The b cytochromes in succinate--cytochrome c reductase from pigeon breast mitochondria

A quick and efficient method for the preparation of succinate-cytochromc c reductase from pigeon breast mitochondria using a mixture of ionic and nonionic detergents is described. The spectral characteristics of the cytochrome components of this preparation obtained during the equilibrium potentiometric titration in a new scanning spectrophotometer show a close resemblance to those of the intact mitochondria. Two b cytochromes are present whose properties can be modified by the detergents and lyotropic anions. The most sensitive cytochrome toward any modification is cytochrome b_T. Bile salts can convert cytochrome b_T into a form spectrally indistinguishable from that of b_K, and the lyotropic anions cause disappearance of cytoehrome b_T spectrum.     

Multiple cytochromes b in Mycobacterium phlei

Electron transport particles from $\text{M. phlei}$ contain at least 3 different active forms of cytochrome b, one reduced by NADH, with a λ_max at 563 nm (b_N563), and the other two reduced by either succinate or NADH, with λ_max at 559 and 563 nm (b_S559) and (b_S563). Low temperature λ_max for cytochrome b reduction with NADH or succinate are described. During steady state only b_S563 was observed with succinate. In the presence of ATP, succinate reduced an increased amount of a b563. A branching of the NAD⁺-linked pathway and a convergence at the level of cytochrome c is suggested, with only one branch accessible to succinate.     

Effects of bc1-site electron transfer inhibitors on the absorption spectra of mitochondrial cytochromes b

Changes are described that are brought about by antimycin, NoHOQnO, funiculosin, myxothiazol and mucidin in the alpha-, beta- and gamma-absorption bands of reduced and oxidized cytochromes b in the isolated complex bc1 form beef heart mitochondria. The inhibitors can be divided into 2 groups. Antimycin, funiculosin and NoHOQnO are likely to shift the spectrum of b-562 and compete for specific binding with complex bc1, with each other but not with myxothiazol and mucidin. The spectral effects of the latter two inhibitors are more difficult to interpret and may involve contributions not only from b-562 but from b-566 as well. The existence of 2 independent inhibitor binding-sites in the complex bc1 corroborates the Q-cycle hypothesis.     

Multiple light-induced reactions of cytochromes b and c in Rhodopseudomonas spheroides

Illumination of chromatophore preparations from Rhodopseudomonas spheroides causes the oxidation of a cytochrome c and a slight oxidation of a cytochrome b with a maximum at 560nm. When illuminated in the presence of antimycin A the oxidation of cytochrome c was more pronounced and cytochrome b(560) was reduced; the dark oxidation of cytochrome b(560) was biphasic in the presence of succinate, but not in the presence of NADH, a less effective reductant. Split-beam spectroscopy showed that, in addition to the reduction of cytochrome b(560), another pigment with maxima at 565 and 537nm. was reduced and was more rapidly oxidized in the dark than cytochrome b(560). This pigment, tentatively identified as cytochrome b(565), was also detected in spectra at 77 degrees k, after brief illumination at room temperature; the maxima at 77 degrees k were at 562 and 536nm. In the absence of antimycin A, light caused a transient reduction of cytochrome b(565) and an oxidation of cytochrome b(560). Dark oxidation of b(565) was rapid, even in the presence of antimycin A and succinate. Difference spectra, at 77 degrees k, of ascorbate-reduced minus succinate-reduced chromatophores or of anaerobic succinate-reduced minus aerobic succinate-reduced chromatophores suggested that two cytochromes c were present, with maxima at 547 and 549nm. When chromatophores frozen at 77 degrees k were illuminated both these cytochromes c were oxidized, indicating a close association with the photochemical reaction centre. A scheme involving two reaction centres is proposed to explain these results.     

Three functionally different cytochrome b redox centres in pigeon heart mitochondria.

Three functionally different cytochrome b redox centres, apparently of high metabolic activity, were detected in intact pigeon heart mitochondria; cytochrome b(1), b(m) and b(h), with maxima of absorption at 556.6 (State 5), 560.6, and 564.5 nm, respectively (alpha-bands, 77K). 2. Cytochrome (b(l) was reduced in the presence of either antimycin or HQNO (2-heptyl-4-hydroxyquinoline N-oxide). The absorption maximum was shifted by dithionite, cyanide, NNN'N'-tetramethyl-p-phenylenediamine + ascorbate, HQNO and antimycin. The spectra obtained on simultaneous or successive addition of HQNO and antimycin favoured the assumption of a common binding site for the two inhibitors. 3. Cytochrome b(m) was reduced in the presence of HQNO, but not in the presence of antimycin. No shifts of absorption maximum was observed. 4. Cytochrome b(h) was reduced in the presence of antimycin. HQNO was unable to cause reduction of this cytochrome by endogenous substrates. The absorption maximum was shifted to lower wavelength by organic solvents. It was inseparable from that of cytochrome b(m) in the presence of 0.4% ethanol. 5. The pattern of reduction in the presence of HQNO or antimycin demonstrates the functional difference of the three redox centres and appears incompatible wih a linear respiratory chain.     

