Effects of dibromothymoquinone and bathophenanthroline on flash-induced cytochrome f oxidation in spinach chloroplasts

The effects of several electron transport inhibitors on themagnitude and kinetics of cytochrome f oxidation induced byflash illumination were studied in the - and -band regions.On the flash excitation only a fraction of cytochrome f presentin the chloroplasts was oxidized with a half time of 0.1 to0.3 msec and then reduced with a half time of 10 to 25 msec. Dibromothymoquinone (DBMIB) at concentrations which severelysuppressed the reduction of cytochrome f approximately doubledthe magnitude of cytochrome f oxidation caused by a flash, mainlyby inducing an additional slow oxidation of cytochrome f witha half time longer than 1 msec. Enhancement of the cytochromef oxidation was also observed in the presence of bathophenanthroline.Such enhanced oxidation in duced by the two inhibitors was largelydiminished with the addition of reduced 2,6-dichlorophenolindophenolwhich accelerated cytochrome f reduction. In contrast, the inhibitionof cytochrome f reduction by 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU) was not associated with an increase in the magnitudeof cytochrome f oxidation. However, addition of DBMIB to theDCMU-poisoned chloroplasts enhanced cytochrome f oxidation,suggesting that this is related to a block of the electron transportbetween plastoquinone and cytochrome f. The results are explainedby assuming the occurrence of an electron carrier between plastoquinoneand cytochrome f. (Received May 10, 1978; )     

NADH-Linked Ferricyanide and Cytochrome c Reduction Activities in Corn Root Plasma Membrane

Corn root plasma membrane catalyzed NADH reduction of ferricyanideand cytochrome c over a wide pH range. At pH 7.5, apparent K_msof NADH-cytochrome c pair were significantly lower than thoseof NADH-ferricyanide pair. FMN and polylysine respectively enhancedthe reduction of ferricyanide and cytochrome c. Yet, polyaspartatedecreased the ferricyanide reduction. NADH oxidation observedin the presence of both ferricyanide and cytochrome c was significantlyslower than the sum of rates obtained with individual acceptors.The results suggest that the membrane may contain differentbut not totally independent reduction sites for cytochrome cand ferricyanide. (Received April 13, 1993; Accepted August 23, 1993)     

Proton pump coupled to cytochrome c oxidase in Paracoccus denitrificans

The proton translocating properties of cytochrome c oxidase in whole cells of Paracoccus denitrificans have been studied with the oxidant pulse method. leads to H+/2e- quotients have been measured with endogenous substrates, added methanol and added ascorbate (+TMPD) as reductants, and oxygen and ferricyanide as oxidants. It was found that both the observed leads to H+/O with ascorbate (+TMPD) as reductant, and the differences in proton ejection between oxygen-and ferricyanide pulses, with endogenous substrates or added methanol as a substrate, indicate that the P. denitrificans cytochrome c oxidase translocates protons with a stoichiometry of 2H+/2e-. The results presented in this and previous papers are in good agreement with recent findings concerning the mitochondrial cytochrome c oxidase, and suggest unequal charge separation by different coupling segments of the respiratory chain of P. denitrificans.     

Cytochrome b561 spectral changes associated with electron transfer in chromaffin-vesicle ghosts

The involvement of cytochrome b561, an integral membrane protein, in electron transfer across chromaffin-vesicle membranes is confirmed by changes in its redox state observed as changes in the absorption spectrum occurring during electron transfer. In ascorbate-loaded chromaffin-vesicle ghosts, cytochrome b561 is nearly completely reduced and exhibits an absorption maximum at 561 nm. When ferricyanide is added to a suspension of these ghosts, the cytochrome becomes oxidized as indicated by the disappearance of the 561 nm absorption. If a small amount of ferricyanide is added, it becomes completely reduced by electron transfer from intravesicular ascorbate. When this happens, cytochrome b561 returns to its reduced state. If an excess of ferricyanide is added, the intravesicular ascorbate becomes exhausted and the cytochrome b561 remains oxidized. The spectrum of these absorbance changes correlates with the difference spectrum (reduced-oxidized) of cytochrome b561. Cytochrome b561 becomes transiently oxidized when ascorbate oxidase is added to a suspension of ascorbate-loaded ghosts. Since dehydroascorbate does not oxidize cytochrome b561, it is likely that oxidation is caused by semidehydroascorbate generated by ascorbate oxidase acting on free ascorbate. This suggests that cytochrome b561 can reduce semidehydroascorbate and supports the hypothesis that the function of cytochrome b561 in vivo is to transfer electrons into chromaffin vesicles to reduce internal semidehydroascorbate to ascorbate.     

