Reiko Oshino

Mitochondrial function under hypoxic conditions: The steady states of cytochrome a+a3 and their relation to mitochondrial energy states

The oxidation-reduction states of mitochondrial cytochromes were studied under low O2 concentrations. 1. 1. Relative oxidation states of cytochromes caused by increasing concentrations of O2 apparently followed the sequence of their half reduction potentials only under the uncoupled conditions. 2. 2. In the presence of antimycin A, the O2-induced reduction of cytochrome bT was seen in the difference spectrum. 3. 3. The O2 dependency of the relative reduction state of cytochrome a+a3 with respect to that of cytochrome c altered significantly depending upon the presence or absence of ATP. The most significant change in the O2 dependency was that due to cytochrome a3. 4. 4. When compared at a given low O2 concentration below 0.5 μM, the reduction states of cytochrome a+a3, as well as that of cytochrome c, were higher in the presence of ADP or uncoupler than in the presence of ATP. 5. 5. Whereas the O2 concentration required for 50% oxidation of cytochrome c (P50c) depended upon the respiratory rate, the O2 concentration required for 50% oxidation of cytochrome a3 (P50a3) required information on the energy state of mitochondria. Under conditions where the redox states of cytochrome c and a+a3 are measured continuously and as a function of the O2 concentration it may be possible to evaluate the energetic state of the mitochondria.

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

Abstract 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 bT. Bile salts can convert cytochrome bT into a form spectrally indistinguishable from that of bK, and the lyotropic anions cause disappearance of cytoehrome bT spectrum.

Reduction of mitochondrial components by durohydroquinone

1. Durohydroquinone (H2DQ) has been used to provide pulses of reducing equivalents to suspensions of pigeon heart mitochondria and submitochondrial particles. 2. H2DQ oxidation in the presence of pentachlorophenol was 95% sensitive to antimycin A and 99% sensitive to cyanide. 3. Pulses of 150 μM H2DQ produced reduction of cytochromes b (60–90% of total absorption), fluorescent flavoprotein (25% of total fluorescence) and pyridine nucleotide (60% of total fluorescence). Reduction of the latter two components was mediated through reversed electron transfer and was energy dependent. 4. Pulses of 10–30 μM H2DQ brought about redox cycles of ubiquinone, cytochrome(s) b and absorbing flavoprotein (succinate dehydrogenase). Addition of antimycin A altered the kinetics of electron equilibration between ubiquinone and cytochrome(s) b. Antimycin A enhanced the efficiency of reduction of cytochrome(s) b and flavoprotein by 3–20 μM H2DQ, but diminished the efficiency of ubiquinone reduction under the same experimental conditions. In the absence of inhibitor, ubiquinone was quantitatively the most important acceptor for the electrons arising from H2DQ. In the presence of antimycin A, cytochrome(s) b was the most important acceptor for H2DQ. 5. In the presence of antimycin A, ubiquinone did not show redox cycle changes synchronous with the redox cycles of flavoprotein and cytochromes b, and thus seem not to be an obligatory member for electron transfer in the main pathway from succinate dehydrogenase-cytochrome(s) b to oxygen. This antimycin A effect is interpreted as the result of a conformational change of cytochrome(s) b or an alteration of the hydrocarbon core of the membrane that ubiquinone occupies. 6. 1.2–15 μM additions of H2DQ to cyanide-blocked mitochondria or submitochondrial particles brought about pulses of reducing equivalents which were distributed among the oxidized carriers according to the redox potential of these components. Cytochromes a + a3 and c, absorbing flavorprotein and ubiquinone titrated in the presence or absence of uncoupler, as homogeneous pools in a sequence according to their reported potential values. Cytochrome(s) b titrated in the absence of uncoupler as a heterogeneous pool and in the presence of uncoupler as a homogeneous pool in the latter case the absorbance reaching 50% of the total. 7. In the aerobic steady state (plus or minus ATP) a pulse of 250 μM H2DQ reduces cytochromes b absorbing at 562 and 557 nm. The form absorbing at 557 nm is postulated to be cytochrome b555.

