Circular dichroism of cytochrome oxidase, cytochrome b566, and cytochrome c in beef heart mitochondrial membrane fragments

1. Circular dichroism spectra of the cytochromes in membrane fragments derived from sonicated beef heart mitochondria have been obtained in the wavelength region 400–480 nm in which the major absorbance maxima of the heme prosthetic groups are found.

2. 2. Cytochrome oxidase in the mitochondrial membrane fragments has a band of positive ellipticity at 426 nm in the oxidized form and a pronounced band of positive ellipticity at 445 nm in the reduced form. The reduced-minus-oxidized difference molar ellipticity at 445 nm, Δ[θ]₄₄₅ is 3.0·10⁵ degree·cm⁻²·dmole⁻¹ heme a for membrane-bound oxidase compared to 1.6·10⁵ degree·cm⁻²·dmole⁻¹ heme a for the purified oxidase. The membrane-bound oxidase in the reduced form also appears to have a band of negative ellipticity at 426 nm not found in the purified oxidase.

3. 3. When reduced with succinate in the presence of cyanide and oxygen, cytochrome oxidase in the membrane fragments has a positive band at 442 nm very similar to that observed with the purified oxidase.

4. 4. Cytochrome c, which has a positive band at 426 nm in the purified form when reduced, appears to have a negative band at this wavelength in the mito-chondrial membrane fragments which contributes to the pronounced negative band at 426 nm observed in the membrane fragments reduced with succinate in anaerobiosis. There is no evidence for a contribution to the CD spectra of the membrane fragments from cytochrome c₁ or from cytochrome b₅₆₁ in either the oxidized or the reduced form.

5. 5. Cytochrome b₅₆₆ in the mitochondrial membrane fragments has no detectable CD spectrum in the oxidized form, but has a small positive band at 427 nm and a small negative band at 436 nm in the reduced form. The same CD spectrum is observed with cytochrome b₅₆₆ reduced with succinate in the presence of antimycin A or 2-heptyl-4-hydroxyquinoline-N-oxide. The same increase in positive ellipticity is observed at 427 nm in the mitochondrial membrane fragments, treated with oligomycin to restore energy coupling, when cytochrome b₅₆₆ is reduced with succinate in the energized membrane, as is observed in the inhibitor-treated membrane fragments. The absence of a pronounced conformational change in cytochrome b₅₆₆ on energization, as revealed by its CD spectrum, favors the concept that its reduction by succinate in the energized state is due to reversed electron transport rather than an intrinsic shift in the cytochrome's midpoint redox potential.

Oxidoreduction of cytochrome b in the presence of antimycin

1. The effect of oxidizing equivalents on the redox state of cytochrome b in the presence of antimycin has been studied in the presence and absence of various redox mediators.

2. The antimycin-induced extra reduction of cytochrome b is always dependent on the initial presence of an oxidant such as oxygen. After removal of the oxidant this effect remains or is partially (under some conditions even completely) abolished depending on the redox potential of the substrate used and the leak through the antimycin-inhibited site.

3. The increased reduction of cytochrome b induced by oxidant in the presence of antimycin involves all three spectroscopically resolvable b components (b-562, b-566 and b-558.

4. Redox mediators with an actual redox potential of less than 100–170 mV cause the oxidation of cytochrome b reduced under the influence of antimycin and oxidant.

5. Redox titrations of cytochrome b with the succinate/fumarate couple were performed aerobically in the presence of cyanide. In the presence of antimycin two b components are separated potentiometrically, one with an apparent midpoint potential above 80 mV (at pH 7.0), outside the range of the succinate/fumurate couple, and one with an apparent midpoint potential of 40 mV and an n value of 2. In the absence of antimycin cytochrome b titrates essentially as one species with a midpoint potential of 39 mV (at pH 7.0) and n = 1.14.

6. The increased reducibility of cytochrome b induced by antimycin plus oxidant is considered to be the result of two effects: inhibition of oxidation of ferrocytochrome b by ferricytochrome c₁ (the effect of antimycin), and oxidation of the semiquinone form of a two-equivalent redox couple such as ubiquinone/ubiquinol by the added oxidant, leading to a decreased redox potential of the QH₂/QH^• couple and reduction of cytochrome b.     

