Does the low respiratory rate in unfertilized eggs result mainly from depression of the redox reaction catalyzed by flavoproteins? Analysis of the respiratory system by light-induced release of CO-mediated inhibition

In unfertilized eggs of the sea urchin, the quite low respiratory rate is enhanced by tetramethyl- p -phenylenediamine (TMPD), phenazine methosulfate (PMS) and sperm and this augmentation is completely inhibited by carbon monoxide (CO). Exposure to light releases eggs from this CO-mediated inhibition. The action spectra for photoreactivation of CO-inhibited cytochrome c oxidase in isolated mitochondria and CO-blocked respiration in TMPD-treated eggs were found to be similar to the absorption spectrum of CO-bound cytochrome aa ₃. In PMS-treated eggs and fertilized eggs, the maximum photoreactivation of CO-inhibited respiration occurred at a light fluence rate higher than that for maximum photoreactivation of CO-inhibited respiration in TMPD-treated eggs, with peaks at the same wavelengths as those in the absorption spectrum of reduced cytochrome b. A similar phenomenon was seen for NADH cytochrome c reductase in mitochondria. Thus, cytochrome c oxidase and NADH cytochrome c reductase, whose activities are not altered by fertilization, seem to be functional, even in unfertilized eggs. In unfertilized eggs, difference spectra indicated that PMS and sperm augmented cytochrome b reduction and that TMPD accelerated cytochrome c reduction without cytochrome b reduction. Therefore, it is likely that depression of electron transport to cytochrome b , which is augmented by PMS and sperm, is responsible for the low respiratory rate in unfertilized eggs.     

Photo-activation of respiration in the presence of CO in sperm of several marine invertebrates

• 1.1. The light irradiation at examined wavelengths between 360 and 630 nm enhanced the respiratory rate in the presence of CO in sperm, as well as eggs, of the sea urchins, Anthocidaris crassispina and Hemicentrotus pulcherrimus, the starfish, Asterina pectinifera, and the echiuroid, Urechis unicinctus.
• 2.2. The maximum peaks of stimulating effects of light irradiation on CO-insensitive respiration were found at wavelengths of 430, 530 and 570 nm in sperm of these species.
• 3.3. The respiration in the presence of CO was insensitive to light irradiation in sperm and eggs of the oyster, Crassostrea gigas and the tunicate, Ciona intestinaris.

     

Light Effects in Yeast: Evidence for Participation of Cytochromes in Photoinhibition of Growth and Transport in Saccharomyces cerevisiae Cultured at Low Temperatures

Visible light of moderate intensity inhibits growth, respiration, protein synthesis, and membrane transport in bakers' yeast and has a deleterious effect on membrane integrity. The results of this study indicate that these effects require the presence of cytochromes b and a/a(3). The light sensitivities of growth rate and [(14)C]histidine uptake in wild-type rho(+) Y185 and D225-5A strains of Saccharomyces cerevisiae were compared with those in a variety of mutants lacking cytochrome b or a/a(3) or both; a close correlation was found between the presence of these respiratory pigments and photosensitivity. Thus, strain TL5-3C, a nuclear petite lacking cytochromes b, a, and a(3), was resistant to light; strain GL5-6A, another nuclear petite having reduced amounts of cytochromes a and a(3), was partially resistant; strains MB127-20C and MB1-6C, nuclear petites lacking only cytochrome b, were also only partially resistant to light; whereas mutants containing all three cytochromes but having their respiratory chain either nonfunctional (strain ZK3-6B) or uncoupled (strain 18-27/t12) were fully sensitive to light. Finally, an equal-energy, broad-band action spectrum for the light inhibition of growth and transport indicated that blue light (408 nm) was most effective; these wavelengths correspond to the Soret region of the cytochrome absorption spectrum. The results suggest, therefore, that the yeast cytochromes b, a, and a(3) are the primary photoreceptors for the inhibitory effects of light and, perhaps, for other processes, such as the entrainment of biological rhythms in this species.     

