Cytochrome electron injection

With a method in hand for routinely constructing cytochrome c oxidase modified electrodes that exhibited direct electron transfer between the electrode and the oxidase, amperometry was used to detect reduced cytochrome c in solution at the oxidase-modified electrodes in a flow injection analysis format [69]. The dialysis cell was equipped with a wall jet inlet to direct cytochrome c solution past the oxidase-modified electrodes. Figure 12 shows the current response for three sequential reduced cytochrome c injections. Control experiments conducted at bilayer modified electrodes containing no oxidase showed current responses that are about 2% of those shown in Figure 12. This response may be due to changes in electrode capacitance and/or cytochrome c reacting at bilayer defect sites on the electrode. QCM measurements showed that no cytochrome c incorporated into the bilayer. However, cytochrome c was electrostatically held at the surface of the bilayer membrane at lower ionic strength [69]. 

Zhang Z, Nassar A-EF, Lu Z, Schenkman JB, Rusling JF (1997) Direct electron injection from electrorles to cytochrome P450 cam in biomembrane-like films. J Chem Soc Faraday Trans 93 1769-1774... 

Figure 5.10. Accumulation of a radiolabelled LMWP in the lysosomes of the proximal tubular cell. Electron microscope autoradiography of renal proximal tubular cells from a rat injected i.v. with [1251]-tyramine-cellobiose-labelled cytochrome-c, 4 h prior to fixation throngh the abdominal aorta. An intense lysosomal accumulation of the protein is observed in three dark electron-dense lysosomes. A few grains are seen over the apical endocytic apparatus. Part of the luminal brush border is found in the upper right hand corner. Magnification, x 25 000. Unpublished data from E. I. Christensen, Arhus, Denmark, and M. Haas, Groningen, Netherlands.

Microsomal reduction of chromium(VI) can also result in the formation of chromium(V), which involves a one-electron transfer from the microsomal electron-transport cytochrome P450 system in rats. The chromium(V) complexes are characterized as labile and reactive. These chromium(V) intermediates persist for 1 hour in vitro, making them likely to interact with deoxyribonucleic acid (DNA), which may eventually lead to cancer (Jennette 1982). Because chromium(V) complexes are labile and reactive, detection of chromium(V) after in vivo exposure to chromium(VI) was difficult in the past. More recently, Liu et al. (1994) have demonstrated that chromium(V) is formed in vivo by using low-frequency electron paramagnetic resonance (EPR) spectroscopy on whole mice. In mice injected with sodium dichromate(VI) intravenously into the tail vein, maximum levels of chromium(V) were detected within 10 minutes and declined slowly with a life time of about 37 minutes. The time to reach peak in vivo levels of chromium(V) decreased in a linear manner as the administered dose levels of sodium... 

The EPR and ENDOR spectroscopy was used for studies of catalytic intermediates in native and mutant cytochrome P450cam in cryogenic temperatures (6 and 77K) (Davydov et al., 2001). The ternary complex of camphor, dioxygen, and ferrous-enzyme was irradiated with y-rays to inject the second electron. This process showed that the primary product upon reduction of the complex is the end -on intermediate. This species converts even at cryogenic temperatures to the hydroperoxo-ferriheme form and after brief annealing at a temperature around 200 K, causes camphor to convert to the product. In spite of conclusions derived from x-ray analysis (Schlichtich et al., 2000) no spectroscopic evidence for the buildup of a high-valance oxyferryl/porphyrin rc-cation radical intermediate during the entire catalytic circle has been obtained. 

The maturation of fat body mitochondria is affected by the neuroendocrine balance in adult male B. discoidalis. Removal of the CC arrests the maturation of the respiratory enzymes at their partly developed, 5-day level (58, Figure 1). Injections of CC extracts on days 0 to 5 result in precocious, 10-day levels of respiratory activity by 5 days of age. Administration of the uncoupling agent 2,4-dinitrophenol indicates that electron transport, rather than phosphorylation, is the rate-limiting step for respiration in fat bcxiy mitochondria (59). A situation that differs from vertebrate mitochondria where phosphorylation is limiting. This indicates that the levels of cytochrome enzymes available for electron transport in fat body mitochondria determine the respiratory and ATP synthesis capacities of the tissue and influence its biosynthetic potential. 