A thermodynamic characterisation of the cytochromes of chromatophores from Rhodopseudomonas capsulata

1. The cytochromes of chromatophores from photosynthetically grown Rhodopseudomonas capsulata have been characterised both spectrally, using the carotenoid free mutant Ala Pho⁺, and thermodynamically, using the technique of redox titrations. Five cytochromes were present; two cytochromes b, E′₀ = 60 mV at pH 7.0; and three cytochromes c, E′₀ = 340 mV, $\text{E}\text{t}\text{?}{}_0\text{= 120}\text{mV}$, E′₀ = 0 mV at pH 7.0.2. Redox titrations at different values of pH indicated that the mid point potentials of all the cytochromes varied with pH over some parts of the range between pH 6 and 9, with the possible exception of cytochrome c₃₄₀.3. The effects of succinate and NADH on the steady state reduction of the cytochromes are reported. Succinate could reduce cytochromes c₃₄₀, c₁₂₀ and b₆₀; NADH could reduce cytochromes c₃₄₀, c₁₂₀, b₆₀ and b_?25. Cytochrome c₀ could be reduced by dithionite but not by the other substrates tested.     

Alkali-induced reduction of the b-cytochromes in purified Complex III from beef heart mitochondria

Approx. 40–50% of the cytochrome b in purified Complex III is reduced by ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine or phenazine methosulfate at neutral pH. The remaining cytochrome b, including cytochrome b-565, is reduced by increasing the pH. The apparent pK for this reduction is between pH 10 and 11, and is more than two pH units higher than a similar alkali-induced transition in Mg-ATP particles. Alkali-induced reduction of cytochrome b occurs concomitantly with the exposure of hydrophobic tyrosine and tryptophan residues to a more hydrophilic environment. The relationship of these findings to the presence of a substrate accessibility barrier in Complex III is discussed.     

Kinetics of cytochrome c oxidase from yeast. Membrane-facilitated electrostatic binding of cytochrome c showing a specific interaction with cytochrome c oxidase and inhibition by ATP (With an appendix on the occurrence of two-dimensional diffusion of cytochrome c on the membrane surface)

The steady-state kinetics of purified yeast cytochrome c oxidase were investigated at low ionic strength where the electrostatic interaction with cytochrome c is maximized. In 10 mM cacodylate/Tris (pH 6.5) the oxidation kinetics of yeast iso-1-cytochrome c were sigmoidal with a Hill coefficient of 2.35, suggesting cooperative binding. The half-saturation point was 1.14 μM. Horse cytochrome c exhibited Michaelis-Menten kinetics with a higher affinity (K_m = 0.35 μM) and a 100% higher maximal velocity.In 67 mM phosphate the Hill coefficient for yeast cytochrome c decreased to 1.42, and the species differences in Hill coefficients were lessened. Under the latter conditions, a yeast enzyme preparation partially depleted of phospholipids was activated on addition of diphosphatidylglycerol liposomes. When the enzyme was incorporated into sonicated yeast promitochondrial particles the apparent K_m for horse cytochrome c was considerably lower than the value for the isolated enzyme.ATP was found to inhibit both the isolated oxidase and the membrane-bound enzyme. With the isolated enzyme in 10 mM cacodylate/Tris, 3 mM ATP increased the half-saturation point with yeast cytochrome c 3-fold, without altering the maximal velocity or the Hill coefficient. 67 mM phosphate abolished the inhibition of the isolated oxidase by ATP.The increase in affinity for cytochrome c produced by binding the oxidase to the membrane was not observed in the presence of 3 mM ATP, with the result that the membrane-bound enzyme was more sensitive to inhibition by ATP. ADP was a less effective inhibitor than ATP, and did not prevent the inhibition by ATP.It is proposed that non-specific electrostatic binding of cytochrome c to phospholipid membranes, followed by rapid lateral diffusion, is responsible for the dependence of the affinity on the amount and nature of the phospholipids and on the ionic strength.ATP may interfere with the membrane-facilitated binding of cytochrome c by a specific electrostatic interaction with the membrane or by binding to cytochrome c.     

Components of cytochrome c oxidase detectable by EPR spectroscopy

A procedure for the preparation from frozen beef heart mitochondria of cytochrome c oxidase (EC 1.9.3.1) of high heme ( 14 μmoles/mg protein) and low extraneous copper ( 1.1 atoms Cu/mole heme) and low lipid ( 0.05 g phospholipid/g protein) content is described. EPR signals observed with the enzyme between 6 and 100 °K at various states of oxidation and at different conditions of pH and presence of solutes are described in detail. The quantities of paramagnetic species represented by these signals are estimated. Under no conditions does the sum of the EPR detectable species represent more than approx. 50% of the potentially paramagnetic components of the enzyme. Comparisons are made to the corresponding signals as observed in whole tissue, mitochondria and submitochondrial particles from a number of species. The assignment of the observed signals to known components of cytochrome c oxidase is discussed briefly.     

