Mitochondrial polymorphism. IV. Phospholipid composition of mitochondria of a wheat hybrid and its parents

Mitochondria of young seedlings of wheat genotypes 28, 31MS, and 31MS |MX 28 differed in total lipid and phospholipid. Hybrid mitochondria had more lipid and phospholipid than did the parents, and the three genotypes differed in fatty acid composition of the phospholipid fraction. Hybrid mitochondria exhibited heterosis in cytochrome oxidase activity. Although depletion of phospholipid greatly reduced cytochrome oxidase activity in hybrid mitochondria, the heterotic property was preserved. Regulatory aspects of lipid and phospholipid in mitochondria of the hybrid and complementing parental mixture are considered.This research was supported by DeKalb Ag-Research Inc. and in part by grant A-338 from Robert A. Welch Foundation of Houston, Texas.The preceding paper in this series is Sarkissian, I. V., and Srivastava, H. K. (1971). Biochem. Genet. 5:57.     

The participation of cytochromes in the process of nitrate respiration in Klebsiella (Aerobacter) aerogenes

1. The participation of cytochromes in the membrane-bound, nitrate and oxygen respiratory systems of Klebsiella (Aerobacter) aerogenes has been investigated. The membrane preparations contained the NADH, succinate, lactate and formate oxidase systems, and in addition a high respiratory nitrate reductase activity.2. Difference spectra indicated the presence of cytochromes b, a₁, d, and o. Cytochromes of the c-type could not be detected in these membranes. Both cytochrome b content and respiratory nitrate reductase activity were the highest in bacteria grown anaerobically in the presence of nitrate.3. Cytochrome b was the only cytochrome which, after being reduced by NADH, could be partially reoxidized anaerobically in the presence of nitrate. Furthermore, nitrate caused a lower aerobic steady state reduction only of cytochrome b.4. NADH oxidase and NADH-linked respiratory nitrate reductase activities were both inhibited by antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide and KCN. NADH oxidase activity was selectively inhibited by CO, while azide was found to inhibit only the respiratory nitrate reductase. In the presence of azide, nitrate did not affect the level of reduction of cytochrome b.5. The evidence presented suggests that cytochrome b is a carrier in the electron transport systems to both nitrate and oxygen; from cytochrome b branching occurs, with one branch linked to the respiratory nitrate reductase and one branch linked to oxidase systems, containing the cytochromes a₁, d and o.     

Oxidative enzyme activities in rat brain subcellular fractions. The effect of deoxycholate, freeze-thaw, and water

(1) The distributions of four oxidative enzymes were studied in crude brain fractions. (2) Freeze-thaw cycle treatment and frozen storage of homogenate fractions gave apparent enhancement of cytochrome oxidase and NADH cytochrome c reductase activities. (3) Deoxycholate released cytochrome oxidase and NADH cytochrome c reductase activities from low-speed precipitates. The NADH diaphorase was enhanced to a small degree while NADPH cytochrome c reductase was not affected by deoxycholate. (4) Distilled water coupled with a single homogenization released trapped soluble enzymes and mitochondria and gave nearly maximal cytochrome oxidase activity as judged by deoxycholate treatment. The total distilled water activity of NADH cytochrome c reductase was much less than that of deoxycholate-stimulated fractions. The activities of other enzymes were not markedly affected by distilled water although their distribution was changed.     