Purification and properties of a cytochrome b560-d complex, a terminal oxidase of the aerobic respiratory chain of Photobacterium phosphoreum

A cytochrome b560-d complex, a terminal oxidase in the respiratory chain of Photobacterium phosphoreum grown under aerobic conditions, was purified to near homogeneity. The purified oxidase complex is composed of equimolar amounts of two polypeptides with molecular weights of 41,000 and 54,000, as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. It contains 10.2 nmol of protoheme and 22.5 nmol of iron/mg of protein. The enzyme is a "cytochrome bd-type oxidase," showing absorption peaks at 560 and 625 nm in its reduced minus oxidized difference spectrum at 77K. This oxidase combined with CO, and its CO difference spectrum at room temperature in the Soret region showed a peak at 418 nm and a trough at 434 nm. In addition, a trough at 560 nm (cytochrome b), and a trough at 620 nm and a peak at 639 nm (cytochrome d) were observed in the CO-binding spectrum. This cytochrome b560-d complex catalyzed the oxidation of ubiquinol-1 and ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride or phenazine methosulfate. The oxidase activity required phospholipids and was inhibited by the respiratory inhibitors, KCN and NaN3, and the divalent cation, ZnSO4. Formation of a membrane potential by the cytochrome b560-d complex reconstituted into liposomes was observed with the fluorescent dye, 3,3'-dipropylthiodicarbocyanine iodide, on the addition of ubiquinol-1, showing that the enzyme provided a coupling site for oxidative phosphorylation.     

The electron transport chain of Bacterionema matruchotii

The membrane fraction of Bacterionema matruchotii contains an electron transport chain with oxidizing activity for NADH and succinate. Respiration was inhibited by KCN, 2-heptyl-4-hydroxyquinoline-N-oxide, UV light irradiation and CO. UV light irradiation, analysis of membrane extracts, and reconstitution of respiration in UV light treated membranes suggested that respiration is mediated by a menaquinone derivative. The membranes contained cytochromes a, b, and c. Inhibition studies and the effect of KCN and CO on the cytochrome spectrum indicated the presence of an a+a3 cytochrome oxidase and cytochrome o. The membrane fraction from cells grown under O2-limiting conditions contained nitrate reductase activity. In B. matruchotii, electron transport is coupled to oxidative phosphorylation as judged by the effects of substrates and inhibitors on the intracellular ATP concentration.     

Inhibition of Respiration in Prototheca zopfii by Light

Irradiation of cells of Prototheca zopfii with blue light inhibited the respiratory capacity of the cells. The inhibition of respiration was correlated with a photodestruction of cytochrome c(551), cytochrome b(559), and cytochrome a₃. Cytochrome c(549), cytochrome b(555), and cytochrome b(564) were unaffected by the irradiation treatment. The α-band of reduced cytochrome a was shifted from 599 to 603 nm by irradiation, an effect similar to that observed when methanol was added to nonirradiated cells. The presence of oxygen was required during irradiation for both photoinhibition of respiration and photodestruction of the cytochromes. Cytochrome a₃ was protected against photodestruction by cyanide. Photodestruction of these same cytochromes also occurred when washed mitochondria of P. zopfii were irradiated.     

Purification and some properties of ubiquinol oxidase from obligately chemolithotrophic iron-oxidizing bacterium, Thiobacillus ferrooxidans NASF-1

Ubiquinol-oxidizing activity was detected in an acidophilic chemolithotrophic iron-oxidizing bacterium, T. ferrooxidans. The ubiquinol oxidase was purified 79-fold from plasma membranes of T. ferrooxidans NASF-1 cells. The purified oxidase is composed of two polypeptides with apparent molecular masses of 32,600 and 50,100 Da, as measured by gel electrophoresis in the presence of sodium dodecyl sulfate. The absorption spectrum of the reduced enzyme at room temperature showed big peaks at 530 and 563, and a small broad peak at 635 nm, indicating the involvement of cytochromes b and d. Characteristic peaks of cytochromes a and c were not observed in the spectrum at around 600 and 550 nm, respectively. This enzyme combined with CO, and its CO-reduced minus reduced difference spectrum showed peaks at 409 nm and 563 nm and a trough at 431 nm. These results indicated that the oxidase contained cytochrome b, but the involvement of cytochrome d was not clear. The enzyme catalyzed the oxidations of ubiquinol-2 and reduced N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride. The ubiquinol oxidase activity was activated by the addition of albumin and lecithin to the reaction mixture and inhibited by the respiratory inhibitors KCN, HQNO, NaN3, and antimycin A1, although the enzyme was relatively resistant to KCN, and the divalent cation, Zn2+, compared with ubiquinol oxidases of E. coli.     