Stigmatellin induces reduction of iron-sulfur protein in the oxidized cytochrome bc1 complex

Stigmatellin, a Q(P) site inhibitor, inhibits electron transfer from iron-sulfur protein (ISP) to cytochrome c1 in the bc1 complex. Stigmatellin raises the midpoint potential of ISP from 290 mV to 540 mV. The binding of stigmatellin to the fully oxidized complex, oxidized completely by catalytic amounts of cytochrome c oxidase and cytochrome c, results in ISP reduction. The extent of ISP reduction is proportional to the amount of inhibitor used and reaches a maximum when the ratio of inhibitor to enzyme complex reaches unity. A g = 2.005 EPR peak, characteristic of an organic free radical, is also observed when stigmatellin is added to the oxidized complex, and its signal intensity depends on the amount of stigmatellin. Addition of ferricyanide, a strong oxidant, to the oxidized complex also generates a g = 2.005 EPR peak that is oxidant concentration-dependent. Oxygen radicals are generated when stigmatellin is added to the oxidized complex in the absence of the exogenous substrate, ubiquinol. The amount of oxygen radical formed is proportional to the amount of stigmatellin added. Oxygen radicals are not generated when stigmatellin is added to a mutant bc1 complex lacking the Rieske iron-sulfur cluster. Based on these results, it is proposed that ISP becomes a strong oxidant upon stigmatellin binding, extracting electrons from an organic compound, likely an amino acid residue. This results in the reduction of ISP and generation of organic radicals.     

beta-Oxidation and Glyoxylate Cycle Coupled to NADH: Cytochrome c and Ferricyanide Reductases in Glyoxysomes

Glyoxysomes isolated from castor bean (Ricinus communis L., var Hale) endosperm had NADH:ferricyanide reductase and NADH:cytochrome c reductase activities averaging 720 and 140 nanomole electrons/per minute per milligram glyoxysomal protein, respectively. These redox activities were greater than could be attributed to contamination of the glyoxysomal fractions in which 1.4% of the protein was mitochondrial and 5% endoplasmic reticulum. The NADH:ferricyanide reductase activity in the glyoxysomes was greater than the palmitoyl-coenzyme A (CoA) oxidation activity which generated NADH at a rate of 340 nanomole electrons per minute per milligram glyoxysomal protein. Palmitoyl-CoA oxidation could be coupled to ferricyanide or cytochrome c reduction. Complete oxidation of palmitoyl-CoA, yielding 14 nanomole electrons/per nanomole palmitoyl-CoA, was demonstrated with the acceptors, NAL, cytochrome c, and ferricyanide. Malate was also oxidized by glyoxysomes, if acetyl-CoA, ferricyanide, or cytochrome c was present. Glyoxysomal NADH:ferricyanide reductase activity has the capacity to support the combined rates of NADH generation by β-oxidation and the glyoxylate cycle.     

Catalysis of the oxidation of steroid and stilbene estrogens to estrogen quinone metabolites by the beta-naphthoflavone-inducible cytochrome P450 IA family.