Derepression of mitochondria and their enzymes in yeast: regulatory aspects

We have performed a detailed analysis of the properties of glucose-repressed cells of a commercial strain of Saccharomyces cerevisiae. They contain measurable amounts of the respiratory enzymes NADH oxidase, cytochrome c oxidase, succinate dehydrogenase, succinate:cytochrome c reductase and NADH:cytochrome c reductase (antimycin A-sensitive) as well as the dehydrogenases for l-malate, l-glutamate, and l₈-isocitrate. Cytochromes b, c₁, and aa₃ are present in amounts that may be in excess of those required for cytochrome-linked enzyme activities. Enzymes and cytochromes are localized in large, presumably mitochondrial organelles among which no compositional or functional heterogeneity could be detected.We have also analyzed the kinetics of synthesis of respiratory enzymes and cytochromes during the release from catabolite(glucose) repression. All activities assayed except for cytochrome c oxidase begin their derepression before the external glucose concentration falls below 0.4%; derepression of cytochrome oxidase occurs only after the glucose concentration falls below 0.1%. The earlier events comprise the “fermentative” phase of derepression while the later events comprise the “oxidative” phase. The two phases can be distinguished operationally by their sensitivity to antimycin A. Only the oxidative phase is blocked by the inhibitor. Respiratory enzymes and cytochromes appear to fall into two classes distinguishable by their increase during derepression. An apparently constitutive one consists of cytochrome c oxidase, ATPase, and cytochromes aa₃, b, and c₁; these entities increase in amount per cell but not in amount per unit of mitochondrial mass and are of the order of 5-fold or less. The second class consists of those activities that increase by more than 6-fold and may be considered derepressible in the strict sense. Thus, proliferation and differentiation of mitochondria both contribute to the cellular changes associated with derepression.The fermentative phase of derepression does not require mitochondrial function, mitochondrial protein, or RNA synthesis, or the gradual accumulation of regulatory elements for either its initiation or persistence. This phase of derepression also occurs in cytoplasmic petites. In contrast, the oxidative phase of derepression requires mitochondrial function. Mitochondrial gene expression is required for the biogenesis of fully functional mitochondria but, except for cytochrome c, it plays little or no role in regulating the expression of nuclear genes the products of which are localized in mitochondria.     

Partial purification of the superoxide-generating system of macrophages. Possible association of the NADPH oxidase activity with a low-potential (−247 mV) cytochrome b

NADPH oxidase activity was solubilized by detergent treatment of subcellular particles obtained from guinea-pig peritoneal macrophages stimulated with phorbol myristate acetate. Gel filtration of the material containing the NADPH oxidase activity gave two peaks of proteins, one of which eluted with the void and the other with the included volume of an AcA 22 column. The material eluted in the void volume contained more than 50% of the NADPH oxidase activity and less than 10% of the NAD(P)H cytochrome c reductase activity. A b-type cytochrome with peaks of absorption at 558, 528 and 426 nm was also enriched in the fraction which contained the NADPH oxidase activity. The distribution of flavoproteins as revealed by the measurement of FAD was different from that of NADPH oxidase and cytochrome b, and followed the elution profile of NADH cytochrome c reductase. Studies in subcellular particles showed that the b cytochromes of mitochondria and endoplasmic reticulum reduced by selective biochemical means accounted for only a minor part of the total b-type cytochromes and that the new cytochrome b previously described in neutrophils is the major chromophore also in macrophages. Oxidation-reduction midpoint potential of the partially purified cytochrome b was shown to be −247 mV. Association of cytochrome b with the NADPH oxidase activity and its very low E_m7.0 makes it a suitable candidate to be part of the superoxide-generating system also in macrophages.     

The Respiratory Chain of Plant Mitochondria: VIII. Reduction Kinetics of the Respiratory Chain Carriers of Mung Bean Mitochondria with Reduced Nicotinamide Adenine Dinucleotide