The cytochromes of Acanthamoeba castellanii.

1. Low-temperature difference spectra of gradient-purified mitochondria of Acanthamoeba castellanii reveal the presence of cytochromes b-555, b-562 and c-549, with a-type cytochromes having a broad asymmetrical maximum at 602 nm; these components were also observed in specta of whole cells. 2. The a-type cytochromes are unusual in that they have split Soret absorption maxima (at 442 and 449 nm) and an uncharacteristic CO difference spectrum. 3. CO difference spectra of whole cells and 'microsomal' membranes show large amounts of cytochrome P-420 compared with cytochrome P-450. 4. Difference spectra in the presence of cyanide indicate the presence of an a-type cytochrome and two cyanide-reacting components, one of which may be cytochrome a3. 5. Whole-cell respiration in a N2/O2 (19:1) atmosphere was decreased by 50%, suggesting the presence of a low-affinity oxidase. This lowered respiration is inhibited by 50% by CO, and the inhibition is partially light-reversible; photochemical action spectra suggest that cytochrome a3 contributes to this release of inhibition. Other CO-reacting oxidases are also present. 6. The results are discussed with the view that cytochrome a3 is present in A. castellanii, but its identification in CO difference spectra is obscured by other component(s).     

Examination of the oxidase function of the b-type cytochrome in human polymorphonuclear leucocytes

The spectral properties of a particulate fraction of human polymorphonuclear neutrophils capable of oxidizing NADPH were studied before and after depletion of myeloperoxidase by KCl treatment. Difference spectra (dithionite reduced minus oxidized) at 77 K of non-extracted particles showed peaks of a b-type cytochrome at 556, 527 and 425 nm and of myeloperoxidase at 636 and 474 nm. Extraction of myeloperoxidase led to a 4-5-fold increase in the size of the cytochrome b peaks. In non-extracted particles, the CO-reduced spectra at 77 K revealed a typical CO-reduced myeloperoxidase complex with new peaks at 625-630 and 462 nm, and a limited shift of the Soret band of reduced cytochrome b from 425 to 424-423 nm. The same shift was observed for cytochrome b in extracted particles. Photoirradiation of the CO-dithionite-reduced particles resulted in a back shift of the CO-reduced peaks to their original positions in the reduced spectrum. Concomitantly, the size of the peaks both for myeloperoxidase and cytochrome b was increased, indicating photoreduction. Cytochrome b and myeloperoxidase in neutrophil particles were poorly reduced by NADPH; reduction occurred upon photoirradiation. FAD and FMN added to particles in the presence of NADPH were photoreduced concomitantly with cytochrome b. Addition of phorbol myristate acetate to intact neutrophils in the presence of glucose resulted in CO- and cyanide-insensitive respiration, accumulation of O-2, and also in reduction of cytochrome b. The lag required to reach the steady-state production of O-2 was equal to the lag required for cytochrome b to reach a plateau of reduction. The data are consistent with the idea that cytochrome b in neutrophils might belong to a branched pathway that is not rate-limiting in the cyanide-resistant respiration of the neutrophils.     

The cytochrome bd terminal oxidase of Azotobacter vinelandii: Low temperature photodissociation spectrophotometry reveals reactivity of cytochromes b595 and d with both carbon monoxide and oxygen

Abstract Cytochromes d and b ₅₉₅ were studied by low temperature photodissociation of CO-ligated Azotobacter vinelandii membranes. White light or He-Ne laser irradiation revealed 436 and 594–597 nm absorption bands to be due to Fe¹¹ cytochrome b ₅₉₅. Oxy-cytochrome d (648 nm) was formed when the CO adduct was photolysed in the presence of oxygen. This was followed by ligand recombination (presumably oxygen) to the high-spin cytochrome b ₅₉₅, with a distinctive shift to shorter wavelengths of the α-band of the cytochrome, and a decrease in the oxygenated form. All spectral changes were light-reversible. We demonstrate the light-reversible binding of CO to both cytochromes b ₅₉₅ and d , and suggest migration of oxygen from cytochrome d to cytochrome b ₅₉₅ at a haem-haem binuclear centre during the oxidase reaction.     