Diethylstilbestrol (DES) or catecholestrogens are metabolized by microsomal enzymes to quinones, DES Q or catecholestrogen quinones, respectively, which have been shown to bind covalently to DNA and to undergo redox cycling. The isoforms of cytochrome P450 catalyzing this oxidation of estrogens to genotoxic intermediates were not known and have been identified in this study by (a) using microsomes of rats treated with various inducers of cytochrome P450; (b) using purified cytochrome P450 isoforms; and (c) examining the peroxide cofactor concentrations necessary for this oxidation by microsomes or pure isoenzymes. The highest rate of oxidation of DES to DES Q was obtained using beta-naphthoflavone-induced microsomes (14.0 nmol DES Q/mg protein/min) or cytochrome P450 IA1 (6.4 pmol DES Q/min/pmol P450). Isosafrole-induced microsomes or cytochrome P450 IA2 oxidized DES to quinone at one-third or one-fifth of that rate, respectively. Low or negligible rates of oxidation were measured when oxidations were catalyzed by microsomal rat liver enzymes induced by phenobarbital, ethanol, or pregnenolone-16 alpha-carbonitrile or by pure cytochromes P450 IIB1, IIB4, IIC3, IIC6, IIE1, IIE2, IIG1, or IIIA6. Cytochrome P450 IA1 also catalyzed the oxidation of 2- or 4-hydroxyestradiol to their corresponding quinones. The beta-naphthoflavone-induced microsomes and cytochrome P450 IA1 had the highest "affinity" for cumene hydroperoxide cofactor (Km = 77 microM). Cofactor concentrations above 250 microM resulted in decreased rates of oxidation. The other cytochrome P450 isoforms required much higher cofactor concentrations and were not inactivated at high cofactor concentrations. The data demonstrate that beta-naphthoflavone-inducible cytochrome P450 IA family enzymes catalyze most efficiently the oxidation of estrogenic hydroquinones to corresponding quinones. This oxidation may represent a detoxification pathway to keep organic hydroperoxides at minimal concentrations. The resulting quinone metabolites may be detoxified by other pathways. However, in cells with decreased detoxifying enzyme activities, quinones metabolites may accumulate and initiate carcinogenesis or cell death by covalent arylation of DNA or proteins.     

Salt-induced redox-independent phosphorylation of light harvesting chlorophyll a/b proteins in Dunaliella salina thylakoid membranes

This study investigated the regulation of the major light harvesting chlorophyll a/b protein (LHCII) phosphorylation in Dunaliella salina thylakoid membranes. We found that both light and NaCl could induce LHCII phosphorylation in D. salina thylakoid membranes. Treatments with oxidants (ferredoxin and NADP) or photosynthetic electron flow inhibitors (DCMU, DBMIB, and stigmatellin) inhibited LHCII phosphorylation induced by light but not that induced by NaCl. Furthermore, neither addition of CuCl(2), an inhibitor of cytochrome b(6)f complex reduction, nor oxidizing treatment with ferricyanide inhibited light- or NaCl-induced LHCII phosphorylation, and both salts even induced LHCII phosphorylation in dark-adapted D. salina thylakoid membranes as other salts did. Together, these results indicate that the redox state of the cytochrome b(6)f complex is likely involved in light- but not salt-induced LHCII phosphorylation in D. salina thylakoid membranes.     

The reactivation of nitrate reductase from spinach (Spinacia oleracea L.) inactivated by NADH and cyanide,using trivalent manganese either generated by illuminated chloroplasts or as manganipyrophosphate

Nitrate reductase of spinach (Spinacia oleracea L.) leaves which had been inactivated in vitro by treatment with NADH and cyanide, was reactivated by incubation with oxidant systems and measured as FMNH₂-dependent activity. Reactivation was produced with trivalent manganese compounds represented either by manganipyrophosphate or produced by oxidation of Mn²⁺ ions in the presence of illuminated chloroplasts and compared with reactivation obtained with ferricyanide. Reactivation in the chloroplast system was equivalent to that with ferricyanide when orthophosphate was used but was variable and weak in the presence of pyrophosphate, although manganipyrophosphate was formed, freely. Reactivation by manganipyrophosphate in dark reaction conditions was less effective than with ferricyanide but was not inhibited by the addition of pyrophosphate. Reactivation with illuminated unheated chloroplasts was dependent on added manganese and oxidation of manganese in the presence of pyrophosphate was abolished by boiling the chloroplasts. In the presence of orthophosphate however, boiled, illuminated chloroplasts reactivated the enzyme in the absence of added manganese. Reactivation occurred spontaneously in air, more slowly than with the other oxidants, but to a similar extent to that produced by manganipyrophosphate. The results provide a possible model for physiological reactivation mechanisms.     