Addition of 90 micromolar reduced nicotinamide adenine dinucleotide (NADH) in the presence of cyanide to a suspension of aerobic mung bean (Phaseolus aureus) mitochondria depleted with ADP and uncoupler gives a cycle of reduction of electron transport carriers followed by reoxidation, as NADH is oxidized to NAD⁺ through the cyanide-insensitive, alternate oxidase by excess oxygen in the reaction medium. Under these conditions, cytochrome b₅₅₃ and the nonfluorescent, high potential flavoprotein Fp_ha of the plant respiratory chain become completely reduced with half-times of 2.5 to 2.8 seconds for both components. Reoxidation of flavoprotein Fp_ha on exhaustion of NADH is more rapid than that of cytochrome b₅₅₃. There is a lag of 1.5 seconds after NADH addition before any reduction of ubiquinone can be observed, whereas there is no lag perceptible in the reduction of flavoprotein Fp_ha and cytochrome b₅₅₃. The half-time for ubiquinone reduction is 4.5 seconds, and the extent of reduction is 90% or greater. About 30% of cytochrome b₅₅₇ is reduced under these conditions with a half-time of 10 seconds; both cytochrome b₅₆₂ and the fluorescent, high potential flavoprotein Fp_hf show little, if any, reduction. The two cytochromes c in these mitochondria, c₅₄₇ and c₅₄₉, are reduced in synchrony with a half-time of 0.8 second. These two components are already 60% reduced in the presence of cyanide but absence of substrate, and they become completely reduced on addition of NADH. These results indicated that reducing equivalents enter the respiratory chain from exogenous NADH at flavoprotein Fp_ha and are rapidly transported through cytochrome b₅₅₃ to the cytochromes c; once the latter are completely reduced, reduction of ubiquinone begins. Ubiquinone appears to act as a storage pool for reducing equivalents entering the respiratory chain on the substrate side of coupling site 2. It is suggested that flavoprotein Fp_ha and cytochrome b₅₅₃ together may act as the branching point in the plant respiratory chain from which forward electron transport can take place to oxygen through the cytochrome chain via cytochrome oxidase, or to oxygen through the alternate, cyanide-insensitive oxidase via the fluorescent, high potential flavoprotein Fp_hf.     

An evaluation of mitochondrial heterosis and in vitro mitochondrial complementation in wheat,barley and maize

Summary Two families each of wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) and maize (Zea mays L.) were studied for mitochondrial heterosis and in vitro mitochondrial complementation. Inbred parents and their hybrids were compared for seedling heights and rate of oxygen uptake by the whole tissue to find out if the hybrids showed greater growth and respiratory activity at the seedling stage. Further comparisons were made by isolating mitochondria from the seedling tissues and measuring their ADP0 ratio, respiratory control ratio and cytochrome c oxidase activity for mitochondrial heterosis. Mixtures of parental mitochondria were similarly compared with parental and hybrid mitochondria for in vitro mitochondrial complementation. No evidence for mitochondrial heterosis or in vitro mitochondrial complementation was found, nor any correlation between the different mitochondrial parameters, seedling heights or rates of oxygen uptake by seedling tissue. The suggested use of mitochondrial heterosis and in vitro mitochondrial complementation for plant breeding is discussed.Data for this paper is taken from the author's dissertation written as a part of Ph.D. degree requirements at the Biology Department, Texas A & M University, College Station, Texas     

Mitochondrial Complementation and Grain Yield in Hybrid Wheat

ADP/O ratios, cytochrome c oxidase and adenosine triphosphatasehave been measured in mitochondria and mixtures of mitochondriaisolated from two day-old shoots of wheat of known F₁ hybridgrain yield performance. Mixtures of mitochondria from two varieties,Peko and Cappelle-Desprez, which have considerable F₁ hybridyield heterosis, showed a significantly increased ADP/O ratioover the mean value for mitochondria from the varieties assayedindividually, i.e. these varieties showed ‘mitochondrialcomplementation’. No mitochondrial complementation wasdetected for cytochrome c oxidase or adenosine triphosphatase.In other mitochondrial mixtures no complementation in ADP/Oratios were found even when the varieties showed F₁ hybrid yieldheterosis. Mitochondrial ADP/O ratios were studied in six varietiesindividually and in mixtures. In only one mixture was any significantcomplementation detected. However, when all the results wereconsidered together, mitochondrial complementation was significantlycorrelated with F₁ hybrid grain yield heterosis when the plantswere grown at a low seed density but not at a high seed density.New hypotheses are offered to account for mitochondrial complementationand its statistical relationship with yield heterosis.     

