A comparison of model heme compounds with isolated and with mitochondrial cytochrome b using MCD and EPR showed that the unusual coordination structure of the cytochrome b heme(s) can be represented by hindered bis-imidazole protoheme.
The midpoint potentials for the b and c(,1) cytochrome were measured using MCD and EPR. A value of 270 mV was obtained for cytochrome c(,1) while the midpoint potentials found for the two species of cytochrome b varied with temperatures, viz. 62 and -20 mV at 23(DEGREES)C (MCD) compared to 116 and -4 mV at about 10 K (EPR). The midpoint potential of the iron-sulfur center obtained by low-temperature EPR was 286 mV. The potentials of the two half reactions of ubiquinone were measured by EPR at 110 and 296(DEGREES)K. Potentials of 176 and 51 mV were found at 110(DEGREES)K, while values of 200 and 110 mV were observed at 296(DEGREES)K.
The potentials of cytochrome b, as that of c, were also independent of pH when titrations were performed in deoxycholate buffers, while a variation of -30 mV per pH unit was observed for both b species in taurocholate buffers. These two detergents also produced different MCD-contributions of the two b hemes.
The oxidative reaction mechanism was investigated through the reoxidation of 2-electron, 4-electron (CoO-depleted) and fully-reduced Complex III by ferricyanide.
The nonlinear first-order plots of the oxidation of c(,1), were obtained for the reaction of 2-electron reduced sample at all tested ferricyanide concentrations. These data were explained by an extremely rapid intramolecular electron equilibration(> 2000 s('-1)) between the iron-sulfur center and cytochrome c(,1). Neither antimycin-A nor CoO had a substantial effect on the reoxidation kinetics of c(,1).
The oxidation of both b and c(,1) cytochromes of 4-electron reduced and fully-reduced samples monitored by the absorbance changes at 561.5 and 553.5 nm exhibited multiphasic kinetic data. The absorbance change at 553.5 nm was preceded by that at 561.5 nm during the first reaction period, indicating a fast depletion of electrons from cytochrome b through c(,1) (or iron-sulfur center) to ferricyanide. Satisfactory simulation for the kinetic data collected at both wavelengths was achieved by a linear scheme:(UNFORMATTED TABLE FOLLOWS)
slow fast fast k(,ox)
b(,H) b(,L) {2Fe/2S} c(,1) oxidant(TABLE ENDS)
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/15790 |
| Date | January 1982 |
| Creators | T'SAI, AH-LIM |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | application/pdf |
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