Diffusion- and reaction rate-limited redox processes mediated by quinones through bilayer lipid membranes.

The mediation of redox reactions through bilayer lipid membranes was studied. With an appropriate choice of electron acceptors the redox process can be limited either by the chemical reaction rate between the mediator and the reactants or by the shuttle frequency of the mediator through the membrane. Both modes were demonstrated for redox reactions mediated by 2,6 dichlorobenzoquinone (DCBQ) and by alpha-tocopherol with ascorbate entrapped inside vesicles using ferricyanide (a mild oxidant) or hexachloroiridate (a strong oxidant) in the external solution. The redox processes were reaction rate-limited and diffusion-limited for ferricyanide and hexachloroiridate, respectively. The kinetics of the redox processes in the diffusion- and the reaction rate-limited modes allows the determination of the shuttle frequencies and of the interfacial reaction rates of the mediators, respectively. The shuttle frequencies of DCBQ and alpha-tocopherol were approximately 8 and 0.08 s-1, respectively, in L-alpha-dipalmitoyl phosphatidylcholine (DPPC) cholesterol vesicles at 25 degrees C. Interfacial reaction rates between the mediators and ferricyanide were about two- and tenfold lower compared with bulk reaction rates for DCBQ (water) and tocopherol (50% ethanol solution), respectively, i.e., tocopherol is relatively less accessible to aqueous oxidants at the membrane interface. Tocopherol and oxidized tocopherol are reversible hydrophobic redox couples that interact very rapidly with strong oxidants. In both modes of mediation DCBQ was more effective than alpha-tocopherol.     

Binding Affinities of Oxidized and Reduced Forms of Tetrahalogenated Benzoquinones to the QB Site in Oxygen-Evolving Photosystem II Particles from Synechococcus elongatus

Binding affinities of the Q_B site for four tetrahalogenatedbenzoquinones (THBQs) were investigated by measuring their abilityto serve as electron acceptors or act as inhibitors of oxygenevolution in Synechococcus photosystem II particles. Iodanil,bromanil and chloranil but not fluoranil induced a rapid oxidationof Q_A^– and doubled the area over the fluorescence inductioncurve, indicating dark oxidation of Q₄₀₀. Analyses of thesetwo THBQ-induced reactions and inhibition of the acceleratedQ_A^– oxidation by DCMU yielded binding constants of thequinones comparable to those determined from measurements ofoxygen evolution. Generally, THBQs bound tightly to the Q_B site.However, the binding affinity varied in a wide range with THBQs.The Q_B site bound iodanil with an extremely high affinity butfluoranil relatively weakly. The hydroquinone forms of the THBQsalso bound to the Q_B site and inhibited Q_A^– oxidationby Q_B. The concentrations of the hydroquinones required for50% inhibition of Q_A^– oxidation suggest that the Q_B sitebinds the hydroquinones more weakly than the corresponding quinonesexcept for fluoranil, which binds to the Q_B site more tightlyin its reduced form than in oxidized one. The abilities of THBQsto function as electron acceptors or inhibitors of oxygen evolution,and as oxidants of Q₄₀₀ in the dark, are discussed in relationto the binding affinities of the quinones to the Q_B site. ⁴Present address: Department of Biology, Faculty of Science,Toho University. Miyama 2-2-1, Funabashi, 274 Japan     