Liver microsomal electron transport systems. Properties of a reconstituted, NADH-mediated benzo[a]pyrene hydroxylation system.

An enzyme system from rat liver microsomes which catalyzes the NADH-mediated hydroxylation of benzo[a]pyrene has been reconstituted. The essential microsomal components of this NADH-dependent pathway were NADH-cytochrome b₅ reductase, cytochrome b₅, cytochrome P-448 and, phosphatidyl choline. Highly purified NADPH-cytochrome c reductase containing small amounts of deoxycholate stimulated this NADH-mediated pathway supported by 0.2 mm NADH whereas boiled reductase had little effect. Part of this stimulation could be attributed to hydroxylation of benzo[a]pyrene via a second pathway; i.e., NADPH-cytochrome c reductase in combination with cytochrome P-448 and phosphatidylcholine also supported a low rate of NADH-dependent hydroxylation. The mechanism of the remaining stimulation is not known. However, the effect of NADPH-cytochrome c reductase on the reconstituted cytochrome b₅-dependent pathway was not unique; high concentrations of deoxycholate also stimulated this pathway, perhaps by facilitating the transfer of electrons from NADH-cytochrome b₅ reductase to cytochrome b₅. The addition of NADPH-cytochrome c reductase to the cytochrome b₅-dependent reconstituted system also affected the apparent K_m of NADH for benzo[a]pyrene hydroxylation. In the absence of NADPH-cytochrome c reductase, the apparent K_m of NADH was 1.3 μm while in its presence a low (1.3 μm) and a high (1700 μm) K_m were observed, consistent with the affinities of the two flavoproteins for NADH. Our results also indicate that the relative contribution of the pathway due to NADPH-cytochrome c reductase in combination with phosphatidyl choline and cytochrome P-448 to the overall rate of NADH-supported benzo[a]pyrene hydroxylation in microsomes would be greatly dependent on the concentration of NADH chosen. The rate of benzo[a]pyrene hydroxylation by these reconstituted components was almost 10-fold greater with 10 mm NADH than with 0.2 mm NADH, a result consistent with the reduction of NADPH-cytochrome c reductase by high concentrations of NADH.     

ANALYTICAL STUDY OF MICROSOMES AND ISOLATED SUBCELLULAR MEMBRANES FROM RAT LIVER : I. Biochemical Methods

The series introduced by this paper reports the results of a detailed analysis of the microsomal fraction from rat liver by density gradient centrifugation. The biochemical methods used throughout this work for the determination of monoamine oxidase, NADH cytochrome c reductase, NADPH cytochrome c reductase, cytochrome oxidase, catalase, aminopyrine demethylase, cytochromes b₅ and P 450, glucuronyltransferase, galactosyltransferase, esterase, alkaline and acid phosphatases, 5'-nucleotidase, glucose 6-phosphatase, alkaline phosphodiesterase I, N-acetyl-β-glucosaminidase, β-glucuronidase, nucleoside diphosphatase, aldolase, fumarase, glutamine synthetase, protein, phospholipid, cholesterol, and RNA are described and justified when necessary.     

An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b₅ reductase and cytochrome b₅.     

Different effects of 2-n-heptyl-4-hydroxyquinoline-N-oxide on oxygen and nitrate respiration in Klebsiella aerogenes