The microbial metabolism of C1 compounds. The electron-transport chain of Pseudomonas AM1

Pseudomonas AM1, Hyphomicrobium X and Pseudomonas MS all contain cytochrome a/a(3) and a b-type cytochrome able to react with CO. Pseudomonas AM1 and Hyphomicrobium X also have a CO-binding cytochrome c. The purified cytochrome c (redox potential 0.26V) of Pseudomonas AM1 was not susceptible to oxidation by molecular oxygen. CO reacted slowly with the reduced form giving a CO difference spectrum with a peak at 412nm and troughs at 420nm and 550nm. Similar results were obtained with the cytochrome c of Hyphomicrobium (aerobically grown or anaerobically grown with nitrate) and with that of Pseudomonas extorquens. The results given in the present paper are incompatible with an oxygenase or oxidase function for the soluble cytochrome c of methylotrophs. Studies with whole cells of Pseudomonas AM1 and a cytochrome c-deficient mutant have demonstrated that cytochrome b (redox potential 0.009V) is the first cytochrome in the electron-transport chain for oxidation of all substrates except methanol (and ethanol) whose oxidation does not involve this cytochrome. All substrates are usually oxidized by way of cytochrome c and cytochrome oxidase (cytochrome a/a(3)), but there is an alternative route for the reduction of cytochrome a/a(3) in the mutant lacking cytochrome c. Results of experiments on cyanide inhibition of respiration and cytochrome oxidation support the suggestion that the susceptibility of cytochrome b to oxidation by molecular oxygen (reflected in its ability to react with CO) is probably irrelevant to the normal physiology of Pseudomonas AM1.     

Energy tranduction in photosynthetic bacteria. XI. Further resolution of cytochromes of b type and the nature of the co-sensitive oxidase present in the respiratory chain of Rhodopseudomonas capsulata.

1. In membranes prepared from dark grown cells of Rhodopseudomonas capsulata, five cytochromes of b type (E'0 at pH 7.0 +413+/-5, +270+/-5, +148+/-5, +56+/-5 and -32+/-5 mV) can be detected by redox titrations at different pH values. The midpoint potentials of only three of these cytochromes (b148, b56, and b-32) vary as a function of pH with a slope of 30 mV per pH unit. 2. In the presence of a CO/N2 mixture, the apparent E'0 of cytochrome b270 shifts markedly towards higher potentials (+355mV); a similar but less pronounced shift is apparent also for cytochrome b150. The effect of CO on the midpoint potential of cytochrome b270 is absent in the respiration deficient mutant M6 which possesses a specific lesion in the CO-sensitive segment of the branched respiratory chain present in the wild type strain. 3. Preparations of spheroplasts with lysozyme digestion lead to the release of a large amount of cytochrome c2 and of virtually all cytochrome cc'. These preparations show a respiratory chain impaired in the electron pathway sensitive to low KCN concentration, in agreement with the proposed role of cytochrome c2 in this branch; on the contrary, the activity of the CO-sensitive branch remains unaffected, indicating that neither cytochrome c2 nor the CO-binding cytochrome cc' are involved in this pathway. 4. Membranes prepared from spheroplasts still possess a CO-binding pigment characterized by maxima at 420.5, 543 and 574 nm and minima at 431, 560 nm in C0-difference spectra and with an alpha band at 562.5 nm in reduced minus oxidized difference spectra. This membrane-bound cytochrome, which is coincident with cytochrome b270, can be classified as a typical cytochrome "0" and considered the alternative CO-sensitive oxidase.     