Effects of chloride ion on HILL reactions in Euglena chloroplasts

Effects of Cl^– and other anions on the rate of HILL reactionin Euglena chloroplasts were investigated. Cl^– acceleratedthe reaction rate with ferricyanide as HILL oxidant; Br^–,F^– and I^– were also effective; NO₃^–, PO₄^2–and SO₄^2– were less effective. Divalent cations, Ca²⁺and Mg²⁺, were also highly effective. The promoting effectsof these ions were highly dependent on pH and the nature andconcentration of the HILL oxidant used. Accelerating effectsof the ion increased with decreasing concentrations of ferricyanide.Generally, the stimulating effect of Cl^– was much moremarked at pH 7–7.5, with little effect at pH 5. Thus,the pH-activity relationship in the HILL reaction is more orless markedly modified by addition of ions. Cl^–, and other anions, accelerated the reaction by affectingonly the dark rate-limiting portion of the HILL reaction; thelight reaction constant remained uninfluenced. We inferred thatsome reaction step, at which ferricyanide receives electronfrom photosystem 2, is accelerated by Cl^– and other ions.Cl^– effects were rather small, or undetectable, with DPIPor p-benzoquinone as oxidants. (Received January 8, 1970; )     

The Effect of Cyanide on the Respiration and the Oxidative Assimilation of Glucose by Chlorella vulgaris

It is shown that low concentrations of cyanide stimulate theendogenous respiration of Chlorella vulgaris. When glucose isadded the respiration rate is much increased but is now reversiblyinhibited by cyanide. Some 30–60 per cent. of the totalrespiration remains uninhibited. One-eighth to one-ninth ofthe glucose added is completely oxidized. Most of the remainderis assimilated to di- or polysaccharide. Low concentrationsof cyanide which inhibit the rate of glucose oxidation alsoinhibit the assimilation of glucose. Two possible interpretationsof this fact are discussed. It is suggested that the assimilationof glucose is coupled with the oxidation of glucose by a cyanide-sensitiverespiratory system. The mathematical consequences of this theoryare considered and shown to agree with the experimental results.The effect of cyanide on the respiratory quotient is also discussed.     

Kinetics of the reoxidation of succinate dehydrogenase.

The reoxidation phase of the catalytic cycle of succinate dehydrogenase was studied in Complex II preparations' by the rapid freeze-electron paramagnetic resonance (epr) technique. With the synthetic water-soluble Q₁ analog, 2,3-dimethoxy-5-methyl-6-pentyl-1, 4-benzoquinone (DPB), as the oxidant, the observed reoxidation of the epr-detectable components, previously reduced with dithionite or succinate, came to completion within a few milliseconds, well within the turnover time of the enzyme. Only ~80% of Fe-S center 1 and the HiPIP (the high-potential cluster) Fe-S center reacted rapidly with DPB, however; similarly incomplete reactions were observed previously in our studies of the reduction of the enzyme by succinate. The subsequent addition of ferricyanide, which appears to act as a chemical oxidant in these experiments, caused immediate reoxidation of the Fe-S centers and of the free radical. Ferricyanide and phenazine methosulfate (PMS) reoxidized all epr-detectable components in Complex II as well as in reconstitutively active, soluble preparations in' <6 ms, even at 0°C. Thus, reoxidation of the purified enzyme by PMS cannot be rate-limiting. Carboxamides and thenoyltrifluoroacetone inhibit strongly the reoxidation of the Fe-S center 1 and the HiPIP center by DPB, but not their reduction by succinate. These and other data suggest that these inhibitors block electron transport from the dehydrogenase to the Q pool on the O₂-side of the HiPIP center, but there is no evidence that they combine directly with the iron. A recent report that Wurster's blue reacts with soluble succinate dehydrogenase much more rapidly than does PMS could not be confirmed. The two oxidants react at equal rates with the purified soluble enzyme before and after it has been reincorporated into membranes.     

Electron transport and photophosphorylation in chloroplasts as a function of the electron acceptor. III. A dibromothymoquinone-insensitive phosphorylation reaction associated with Photosystem II

Dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) is reputed to be a plastoquinone antagonist which prevents the photoreduction of hydrophilic oxidants such as ferredoxin-NADP⁺. However, we have found that dibromothymoquinone inhibits only a small part of the photoreduction of lipophilic oxidants such as oxidized p-phenylenediamine. Dibromothymoquinone-resistant photoreduction reactions are coupled to phosphorylation, about 0.4 molecules of ATP consistently being formed for every pair of electrons transported. Dibromothymoquinone itself is a lipophilic oxidant which can be photoreduced by chloroplasts, then reoxidized by ferricyanide or oxygen. The electron transport thus catalysed also supports phosphorylation and the $\text{P}\text{e}{}_2$ ratio is again 0.4. It is concluded that there is a site of phosphorylation before the dibromothymoquinone block and another site of phosphorylation after the block. The former site must be associated with electron transfer reactions near Photosystem II, while the latter site is presumably associated with the transfer of electrons from plastoquinone to cytochrome f.     