The inhibition of the oxidase and respiratory nitrate reductase activity in membrane preparations from Klebsiella aerogenes by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) has been investigated. Addition of HQNO only slightly affected the aerobic steady-state reduction of cytochrome b₅₅₉ with NADH, but caused a significantly lower nitrate reducing steady-state of this cytochrome. The changes in the redox states of the cytochromes during a slow transition from anaerobic to aerobic conditions in the presence and absence of HQNO showed that the inhibition site of HQNO is located before cytochrome d. Inhibition patterns obtained upon titration of the NADH oxidase and NADH nitrate reductase activity with HQNO indicated one site of inhibitor interaction in the NADH nitrate reductase pathway and suggested a multilocated inhibition of the NADH oxidase pathway. Difference spectra with ascorbate-dichlorophenolindophenol as electron donor indicated the presence of a cytochrome b₅₆₃ component which was not oxidized by nitrate, but was rapidly oxidized by oxygen. The latter oxidation was prevented by HQNO. A scheme for the electron transport to oxygen and nitrate is presented. In the pathway to oxygen, HQNO inhibits both at the electron-accepting side of cytochrome b₅₅₉ and at the electron-donating side of cytochrome b₅₆₃, whereas in the pathway to nitrate, inhibition occurs only at the electron-accepting side of cytochrome b₅₅₉.     

Biochemical heterogeneity of rat liver mitochondria separated by rate zonal centrifugation

1. Rat liver mitochondria were separated on the basis of their sedimentation coefficients in an iso-osmotic gradient of Ficoll–sucrose by rate zonal centrifugation. The fractions (33, each of 40ml) were collected in order of decreasing density. Fractions were analysed by spectral analysis to determine any differences in the concentrations of the cytochromes and by enzyme analyses to ascertain any differences in the activities of NADH dehydrogenase, succinate dehydrogenase and α-glycerophosphate dehydrogenase. 2. When plotted as% of the highest specific concentration, the contents of cytochrome a+a₃ and cytochrome c+c₁ were constant in all fractions but cytochrome b was only 65% of its maximal concentration in fraction 7 and increased with subsequent fractions. As a result, the cytochrome b/cytochrome a+a₃ ratio almost doubled between fractions 7 and 25 whereas the cytochrome c+c₁/cytochrome a+a₃ ratio was unchanged. 3. Expression of the dehydrogenase activities as% of highest specific activity showed the following for fractions 6–26: NADH dehydrogenase activity remained fairly constant in all fractions; succinate dehydrogenase activity was 62% in fraction 6 and increased steadily to its maximum in fraction 18 and then decreased; the activity of α-glycerophosphate dehydrogenase was only 53% in fraction 6 and increased slowly to its peak in fractions 22 and 24. 4. These differences did not result from damaged or fragmented mitochondria or from microsomal contamination. 5. These results demonstrate that isolated liver mitochondria are biochemically heterogeneous. The importance of using a system for separating biochemically different mitochondria in studies of mitochondrial biogenesis is discussed.     

Analytical study of microsomes and isolated subcellular membranes from rat liver. 3. Subfractionation of the microsomal fraction by isopycnic and differential centrifugation in density gradients

Rat liver microsomal fractions have been equilibrated in various types of linear density gradients. 15 fractions were collected and assayed for 27 constituents. As a result of this analysis microsomal constituents have been classified, in the order of increasing median density, into four groups labeled a, b, c, and d. Group a includes: monoamine oxidase, galactosyltransferase, 5''-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and cholesterol; group b: NADH cytochrome c reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b ₅, and cytochrome P 450; group c: glucose 6-phosphatase, nucleoside diphosphatase, esterase, β-glucuronidase, and glucuronyltransferase; group d: RNA, membrane-bound ribosomes, and some enzymes probably adsorbed on ribosomes: fumarase, aldolase, and glutamine synthetase. Analysis of the microsomal fraction by differential centrifugation in density gradient has further dissociated group a into constituents which sediment more slowly (monoamine oxidase and galactosyltransferase) than those of groups b and c, and 5''-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and the bulk of cholesterol which sediment more rapidly (group a2). The microsomal monoamine oxidase is attributed, at least partially, to detached fragments of external mitochondrial membrane. Galactosyltransferase belongs to the Golgi complex. Group a2 constituents are related to plasma membranes. Constituents of groups b and c and RNA belong to microsomal vesicles derived from the endoplasmic reticulum. These latter exhibit a noticeable biochemical heterogeneity and represent at the most 80% of microsomal protein, the rest being accounted for by particles bearing the constituents of groups a and some contaminating mitochondria, lysosomes, and peroxisomes. Attention is called to the operational meaning of microsomal subfractions and to their cytological complexity.     