Evidence for more complex ET processes came from studies in which photo chemically generated reductants injected electrons into preformed Fe-cytochrome b lYt-cytochrome c complexes. In one study, the rate of c ET (1.7 X 10 s ) was reported to depend on viscosity and surface mutations. A later laser-flash photolysis study found a rate-limiting second-order reduction of Fe-cytochrome i s/Fe-cytochrome c complexes and no sign of satmation, suggesting that the intracomplex ET rate was greater than lO s-. ... 

More direct ET into the oxidase has been initiated photochemically with an artificial electron donor, tris(2,2 -bipyridyl)ruthenium [17], electrostatically bound to the enzyme, and with Ru-labeled cytochrome c [18]. An objection to these types of experiments is, of course, that they involve unatural electron donors, whose site of interaction with the oxidase may be different from that of cytochrome c itself. This objection does not apply to experiments in which the strongly reducing triplet state of bound Zn-cytochrome c is generated photochemically to inject an electron into the oxidase [19], since this derivative of cytochrome c has the same structure and has been shown to bind to the same site as the native protein [20],... 

Vanadium is an element, and as such, is not metabolized. However, in the body, there is an interconversion of two oxidation states of vanadium, the tetravalent form, vanadyl (V+4), and the pentavalent form, vanadate (V+5). Vanadium can reversibly bind to transferrin protein in the blood and then be taken up into erythrocytes. These two factors may affect the biphasic clearance of vanadium that occurs in the blood. Vanadate is considered more toxic than vanadyl, because vanadate is reactive with a number of enzymes and is a potent inhibitor of the Na+K+-ATPase of plasma membranes (Harris et al. 1984 Patterson et al. 1986). There is a slower uptake of vanadyl into erythrocytes compared to the vanadate form. Five minutes after an intravenous administration of radiolabeled vanadate or vandadyl in dogs, 30% of the vanadate dose and 12% of the vanadyl dose is found in erythrocytes (Harris et al. 1984). It is suggested that this difference in uptake is due to the time required for the vanadyl form to be oxidized to vanadate. When V+4 or V+5 is administered intravenously, a balance is reached in which vanadium moves in and out of the cells at a rate that is comparable to the rate of vanadium removal from the blood (Harris et al. 1984). Initially, vanadyl leaves the blood more rapidly than vandate, possibly due to the slower uptake of vanadyl into cells (Harris et al. 1984). Five hours after administration, blood clearance is essentially identical for the two forms. A decrease in glutathione, NADPH, and NADH occurs within an hour after intraperitoneal injection of sodium vanadate in mice (Bruech et al. 1984). It is believed that vanadate requires these cytochrome P-450 components for oxidation to the vanadyl form. A consequence of this action is the diversion of electrons from the monooxygenase system resulting in the inhibition of drug dealkylation (Bruech et al. 1984). 

Calcium chromate has been shown to induce cytoplasmic petite mutations in mitochondria of Saccharomyces cerevisiae K Calcium chromate also dramatically depressed the content of the mitochondrial gene products cytochrome aa3 and cytochrome b, in whole yeast cells. Chromate ( 8 nM) was readily taken up by rat thymocytes and after 30 min 9% of the Cr was found in the mitochondria although 62% was found in the nuclei . Isolated rat thymus mitochondria and nuclei readily took up CrOj . After one hour incubation of Erlich ascites tumor cells with CrOj (380 /nuclear fraction and 12% was in the mitochondrial-microsomal fraction. Levels of chromium in rat liver mitochondria reached a plateau six hours after i.v. injection of chromate (0.02 mg/kg) and remained at that level through 5 days. Liver nuclear chromium levels in the same animals, although similar to mitochondrial levels at 6 h, reached a maximum at 12 h and steadily decreased after that time. Therefore the nuclear chromium levels were lower than the mitochondrial chromium levels at later times (24-120 h) after injection. The subcellular distribution of chromium in the liver of rats injected i.v. with chromate (0.56 mg/kg) was also found to be time dependent in another study. The distribution of chromium in rat liver mitochondria increased from 5% at 15 min to 21% at 72 h and also increased in the nuclear fraction from 22% at 15 min to 52% at 72 h. Incubation of isolated rat liver mitochondria with chromate (0.3-16.6 electron transport chain of the mitochondrial iner membrane. 

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