The aerobic electron transport system of Eikenella corrodens

The respiratory system of the fastidious beta-proteobacterium Eikenella corrodens grown with limited oxygen was studied. Membranes showed the highest oxidase activity with ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) or succinate and the lowest activity with NADH and formate. The presence of a bc1-type complex was suggested by the inhibition exerted by 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), myxothiazol, and antimycin A on respiration with succinate and by the effect of the latter two inhibitors on the succinate-reduced difference spectra. Respiration with succinate or ascorbate-TMPD was abolished by low KCN concentrations, suggesting the presence of a KCN-sensitive terminal oxidase. Cytochromes b and c were spectroscopically detected after reduction with physiological or artificial electron donors, whereas type a and d cytochromes were not detected. The CO difference spectrum of membranes reduced by dithionite and its photodissociation spectrum (77 K) suggested the presence of a single CO compound that had the spectral features of a cytochrome o-like pigment. High-pressure liquid chromatography analysis of membrane haems confirmed the presence of haem B; in contrast, haems A and O were not detected. Peroxidase staining of membrane type c cytochromes using SDS-PAGE revealed the presence of five bands with apparent molecular masses of 44, 33, 30, 26, and 14 kDa. Based on our results, a tentative scheme of the respiratory chain in E. corrodens, comprising (i) dehydrogenases for succinate, NADH, and formate, (ii) a ubiquinone, (iii) a cytochrome bc1, and (iv) a type-cbb' cytochrome c oxidase, is proposed.     

Inhibition of Respiration in Sea Urchin Spermatozoa Following Interaction with Fixed Unfertilized Eggs V. Inhibition of Electron Transport in a Span of Mitochondrial Respiratory Chain between Cytochrome b and Cytochrome c in Sea Urchin Spermatozoa, Induced by the Interaction with Glutaraldehyde Fixed Eggs

In the sea urchin, Anthocidaris crassispina , α and γ peaks of reduced cytochrome b were distinctly observed but no peaks of cytochrome a and cytochrome c were found in the difference spectra between H₂O₂ oxidized and the aerobic suspensions of the immotile spermatozoa, which were obtained by an incubation of the suspension of spermatozoa and the glutaraldehyde-fixed eggs for 15 min at 20°C. A similar profile of difference spectrum was also obtained between the aerobic sperm suspension containing antimycin A and the H₂O₂ oxidized one. In Hemicentrorus pulcherrimus , faint peaks of reduced cytochrome a and cytochrome c , as well as evident peaks of cytochrome b , were also found in the difference spectra between aerobic suspension of the fixed-egg-reacted spermatozoa and the H₂O₂ oxidized one. In intact swimming spermatozoa of A. crassispina as well as H. pulcherrimus , no peaks of reduced cytochromes were found under aerobic condition. These results suggest that the inhibition of sperm respiration by the fixed eggs is due, at least in part, to the blockage of electron transport in a span between cytochrome b and cytochrome c.     

Effect of illumination on the redox state of cytochrome c oxidase in wheat leaves in vivo

The redox states of cytochrome a ₃ in wheat leaves (Triticum aestivum L.), in light and in the dark were monitored by its reaction with CO, which resulted in the stimulation of the in vivo reduction of nitrate to nitrite by nitrate reductase under aerobic conditions. Illumination of the leaves for 10 min markedly stimulated the steady state reduction of cytochrome a ₃, probably associated with a decreased energization of the mitochondria in light. In the dark, during steady state respiration of the tightly coupled mitochondria, cytochrome oxidase was in a more oxidised state than in the light, as judged by its reaction with CO. It is also likely that in light, intra mitochondrial NAD⁺ will be highly reduced on account of a high phosphate potential.     

Cytochrome spectrum of an obligate anaerobe, Eubacterium lentum.

An obligately anaerobic bacterium, Eubacterium lentum, was shown to contain cytochromes a, b, and c and a carbon monoxide-binding pigment. Extracts of cells grown with hemin gave a typical absorption spectrum for cytochrome c with maxima at 424, 525, and 553 nm. Extracts from cells grown in the absence of hemin also had an absorption peak corresponding to cytochrome b (562 nm) in their reduced versus oxidized spectrum. Extraction of hemes and formation of pyridine hemochromes allowed quantitation of protoheme IX and heme c. Large amounts of cytochrome c masked the presence of cytochrome b in cells grown in medium containing hemin. When cells were grown in the presence of 50 mM nitrate, cytochrome A (606 nm) was detected. In anaerobic extracts of cells grown either with or without nitrate, cytochromes b and c were reduced by formate and oxidized by NO3. Cytochrome a appeared to be partially oxidized by NO3 and completely oxidized by air.     