Bioenergetics of mitochondria from flight muscles of aging blowflies: partial reactions of oxidation and phosphorylation.

Mitochondria from flight muscles of senescent blowflies, Phormia regina, which exhibit decreased rates of coupled, or ADP-stimulated, oxidation of α-glycerolphosphate or pyruvate show similar decreased rates of uncoupled, or carbonylcyanide-p-trifluoromethoxyphenylhydrazone-stimulated oxidation. Thus, uncoupled α-glycerolphosphate oxidation is decreased by 20% and uncoupled pyruvate oxidation by 39% in mitochondria isolated from 31- to 33-day-old blowflies as compared with mitochondria from 7- to 9-day-old flies. The finding of nearly equal decreases with age in coupled and uncoupled respiration suggests that the age-dependent defect lies within the oxidative or electron transport pathway, and not associated with phosphorylation. However, no such change with age is observed in any of the partial reactions of electron transport that were examined. These include the following partial reactions: (a) pyruvic dehydrogenase; (b) pyruvate-ferricyanide reductase: (c) α-glycerolphosphate dehydrogenase; (d) α-glycerolphosphate-ferricyanide reductase; and (e) cytochrome oxidase. In addition, no age-associated decline is observed in the content of cytochromes b, c + c₁, a, or a₃ or in the specific activity of the fully activated mitochondrial ATPase (assayed in the presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone). However, the specific activity of the masked ATPase (assayed in the absence of uncoupler) is increased 38% in mitochondria from senescent blowflies.     

Reduced Pyridine Nucleotide Oxidases of Euglena gracilis var. bacillaris*

SYNOPSIS. Cell-free extracts of a streptomycin-bleached strain of Euglena gracilis var. bacillaris have been examined for enzyme systems primarily responsible for the oxidation of reduced pyridine nucelotides. NADH lipoyl dehydrogenase, NADH and NADPH oxidase, NADH and NADPH diaphorase, and NADH and NADPH cytochrome c reductase have been demonstrated. The NADPH-linked enzymes had lower activity rates and were less sensitive to N-ethyl maleimide and p-hydroxymercuribenzoate than their NADH-linked counterparts. NADH cytochrome c reductase was the most sensitive to antimycin A. Michaelis-Menten constants (K_m) determined were as follows: NADH diaphorase, 350 μM; NADPH diaphorase, 200 μM; NADH cytochrome c reductase, 13 μM; NADPH cytochrome c reductase, 9 μM; NADH oxidase, 100 μM; NADPH oxidase 150 μM; NADH lipoyl dehydrogenase, 0.35 μM. Enzyme activities after storage at –5 C indicate that the diaphorases are less labile than the other tested enzymes, and the differential activities of the NADH and NADPH linked enzymes suggest that functionally they may have different roles.     

Cytochrome b type oxidases in the respiratory chains of the phytopathogenic fluorescent bacteria Pseudomonas cichorii and Pseudomonas aptata

Membrane fragments from the phytopathogenic bacteria Pseudomonas cichorii and Pseudomonas aptata have been examined. A branched respiratory chain is operative in P. cichorii whereas a linear electron transport system characterizes the related bacterium P. aptata. Both species contain several b type cytochromes resolved by redox titration analysis, but no a type components may be detected. In contrast, only P. cichorii is endowed with c type cytochromes and hence with cytochrome c oxidase activity. Among the b type cytochromes, two high-potential components, with Em_7.0 at +250 mV and +380 mV, have been kinetically characterized and tentatively associated with cyanideresistant and cytochrome c oxidase activities, respectively. Cytochrome b-250 should correspond to the spectrally detectable cytochrome o whereas cytochrome b-380 is functionally similar to cytochrome b-410 described in Rhodopseudomonas capsulata. This conclusion seems to blur previous reported data on other obligate aerobes in which cytochrome o has been generally associated with cytochrome c oxidase and also suggests that a more accurate reconsideration of the actual physiological role of cyt. o in bacterial respiration is necessary. Furthermore the question arises whether cyt. b-410 like oxidases, i. e. high-potential b's similar to cyt. b-410 of R. capsulata, may be widely distributed among aerobes rather than restricted to facultative photosynthetic prokaryotes.     