Ascorbic acid decreases oxidant stress in endothelial cells caused by the nitroxide tempol

Stable nitroxide radicals have been considered as therapeutic antioxidants because they can scavenge more toxic radicals in biologic systems. However, as radicals they also have the potential to increase oxidant stress in cells and tissues. We studied the extent to which this occurs in cultured EA.hy926 endothelial cells exposed to the nitroxide Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl). Tempol was rapidly reduced by the cells, as manifest by an increase in the ability of the cells to reduce extracellular ferricyanide and by disappearance of the Tempol EPR signal. Cells loaded with ascorbic acid, which directly reacts with Tempol, showed increased rates of Tempol-dependent ferricyanide reduction, and a more rapid loss of the Tempol EPR signal than cells not containing ascorbate. In this process, intracellular ascorbate was oxidized, and was depleted at lower Tempol concentrations than was GSH, another important intracellular low molecular weight antioxidant. Further evidence that Tempol concentrations of 100-1000 μM induced an oxidant stress was that it caused an increase in the oxidation of dihydrofluorescein in cells and inhibited ascorbate transport at concentrations as low as 50-100 μM. The presence of intracellular ascorbate both prevented dihydrofluorescein oxidation and spared GSH from oxidation by Tempol. Such sparing was not observed when GSH was depleted by other mechanisms, indicating that it was likely due to protection against oxidant stress. These results show that whereas Tempol may scavenge other more toxic radicals, care must be taken to ensure that it does not itself induce an oxidant stress, especially with regard to depletion of ascorbic acid.     

Human neutrophils oxidize low-density lipoprotein by a hypochlorous acid-dependent mechanism: the role of vitamin C

Oxidatively modified low-density lipoprotein (LDL) has been strongly implicated in the pathogenesis of atherosclerosis. Peripheral blood leukocytes, such as neutrophils, can oxidize LDL by processes requiring superoxide and redox-active transition metal ions; however, it is uncertain whether such catalytic metal ions are available in the artery wall. Stimulated leukocytes also produce the reactive oxidant hypochlorous acid (HOCl) via the heme enzyme myeloperoxidase. Since myeloperoxidase-derived HOCl may be a physiologically relevant oxidant in atherogenesis, we investigated the mechanisms of neutrophil-mediated LDL modification and its possible prevention by the antioxidant ascorbate (vitamin C). As a sensitive marker of LDL oxidation, we measured LDL thiol groups. Stimulated human neutrophils (5x10(6) cells/ml) incubated with human LDL (0.25 mg protein/ml) time-dependently oxidized LDL thiols (33% and 79% oxidized after 10 and 30 min, respectively). Supernatants from stimulated neutrophils also oxidized LDL thiols (33% oxidized after 30 min), implicating long-lived oxidants such as N-chloramines. Experiments using specific enzyme inhibitors and oxidant scavengers showed that HOCl, but not hydrogen peroxide nor superoxide, plays a critical role in LDL thiol oxidation by neutrophils. Ascorbate (200 microM) protected against neutrophil-mediated LDL thiol oxidation for up to 15 min of incubation, after which LDL thiols became rapidly oxidized. Although stimulated neutrophils accumulated ascorbate during oxidation of LDL, pre-loading of neutrophils with ascorbate did not attenuate oxidant production by the cells. Thus, activated neutrophils oxidize LDL thiols by HOCl- and N-chloramine-dependent mechanisms and physiological concentrations of vitamin C delay this process, most likely due to scavenging of extracellular oxidants, rather than by attenuating neutrophil oxidant production.     

A high-potential redox component located within cyanobacterial photosystem II.