Inhibition by carbon monoxide of the secondary-amine mono-oxygenase of Pseudomonas aminovorans and the photochemical action spectrum for its reversal.

The secondary-amine mono-oxygenase (EC 1.14.99.--) of Pseudomonas aminovorans is potently inhibited by carbon monoxide. The degree of inhibition of the purified enzyme was determined by the CO:O2 ratio rather than by the absolute concentration of carbon monoxide. The partition constant (the CO:O2 ratio causing 50% inhibition of activity) was 9.2 X 10(-4). The inhibition could be reversed by light, and the extent of reversal was proportional to the light intensity. With monochromatic light of wavelength 417 nm, the light sensitivity, L, was determined to be 2.5 X 10(8) cm2 min/mol quantum. The photochemical action spectrum for the light reversal of inhibition showed a single maximum of effectiveness at about 420 nm. The difference spectrum of the enzyme (reduced by NADH) on bubbling with CO (compared with an NADH-reduced reference sample) showed a peak at 426 nm. The preparations showed none of the spectral properties to cytochrome P-450 mono-oxygenase preparations, and was much more sensitive to carbon monoxide. The enzyme behaves as a typical o-type cytochrome (i.e. a carbon-monoxide-reactive b-type cytochrome), and its sensitivity to carbon monoxide as well as in its spectral properties, shows close resemblances to haemoglobin.     

Cytochrome P-450 revealed: the effect of the respiratory cytochromes on the spectrum of bacterial cytochrome P-450

Soluble extracts of Bacillus megaterium ATCC 14581 prepared by centrifuging a sonicated cell suspension at 40,000 xg for 30 min apparently contained no cytochrome P-450 unless the culture had been grown in the presence of an inducer: a reduced+CO minus reduced spectrum was used to measure cytochrome P-450 concentration. When the 40,000 xg supernatants from the uninduced cultures were recentrifuged at 105,000 xg the respiratory cytochromes, including one like cytochrome a1, were sedimented, and cytochrome P-450 was observed to be 100 nM or 30 +/- 9 p mol cytochrome P-450/mg protein (n=9). Measurements of cytochrome P-450 in cultures induced with phenobarbital were always higher after ultracentrifugation. There was soluble cytochrome o in all extracts. When cytochrome a1 was present a deep trough at 441 nm developed in the reduced +CO minus reduced difference spectrum of the 40,000 xg supernatant of both the uninduced and the induced cultures. The 40,000 xg supernatant obtained after lysing protoplasts of B. megaterium did not contain cytochrome a1 and always gave a good measure of cytochrome P-450.     

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.     

Respiration in Eggs of the Echiuroid, Urechis unicinctus, Before and After Fertilization

In eggs of the echiuroid Urechis unicinctus the respiration rate, which is not altered by fertilization, is inhibited by rotenone, antimycin A and cyanide. The respiration in echiuroid eggs is probably mediated by the mitochondrial respiratory chain. In fertilized eggs, the respiration was inhibited by oligomycin and stimulated by the uncouplers of oxidative phosphorylation 2,4-dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone, whereas respiration in unfertilized eggs was insensitive to these compounds. Insemination increased the respiratory rate in eggs in the presence of uncouplers and reduced it in the presence of oligomycin. These findings suggest that the capacity of electron transport in mitochondira is elevated by fertilization but becomes latent on fertilization-induced coupling of respiration with oxidative phosphorylation. Strong stimulation of the respiration in unfertilized eggs was induced by dichlorophenol indophenol, phenazine methosulfate and tetramethyl p-phenylenediamine, suggesting possible sites at which electron transport is regulated in unfertilized eggs. The resulting stimulation of respiration in unfertilized eggs was insensitive to uncouplers and oligomycin, but became sensitive to them after fertilization simultaneously with considerable decrease in its rate. Fertilization-induced coupling of the respiration seemed to reduce the respiratory rate enhanced artificially by these redox compounds.