The properties of the mitochondrial succinate-cytochrome c reductase

The cytochromes b and b_T of pigeon heart mitochondria have half-reduction potentials (Em's) of +30 mV and −30 mV at pH 7.2. The midpoint potentials of these cytochromes become more negative by 30–60 mV per pH unit when the pH is made more alkaline. Detergents may be used to prepare a succinate-cytochrome c reductase free of cytochrome oxidase in which the activation of electron transport induced by oxidation of cytochrome c₁ causes the half-reduction potential of cytochrome b_T to become at least 175 mV more positive than in the absence of electron transport. This change is interpreted as indicating that the primary energy conservation reaction at site 2 remains fully functional in the purified reductase. Preliminary electron paramagnetic resonance spectra of the succinate-cytochrome c reductase as measured at near liquid helium temperatures are presented.     

The cytochromes of mitochondria from Tetrahymena pyriformis strain ST

1. Mitochondria of the obligately aerobic ciliate protozoon, Tetrahymena pyriformis strain ST, are unusual in that they possess a cytochrome oxidase system that does not react with reduced mammalian cytochrome c; the presence of cytochromes a₆₀₃+a₃ is masked in the α-band region of spectra by the broad absorption band of cytochrome a₆₂₀. 2. Other haemoproteins present include cytochromes b₅₆₀, b₅₅₆, c₅₅₃ and c₅₄₉. 3. The reaction of reduced cytochrome a₃ with CO is reversed by flash photolysis, and in the presence of O₂ the subsequent oxidation of this cytochrome is followed by that of cytochrome a₆₀₃. 4. Cytochromes a₆₂₀ and b₅₆₀ also react with CO and with KCN; the latter cytochrome corresponds with that designated cytochrome o by other workers. 5. The contribution of cytochrome a₆₀₃ to difference spectra is revealed by making use of the fact that it does not react with KCN. 6. Cytochrome a₆₂₀ is unstable, and its α-absorption band is lost from spectra of mitochondria which have been aged or treated with ultrasound, detergents or organic solvents. 7. Possible pathways of electron transport via the several different terminal oxidases in Tetrahymena mitochondria are proposed.     

Succinate oxidation by coupled potato tuber mitochondria followed by rapid scan spectrometry

Oxidation of succinate by potato tuber mitochondria has been investigated from aerobiosis to complete anuerobiosis. Difference spectra of the various steps were recorded by a rapid scan spectrometer delivering averaged spectra every 3 s in the range 380 to 630 mm. The transitions between state 3 and 4 resulted in large redox changes, essentially for the b cytochromes, and in significant changes in the spectral baseline (light scattering). At anaerobiosis the cytochromes c, c₁ and a were reduced while cytochrome a, remained oxidized. – Addition of uncouplers in aerobiosis induced oxidation of the b cytochromes, and when anaerobiosis occurred cytochromes c, c₁a and a₃ were reduced simultaneously. When uncouplers were added in anaerobiosis a partial oxidation of the b cytochromes and the reduction of cytochrome a₃ were observed. These results are interpreted as the building up of a membrane potential, maximal in state 4 and stable after anaerobiosis. The cytochromes buried in the membrane equilibrate with the membrane potential, and their redox states are sensitive to the changes. Variations of membrane potential also induce changes in the light scattering by the mitochondrial membrane.