Recombination and mating-type switching in a ligase-defective mutant of Schizosaccharomyces pombe

Summary The ligase-defective cdc17-L16 mutant of Schizosaccharomyces pombe var. pombe was tested for genetic recombination and mating-type switching. Mitotic recombination was studied in both haploid and heteroallelic diploid cells. Cells carrying a heteroallelic ade6 duplication constructed by Schuchert and Kohli were tested for ectopic genetic recombination. We have found that cdc17-L16 is a mitotic hyper-rec mutant, as it increases the instability of the duplication by a factor of about 6 even at the permissive temperature of 23° C. In diploid cells, the enhancement of recombination rates detected was to that of cdc17⁺ cells. The temperature-sensitive cell cycle defect is also associated with a reduced level of mating and sporulation but does not significantly affect mating-type switching and intragenic meiotic recombination. It is supposed that the mitotic hyper-rec phenotype is a secondary result of insufficient repair of DNA breaks, while the lack of influence of the reduced ligase activity on the latter two processes might be attributed to their peculiar initiation mechanisms.     

Indirect suppression of the wee1 mutant phenotype in Schizosaccharomyces pombe

For S. pombe cells mutations in the wee1 regulatory gene have been shown previously to allow cells to be smaller than normal at cell division, to endow the cell with a significantly long G1 cell cycle interval, and to alter the timing in the cell cycle of certain mutationally-defined cell cycle steps in G2. We show here that situations which lengthen S phase in proliferating wee1 mutant cells 'suppress' to varying degrees these wee1-mediated cell cycle alterations. Conditions chosen to protract S phase were use of cdc22.M45 mutant cells at semipermissive temperatures, and the presence of sub-arresting concentrations of the S phase inhibitors hydroxyurea or deoxyadenosine. Proliferation in the presence of each of these inhibitors was shown directly to result in protracted S phase. Residual cell division measurements were used to measure the cell cycle timing of G1 and G2 cell-cycle steps. The indirect suppression of the wee1 phenotype shown here can be understood in terms of the proposed role of the wee1+ gene product in coordinating cell division with cellular growth.     

Absorption changes of carotenoids and bacteriochlorophyll in energized chromatophores of Rhodospirillum rubrum

The energization of Rhodospirillum rubrum chromatophores by the light, ATP, PP_i, by dark electron transfer via energy-coupling sites of the redox chain, by the combination of KC1 and valinomycin causes absorption changes of carotenoids and bacteriochlorophyll. These changes due to the absorption-band shifts of the pigments are sensitive to the uncoupler p-trifluoromethoxycarbonyl cyanide phenylhydrazone (FCCP) but not to the combination of KC1 and nigericin, which abolishes fluorescence changes of atebrin. Dithionite and ferricyanide depress the light-induced absorption changes of bacteriochlorophyll but have no inhibitory effect on the PP_i-induced changes. Analysis of bacteriochlorophyll absorption changes in the infra-red region shows that the photooxidation of bacteriochlorophyll reaction centers with the negative peak in the region of 890 nm is accompanied by red and blue shifts of bacteriochlorophyll absorption bands. These shifts are due to a transmembrane electrochemical gradient of H⁺ and a local electric field arising as a result of oxidation of the reaction centers. It appears that the superposition of the (1) red shift which is characterized by negative and positive peaks at 865 and 895 nm, respectively, and (2) photobleaching of bacteriochlorophyll reaction centers in the region of 890 nm cause overall absorption changes with the negative peak at 865 nm.     

Cell cycle regulation in Schizosaccharomyces pombe

Cdc2, a cyclin-dependent kinase, controls cell cycle progression in fission yeast. New details of Cdc2 regulation and function have been uncovered in recent studies. These studies involve cyclins that associate with Cdc2 in G1-phase and the proteins that regulate inhibitory phosphorylation of Cdc2 during S-phase and G2-phase. Recent investigations have also provided a better understanding of proteins that regulate DNA replication and that are directly or indirectly controlled by Cdc2.     

