rutin on superoxide production by xanthine oxidase and microsomes (measured via cytochrome c reduction and by lucigenin-amplified CL, respectively) with their effects on microsomal lipid peroxidation [166]. An excellent correlation between the inhibitory effects of both compounds on superoxide production and the formation of TBAR products was found, but at the same time the effect of copper rutin complex was five to nine times higher due to its additional superoxide dismuting capacity. 

Values (p.mol I-1) for Inhibitory Effects of Metal-Rutin Complexes and Rutin on Cytochrome c Reduction by Xanthine Oxidase (I), Iron-Catalyzed Microsomal Lipid Peroxidation (II), and Lucigenin-Amplified Microsomal CL (III) [167]... 

FIGURE 31.1 Effects of rutin and LI on menadione-stimulated cytochrome c reduction normal and T1 erythrocytes. (From IB Afanas ev et al.Transfusion Sci 23 237-238, 2000.)... 

Early methods of superoxide detection are well known and described in many books and reviews. They include cytochrome c reduction, nitroblue tetrazolium reduction, spin trapping, etc. (see, for example, Ref. [1]). The most efficient assays are based on the ability of superoxide to reduce some compounds by one-electron transfer mechanism because such processes (Reaction (1)) proceed with high rates [2] ... 

Possible errors due to the competition of cytochrome c reduction with the reversible reduction of quinones by superoxide are frequently neglected. For example, it has been found that quinones (Q), benzoquinone (BQ), and menadione (MD) enhanced the SOD-inhibitable cytochrome c reduction by xanthine oxidase [6]. This seems to be a mystery because only menadione may enhance superoxide production by redox cycling ( °p)"]/ [MD] =-0.20 V against ,0[02 ]/[02] 0.16 V) via Reactions (3) and (4), whereas for... 

Increase in cytochrome c reduction in the presence of benzoquinone could be due to Reaction (5). 

However, a more discouraged fact is that benzoquinone accelerated SOD-inhibitable part of cytochrome c reduction, which is usually considered as a reliable proof of superoxide formation. Such a phenomenon has been first shown by Winterbourn [7], who suggested that SOD may shift the equilibrium of Reaction (4) to the right even for nonredox cycling quinones. The artificial enhancement of superoxide production by SOD in the presence of quinones was demonstrated in the experiments with lucigenin-amplified CL, in which benzoquinone was inhibitory [6],... 

The efficiency of superoxide assays strongly depend on the nature of superoxide producers. Significant difficulties arise in the detection of superoxide in cells and tissue. Cytochrome c is unable to penetrate cell membranes and therefore, can be used only for the measurement of extracellular superoxide. Furthermore, SOD-inhibitable cytochrome c reduction is difficult to apply in nonphagocytic cells and tissue due to the complications of measuring low rates of superoxide release, direct reduction of cytochrome c by cellular enzymes, the reoxidation of reduced cytochrome by hydrogen peroxide, etc. [8], Moreover, in nonphagocytic cells superoxide is formed exclusively inside the cells and is not released outside as in phagocytes. These circumstances severely limit the number of analytical methods, which can be used for superoxide detection in vasculature. 

Correlations between Lucigenin-Amplified CL and SOD-Inhibited Cytochrome c Reduction or Epinephrine Oxidation... 

FIGURE 32.2 Correlations between maximal chemiluminescence intensity (CL) and the rates of cytochrome c reduction by the xanthine-XO and NADH-XO systems [61]. Line 1, xanthine-XO and line 2, NADH-XO (in this case CL values were multiplied by 10.)... 

Figure 5.10. Cytochrome c reduction by 02 Production of 02 from activated neutrophils may be assayed using cytochrome c. Oxidised (Fe3+) cytochrome c can be reduced by 02" to form Fe2+-cytochrome c, which absorbs at 550 nm thus, in a mixture of activated neutrophils and cytochrome, absorption increases at 550 nm are due to 02 production. Superoxide dismutase (SOD) has a higher affinity for 02 than does cytochrome c thus, the addition of SOD to activated neutrophil suspensions will prevent the reduction of cytochrome c. SOD-inhibitable cytochrome c reduction is therefore a direct measure of the rate of 02 formation.

Hence, this assay is an extremely useful and selective assay to measure O2 secretion. Because of this selectivity and because it measures the initial product of O2 reduction, it is often used as the method of choice to detect NADPH oxidase activity. It is suitable for semi-automation because assays can be performed in 96-well microtitre plates (using ELISA plate readers with a suitable filter), or cytochrome c reduction can be detected using simple spectrophotometers. The assay, however, is not suitable for measuring O2 that may be generated intracellularly within activated neutrophils. 

Because electrode measurements of O2 uptake can detect intra- and extracellular oxidase activity, this assay can be used to measure the respiratory burst elicited by soluble and particulate stimuli. What is somewhat surprising is that, during stimulation of neutrophils with agonists such as fMet-Leu-Phe, the activated O2 uptake profile is biphasic (Fig. 5.11c). A rapid burst of O2 uptake (which coincides with measurements of cytochrome c reduction) is followed by a more sustained activity of lower magnitude. 

To evaluate the functional role(s) of this residue, six mutations, Lys, His, Glu, Asn, Leu, and Ala, were introduced at this site, and the electrochemical and NMR properties of the resulting proteins examined [134]. Contrary to expectation, removal of Arg-38 did not result in a change in the dependence of the cytochrome c reduction potential on pH. Instead, as the electron-withdrawing ability of the residue substituted at position-38 decreased, the reduction potential also decreased, with the greatest decrease (50 mV) observed for the Ala mutant. The variation of reduction potential with pH, however, remained essentially the same as that previously observed for the wild-type protein. 

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