Cytochrome b reduction

When the second-site revertants were segregated from the original mutations, the bci complexes carrying a single mutation in the linker region of the Rieske protein had steady-state activities of 70-100% of wild-type levels and cytochrome b reduction rates that were approximately half that of the wild type. In all these mutants, the redox potential of the Rieske cluster was increased by about 70 mV compared to the wild type (51). Since the mutations are in residues that are in the flexible linker, at least 27 A away from the cluster, it is extremely unlikely that any of the mutations would have a direct effect on the redox potential of the cluster that would be observed in the water-soluble fragments. However, the mutations in the flexible linker will affect the mobility of the Rieske protein. Therefore, the effect of the mutations described is due to the interaction between the positional state of the Rieske protein and its electrochemical properties (i.e., the redox potential of the cluster). 

Production of superoxide radical anion (02 ) during oxidation of dihydroorotate in rat hver mitochondria was not affected by antimycin A, thenoyl-trifluoroacetone, or added ubiquinone but was inhibited by orotate, a product inhibitor of dihydroorotate dehydrogenase (Forman and Kennedy 1975). It appears likely that superoxide is generated at the primary dehydrogenase. Dihydroorotate dehydrogenase differs from succinate dehydrogenase both in its utilisation of ubiquinone and in the mechanism of cytochrome b reduction. Formation of orotate is only partially inhibited by thenoyltri-fluoroacetone and the inhibitor does not prevent the reduction of cytochrome by dihydroorotate. 

This impressive reaction is catalyzed by stearoyl-CoA desaturase, a 53-kD enzyme containing a nonheme iron center. NADH and oxygen (Og) are required, as are two other proteins cytochrome 65 reductase (a 43-kD flavo-protein) and cytochrome 65 (16.7 kD). All three proteins are associated with the endoplasmic reticulum membrane. Cytochrome reductase transfers a pair of electrons from NADH through FAD to cytochrome (Figure 25.14). Oxidation of reduced cytochrome be, is coupled to reduction of nonheme Fe to Fe in the desaturase. The Fe accepts a pair of electrons (one at a time in a cycle) from cytochrome b and creates a cis double bond at the 9,10-posi-tion of the stearoyl-CoA substrate. Og is the terminal electron acceptor in this fatty acyl desaturation cycle. Note that two water molecules are made, which means that four electrons are transferred overall. Two of these come through the reaction sequence from NADH, and two come from the fatty acyl substrate that is being dehydrogenated. 

The cytochromes are iron-containing hemoproteins in which the iron atom oscillates between Fe + and Fe + during oxidation and reduction. Except for cytochrome oxidase (previously described), they are classified as dehydrogenases. In the respiratory chain, they are involved as carriers of electrons from flavoproteins on the one hand to cytochrome oxidase on the other (Figure 12-4). Several identifiable cytochromes occur in the respiratory chain, ie, cytochromes b, Cp c, a, and (cytochrome oxidase). Cytochromes are also found in other locations, eg, the endoplasmic reticulum (cytochromes P450 and h, and in plant cells, bacteria, and yeasts. 

Estabrook, R.W., Hildebrandt, A.G., Baron, J., Netter, K.J. and Leibman, K. (1971) A new spectral intermediate associated with cytochrome P-450 function in liver microsomes. Biochemical and Biophysical Research Communications, 42 (1), 132-139. Pompon, D. and Coon, M.J. (1984) On the mechanism of action of cytochrome P-450. Oxidation and reduction of the ferrous dioxygen complex of liver microsomal cytochrome P-450 by cytochrome b5. Journal of Biological Chemistry, 259 (24), 15377-15385. Hildebrandt, A. and Estabrook, R.W. (1971) Evidence for the participation of cytochrome b 5 in hepatic microsomal mixed-function oxidation reactions. Archives of Biochemistry and Biophysics, 143 (1), 66-79. 

Flavoprotein dehydrogenases usually accept electrons from reduced pyridine nucleotides and donate them to a suitable electron acceptor. The oxidation-reduction midpoint potential of the FAD of the oxidase has been determined by ESR spectroscopy and shown to be -280 mV. The NADP+/ NADPH redox potential is -320 mV and that of the cytochrome b is -245 mV hence, the flavin is thermodynamically capable of accepting electrons from NADPH and transferring them to cytochrome b. As two electrons are transferred from NADPH, although O2 reduction requires only one electron, the scheme of electron transfer shown in Figure 5.8 has been proposed by Cross and Jones (1991). 

Cross, A. R., Harper, A. M., Segal, A. W. (1981). Oxidation-reduction properties of the cytochrome b found in the plasma-membrane fraction of human neutrophils. Biochem. J. 194, 599-606. 

The enzymic reduction and kinetics of oxidation of cytochrome b.245 of neutrophils. Biochem. J. 204, 479-85. 

This was confirmed by Keilin and Hartree using antimycin A as an inhibitor. The antibiotic blocked the reduction of cytochrome cx by NADH or succinate but did not block the reduction of cytochrome b. This site-specific inhibition brought antimycin A into popular use by biochemists in the analysis of electron transfer and oxidative phosphorylation. 