The characterisation of inducible dehydrogenases specific for the oxidation of d-alanine,allohydroxy-d-proline,choline and sarcosine as peripheral membrane proteins in Pseudomonas aeruginosa

The interaction with the cytoplasmic membrane of the inducible, membrane-bound, cytochrome-linked dehydrogenases specific for the oxidation of d-alanine, allohydroxy-d-proline, choline and sarcosine in Pseudomonas aeruginosa was investigated. The susceptibility of d-alanine dehydrogenase to solubilisation by cation depletion or by washing with high ionic strength buffers indicated that it was a peripheral membrane protein. The effect of various divalent cations in reducing the amount of enzyme released by cation depletion suggests a requirement for Mg²⁺ in the binding of d-alanine dehydrogenase to the cytoplasmic membrane. The peripheral nature of all four dehydrogenases was confirmed by examination of the molecular properties and phospholipid content of preparations of the enzymes solubilised with 1 M phosphate buffer (pH 7.0). Additional confirmatory evidence was provided by Arrhenius plots of membrane-bound activity of d-alanine and allohydroxy-d-proline dehydrogenases which were monophasic and independent of the discontinuities attributable to membrane lipid phase separations which characterise such plots of the activity of integral membrane-bound enzymes. The shape of the Arrhenius plots obtained for the activities of known integral respiratory proteins of P. aeruginosa suggests that these enzymes may remain in a fluid environment throughout the course of the phase separation.     

The carotenoid shift in Rhodopseudomonas sphaeroides. The flash induced change

A mutant, Rhodopseudomonas sphaeroides G1C, having only one major carotenoid, neurosporene, is described. The spectrum of the carotenoid shift in this mutant is analysed and it is concluded that only 7–11% of the pigment is involved under conditions of steady-state illumination and that this pigment undergoes a shift of 7 nm.The spectrum of the carotenoid shift under conditions of multi-flash illumination is examined for changes in shape concordant with a progressive red shift of the pigment with increasing membrane potential; the spectra of the fast change after each of three flashes does not agree well with predictions from a model involving a progressive shift of the pigment, the slow change shows qualitative agreement with such a model but the small size of the signal and the presence of more than one phase makes analysis of this phase more difficult.No separate pool of carotenoid, that might correspond to that postulated to participate in the carotenoid shift, could be identified by fourth derivative analysis of, or curve fitting to, the spectrum of the neurosporene.     

hCG-Increased phosphorylation of proteins in primary culture of Leydig cells: further characterization

The level of fructose 2,6-bisphosphate is markedly decreased in the rat V.renal gland in diabetes, falling to 23% of the control value. There is parallel decrease in the flux of 14C-labelled glucose through the glycolytic route and tricarboxylic acid cycle. Only minimal changes in hexokinase (EC 2.7.1.1.), a 22% decrease in Type I hexokinase of the soluble fraction, were observed, highlighting the probable significant involvement of fructose 2,6-bisphosphate in the regulation of glycolysis in the adrenal. In contrast, there was evidence for a marked rise in the flux of glucose through the pentose phosphate pathway, which may be linked to enhanced corticoid synthesis in the diabetic state.     

Functional expression of human P-glycoprotein in Schizosaccharomyces pombe

Human MDR1 cDNA was introduced into the human cultured cells KB-3-1 and Schizosaccharomyces pombe pmdI null mutant KN3. The drug sensitivity of KB-G2 and KN3/pgp, expressing human P-glycoprotein, was examined. KB-G2 was resistant to the peptide antibiotics valinomycin and gramicidin D as well as having a typical multidrug resistance (MDR) phenotype. KN3/pgp was resistant to valinomycin and actinomycin D, but not to adriamycin. The ATP-hydrolysis-deficient mutant did not confer KN3 resistance to these antibiotics. Human P-glycoprotein expressed in S. pombe seemed to lack N-glycosylation. The N-glycosylation-deficient mutant, however, conferred a typical MDR phenotype on KB-3-1. These results suggest that human P-glycoprotein functions as an efflux pump of valinomycin and actinomycin D in the membrane of S. pombe.     

A study of integrative transformation in Schizosaccharomyces pombe

Summary Organ-specific and constitutive expression of the Arabidosis HSP18.2 gene under normal growth conditions (22° C) was observed in transgenic A. thaliana, which carried a fusion gene composed of the promoter region of HSP18.2, one of the genes for low molecular weight heat-shock proteins in Arabidopsis, and the gene for -glucuronidase (GUS) from Escherichia coli. In order to clarify the organ-specific nature of promoter expression, various mutations that affect flower morphology were introduced into the transgenic Arabidopsis line, AHS9. The results show that the pattern of expression observed in sepals, filaments, and styles is regulated in a structure-dependent manner, and suggest that the HSP18.2 gene might have an important role in the process of differentiation of flower buds, as do several other stress-related genes.