The calcium-dependent oxygen evolution activity of preparations of Phormidium luridum shows a marked selectivity in favor of ferricyanide over benzoquinone as Hill oxidant. In addition, the rate of oxygen evolution increases with increasing solution redox potential over the range +350 to +550 mV vs. the standard hydrogen electrode. These properties pertain to both 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive and -insensitive fractions of the total oxygen evolution activity. Neither changes in solution potential nor use of oxidants other than ferricyanide obviate the need for added Ca(2+). To explain these observations, two models are proposed, each of which invokes the existence of a redox component located within Photosystem II and having a midpoint potential greater than +450 mV. In one model, the postulated species is a donor which competes with water for oxidizing equivalents generated by System II. In the other model, the 450 mV species is a high-potential primary acceptor of System II electrons.     

Oxidative phosphorylation and the electron transport system of castor bean mitochondria

The difference spectrum (reduced minus oxidized) of castor bean(Ricinus communis L.) mitochondria showed the presence of cytochromeoxidase (cytochromes a+a₃), b-type cytochromes and cytochromec. The mitochondria actively oxidized succinate, -ketoglutarate,pyruvate and exogenous NADH, and oxidations of these substrateswere stimulated by added ADP, as in mammalian mitochondria.Values for the P/O ratio obtained for succinate, pyruvate and-ketoglutarate were the same as those reported for mammalianmitochondria, indicating that theoretical values are 2, 3 and4, respectively. The theoretical P/O ratio for exogenous NADHseemed to be 2. Oxidations of succinate and exogenous NADH instate 3 were almost completely inhibited by 0.3 mM cyanide and10 µM its antimycin A, while those of NAD⁺-linked substratesin state 3 were not completely suppressed even by excess concentrationsof these inhibitors. There seem to be two types of pathway forelectron transfer in the oxidation of NAD⁺-linked substratesin castor bean mitochondria, i.e. pathways which are sensitiveand insensitive to these inhibitors. Oxidation of exogenousNADH in state 3 was not inhibited by rotenone. Transitions of redox levels of the respiratory components fromstate 4 to state 3 on addition of ADP and from state 3 to state4 on exhaustion of added ADP were observed with a dual-wavelengthspectrophotometer. Effects of inhibitors on redox levels ofthe respiratory components in state 3 were investigated. Cytochromesof b-type and cytochrome c were fully reduced on addition ofcyanide. Cytochromes of b-type were also fully reduced on additionof antimycin A, but cytochrome oxidase (cytochromes a + a₃)and cytochrome c changed to the oxidized forms. The redox levelof the component(s) with an absorption maximum at 465 mµshifted further, but not completely, to the reduced side onaddition of antimycin A. However, this component(s) was oxidizedon addition of cyanide. Cyanide-, or antimycin A-resistant oxidationof NAD⁺-linked substrates seems to occur via an alternate electrontransfer pathway branching from NAD⁺-linked flavoprotein(s)in the mitochondria, not via the normal pathway through thecytochromes-cytochrome oxidase system. (Received June 8, 1970; )     

The influence of cytochrome b559 on the fluorescence yield of chloroplasts at low temperature

The maximum light-induced fluorescence yield, F_M, of spinach chloroplasts at − 196 °C was less when the chloroplasts were oxidized with ferricyanide prior to freezing; the minimum fluorescence yield, F₀, of the dark-adapted chloroplasts at − 196 °C was unaffected. The ratio of the fluorescence yields, F_M/F₀, measured at 695 nm at low temperature was 4.5–5.0 for normal chloroplasts and 2.0–2.5 in the presence of ferricyanide. The oxidative titration curve of F_M followed a 1 electron Nernst equation with a midpoint potential of 365 mV and followed closely to the oxidation of cytochrome b₅₅₉. The photoreduction of C−550 at low temperature was the same at all redox potentials over the range of 200–500 mV. It is suggested that a relatively strong oxidant associated with the water-splitting side of Photosystem II, possibly the primary electron donor, can chlorophyll fluorescence of Photosystem II as well as the primary electron acceptor.