Reviewing the criteria for inclusion of components into the electron transport chain, Slater (1958) highlighted considerations previously advanced by H.A. Krebs as necessary to establish a pathway, namely that the amounts of enzyme present must be commensurate with enzymic activity in the preparation, activity should be fully restored by the reintroduction of the postulated component into an inhibited or depleted preparation, and that the rates of oxidation and reduction of components must be at least as great as those in the system overall. Reduction of cytochrome b by the systems then in use was thought by Chance (1952) and Slater (1958) to be too slow for the inclusion of this cytochrome into the main chain. 

The reduction is mediated by a PCE reductase and hydrogenase, both of which are associated with the cytoplasmic membrane. The hydrogenase faces the periplasm of the cell and thus releases the protons into the periplasm (Schumacher and Holliger 1996). Two electrons are transferred across the membrane to the PCE reductase, via cytochrome b and menaquinone (Fig. 9.13). Since dihydrogen is the only electron donor used by Dehalobacter restrictus, the hydrogenase is a crucial enzyme of this... 

Fe Cytochrome oxidase reduction of oxygen to water Cytochrome P-450 0-insertion from O2, and detoxification Cytochromes b and c electron transport in respiration and photosynthesis Cytochrome f photosynthetic electron transport Ferredoxin electron transport in photosynthesis and nitrogen fixation Iron-sulfur proteins electron transport in respiration and photosynthesis Nitrate and nitrite reductases reduction to ammonium... 

Cytochrome fcs/NADH cytochrome b reductase (human) Reductive detoxification of substituted hydroxylamine carcinogens (K = 200-400 p.M) 27-29, 50... 

Fe oxidation, 36 425-426 Fe release, 36 426-427 metal ion bridges, 36 472 molecule packing, 36 471-472 outer-sphere electron transfer, 36 429-433 comparison of cytochrome c and cytochrome C551, 36 431 cytochrome b 36 429 ferrihemoprotein reduction rates, 36 430-431... 

Hereditary methemoglobinemia arises from a deficiency of the enzyme that catalyzes this reduction, NADH-cytochrome b reductase. 

Q —> cytochrome b —> cytochrome c1 —> cytochrome c —> cytochrome a —> cytochrome a3 —> 02. Note, however, that the order of standard reduction potentials is not necessarily the same as the order of actual reduction potentials under cellular conditions, which depend on the concentration of reduced and oxidized forms (p. 510). A second method for determining the sequence... 

QH2 donates one electron (via the Rieske Fe-S center) to cytochrome c, and one electron (via cytochrome b) to a molecule of Q near the n side, reducing it in two steps to QH2. This reduction also uses two protons per Q, which are taken up from the matrix. 

Bacterial assimilatory nitrate reductases have similar properties.86/86a In addition, many bacteria, including E. coli, are able to use nitrate ions as an oxidant for nitrate respiration under anaerobic conditions (Chapter 18). Tire dissimilatory nitrate reductases involved also contain molybdenum as well as Fe-S centers.85 Tire E. coli enzyme receives electrons from reduced quinones in the plasma membrane, passing them through cytochrome b, Fe-S centers, and molybdopterin to nitrate. The three-subunit aPy enzyme contains cytochrome b in one subunit, an Fe3S4 center as well as three Fe4S4 clusters in another, and the molybdenum cofactor in the third.87 Nitrate reduction to nitrite is also on the pathway of denitrification, which can lead to release of nitrogen as NO, NzO, and N2 by the action of dissimi-latory nitrite reductases. These enzymes873 have been discussed in Chapters 16 and 18. 

This reaction is reminiscent to the autoxidation of cytochrome b5 and described reductions of cytochromes b and c with hyperoxide. In order to study further analogy of osmochrome systems to the cytochromes, water-soluble osmochromes have been prepared from sulfonated precursors. Due to the acidity of pure water, osmochrome systems cannot be held in water, but the osmichrome salt Na3[Os[TPPS4) (1-Meim)2] was isolated [51]. 

The visible absorption spectrum of oxidised cellobiose oxidase is typical of cytochrome b (Fig. 5-15). The flavin in cellobiose oxidase is weakly fluorescent, with emission maxima at 564 nm and excitation maxima at 380 and 444 nm. There are no obvious transient changes on reduction that can be readily ascribed to flavin semiquinone, but the strong absorbance of the cytochrome would make such changes difficult to detect. 

Since other membranes have an integral transmembrane electron transport system, the question arises whether these electron carriers can be involved in an oxidation-reduction driven proton movement. In neutrophil as well as macrophage plasma membranes, the answer is already yes. The superoxide-producing NADPH oxidase in these membranes is associated with a channel for proton movement to accompany the electron flow when internal NADPH is oxidized by external oxygen to produce superoxide (Nanda et al., 1993). This is a relatively simple electron transport system which contains a heterodimeric cytochrome b which also binds flavin. Thus, two proteins in a transmembrane electron transport system can transfer protons across the membrane. 

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