Vibrational equilibration in absorption difference spectra of chlorophyll a.

We describe Franck-Condon simulations of vibrational cooling effects on absorption difference spectra in chlorophyll a (Chl a). The relative contributions of vibrational equilibration in the electronic ground and excited states depend on the pump and probe wavelengths. For Franck-Condon-active vibrational modes exhibiting small Huang-Rhys factors (S < 0.1, characteristic in Chl a pigments), vibrational thermalization causes essentially no spectral changes when the origin band is excited. Significant spectral evolution does occur for S < 0.1 when the 0-1 and 1.0 (hot) vibronic bands are excited. However, vibrational equilibration in these cases causes no spectral shifting in the empirical photobleaching/stimulated emission band maximum. This result bears on the interpretation of time-resolved absorption difference spectra of Chl a-containing antennae such as the Chl a/b light-harvesting peripheral antenna of photosystem II.     

ATP-synthetase activity, respiration and cytochromes of rat heart mitochondria in aging and hyperthyroidism

The ATP-synthetase activity, the rate of oxygen uptake under different metabolic conditions, the tightness of coupling of respiration to oxidative phosphorylation and the cytochrome contents in heart mitochondria of rats from different age groups were studied under normal conditions and in hyperthyroidism. It was found that heart mitochondria of aged animals did not practically differ in terms of their functional activity from those of the young animals. Administration of thyroxin to the animals from all age groups produced no significant effects on the state of mitochondria, increasing the rate of ATP synthesis on alpha-glycerophosphate, which was especially well-pronounced in aged animals, and the cytochrome content in 1-month-old rats.     

Cytochrome P-450 of adrenal mitochondria. Spin states as detected by difference spectroscopy.

Adrenal mitochondrial cytochrome P-450 which functions in cholesterol side chain cleavage (P-450scc) exhibited type I (lambdamax 385, lambdamin 420 nm) and inverse type I (lambdamin 385, lambdamax 420 nm) difference spectra with several steroids. The magnitude and type of response were dependent on the particular steroid and on the extent to which cholesterol was bound to the cytochrome in the intact mitochondrion. the inverse type I difference spectrum induced by 3beta-hydroxy-pregn-5-ene-20-one (pregnenolone) was dependent on the proportion of high spin cholesterol-cytochrome P-450scc complexes. With rat adrenal mitochondria cholest-5-ene-3beta, 20alpha-diol (20alpha-hydroxycholesterol) invariably induced a smaller inverse type I response and, under conditions where cytochrome P-450scc was nearly free of cholesterol, even produced a small type I response. Two distinct steroid binding sites on cytochrome P-450scc were detected by, respectively, the slow type I response to cholest-5-ene-3beta, 25-diol (25-hydroxycholesterol) and the rapid type I response to a subsequent addition of cholest-5-ene-3beta, 20alpha, 22 R-triol (20alpha, 22R-dihydroxycholesterol). The relative proportions of the spectral responses to these steroids were dependent on the previous extent of adrenal activation by adrenocorticotropic hormone (ACTH), because this stimulatory process altered the combination of mitochondrial cholesterol with cytochrome P-450scc. It is proposed that the two steroid binding sites on cytochrome P-450scc interact with steroids in the following way: site I binds cholesterol, 25-hydroxycholesterol, and 20alpha, 22R-dihydroxycholesterol with formation of a partially high spin cytochrome; site II binds both pregnenolone and 20alpha-OH cholesterol resulting in a low spin cytochrome. Interactions between sites I and II are not competitive, and occupancy of site II ensures a low spin state irrespective of the occupancy of site I. A second mode of interaction by 20alpha, 22R-dihydroxycholesterol stabilizes a high spin cytochrome and is competitive with site II binding by 20alpha-hydroxycholesterol or pregnenolone. Formation of a maximally high spin cytochrome follows occupancy by 20alpha, 22R-dihydroxycholesterol at both sites.