A sensitive bacterial luminescence probe for O2 in biochemical systems

Abstract 1. 1. An oxygen assay method applicable at very low oxygen concentrations has been developed using bioluminescence as an oxygen indicator. The method can be applied with accuracy to the oxygen concentration range between 10 −6 and 10 −8 M. 2. 2. The reliability of the method was verified by determining the oxygenation state of horse heart myoglobin as a function of oxygen concentration, and comparing this curve with that obtained polarographically. The luminescence method gave a half-oxygenation concentration of 2.2·10 −6 M (1.1 mm Hg) with a Hill constant n = 1.0, while the polarographic method gave 2·3·10 −6 M (1.2 mm Hg) O 2 with n = 1.0. With a series of heme-substituted myoglobins with half-oxygenation concentrations ranging from 2.2·10 −6 (1.1 mm Hg) to 4·10 −7 M (0.2 mm Hg) O 2 , the luminescence method yielded curves with n = 1.0 in all cases. 3. 3. The oxygenation state of both yeast hemoglobin and Ascaris perienteric fluid hemoglobin were continuously measured as a function of oxygen concentration from the fully oxygenated to the fully deoxygenated states. Both hemoglobins showed a half-oxygenation value of (0.01 mm Hg) 2 · 10 −8 M which is the lowest ever to have been measured. 4. 4. The method was applied to the measurement of the redox change in mitochondrial cytochrome c . The half-reduction of cytochrome c was observed at an oxygen concentration of 7·10 −8 M. 5. 5. The oxygenation state of yeast hemoglobin was compared with the redox state of cytochrome oxidase in yeast cells as a function of oxygen concentration. Half-oxygenation of hemoglobin in yeast cells is observed at an oxygen concentration of 2·10 −8 M, which is identical to that observed in purified yeast hemoglobin and is lower than the oxygen concentration producing a 50% reduction of cytochrome a 3 (3·10 −8 M) and cytochrome a (2·10 −7 M) in yeast cells.

Yeast hemoglobin-reductase complex

Summary Oxygen-binding pigment, so-called yeast hemoglobin was purified from Candida mycoderma in a complex form with a NAD(P)H-specific reductase system. The complex showed a single band by gel filtration on Sephadex G-100, and by electrophoresis either on cellulose acetate strip or SDS-polyacrylamide gel. The complex did not dissociate into hemoglobin and reductase system even in 8 M urea. Three prosthetic groups, protoheme, flavin and non-heme iron were identified by spectrophotometric and/or electron paramagnetic resonance measurements. The heme:flavin:iron ratios were approximately 1:1:1.

The oxygen equilibrium of yeast hemoglobin

Oxygen transport is considered to be the most important function of hemoglobin and myoglobin. Facilitated transport of oxygen may be an important function of myoglobin, and it has been suggested that the hemoglobin of microorganisms functions similarly [l] . Many reports concerning non-circulating hemo globin have appeared in the literature [2-l l] . Keilin originally discovered a hemoglobin-like pigment in yeast [2], but reported that over a period of 4 yr he had detected hemoglobin in bakers yeast only twice [2]. Because hemoglobin so seldom appears in yeast celIs it has been difficult to make any extensive study of the properties of this hemoprotein. During our generalsurvey of the hemoproteins in yeast, we have identified some species of C~ndidu which consistently have a high hemoglobin content. We have subsequently succeeded in purifying this hemoglobin [ 121. In this communication we will present measurements of the oxygen affinity of this hemoglobin as obtained using a sensitive bioluminescent oxygen indicator. The data provides evidence that the yeast hemoglobin binds oxygen non-cooperatively with high affinity and appears to be a “primitive” form of the hemoglobin of higher organisms, Moreover, the data indicates that yeast hemogiobin does not significantly enhance the diffusion of oxygen to the cytrochrome oxidase of the cells in which it is present.

Binding of cytochrome c to cytochrome c - oxidase in intact mitochondria. A study with radioactive photoaffinity-labeled cytochrome c☆

Abstract [3H]-p-Azidophenacylbromide-(methyl-4-mercaptobutyrimidate)-cytochrome c from Saccharomyces cerevisiae was prepared and its properties determined. The radioactive photoaffinity-labeled cytochrome c was linked by irradiation into a covalent complex with cytochrome c oxidase. Analysis of the complex on SDS-polyacrylamide gels showed that cytochrome c bound to one of the smaller subunits of cytochrome c oxidase with an apparent molecular weight of 15,000.

[27] Sensitive oxygen assay method by luminous bacteria

Publisher Summary This chapter describes a sensitive and convenient method to detect changes of oxygen in a range below 10 –6 using luminous bacteria. The emission spectrum of bacterial luminescence has a broad peak around 475 nm; oxygen concentration can be calibrated by measurement of the light intensity near this wavelength using a suitable optical filter. An advantage of the use of intact luminous bacteria, and not using the isolated luciferin-luciferase system, is that otherwise the latter system requires oxygen concentrations above 10 –6 M. Application of the method using the bacteria is most useful for enzyme systems for which spectrophotometric or fluorometric methods for their activity measurement can be used. Also the use of intact bacteria helps to avoid direct interaction of the oxygen indicator (the bacteria) and the oxidase system. The 1–3% NaCl and 0.5 M mannitol required for the bacteria may interfere with the enzyme system under study, and the mannitol is not harmful to mitochondria.