Inhibition of an aa3-Type Two-Subunit Cytochrome c Oxidase from Nitrobacter agilis by N,N'-Dicyclohexylcarbodiimide

The electron transfer activity of an aa₃-type two-subunit cytochromec oxidase of Nitrobacter agilis was inhibited by DCCD. Althoughthe activity of the purified cytochrome c oxidase dissolvedin 1% Triton X- 100 was not affected by DCCD even at a ratioof 1,000 mol DCCD per mol cytochrome aa₃, the activity of theenzyme dissolved in 0.02% Tween 20 or 0.02% Triton X-100 wasinhibited by 60% or more at a ratio of 1,000 mol DCCD per molcytochrome aa₃. The results of SDS polyacrylamide gel electrophoresisof the enzyme incubated with DCCD suggested that subunit IImight be a binding site for DCCD. (Received February 23, 1985; Accepted April 23, 1985)     

aa3-Type Cytochrome c Oxidase Occurs in Halobacterium halobium and Its Activity is Inhibited by Higher Concentrations of Salts

Cytochrome aa₃ was found to occur in Halobacterium halobiumat an early exponential phase of growth. Some of its spectralproperties were determined with a solubilized membrane fractionof the bacterium. Cytochrome c oxidizing activity of the cytochromeaa₃ was not dependent on salt concentration, but decreased steeplywith increasing buffer concentration of the reaction mixture,just as the activity of cytochrome aa₃ from non-halophilic bacteria. (Received July 4, 1986; Accepted October 17, 1986)     

Properties of the Cytochrome c Oxidase Activity of Cytochrome b561 from Photoanaerobically Grown Rhodopseudomonas sphaeroides

Cytochrome b₅₆₁ from Rhodopseudomonas sphaeroides had cytochromec (c2) oxidase activity and a pH optimum at 6.0 for this activity.The activity was affected by the ionic strength of the reactionmixture. The apparent K_m and maximal velocity (V_max) valuesin the absence of addea salts were 14 µM and 120 nmoloxidized per min per mg protein for horse heart cytochrome c.Reduced horse heart cytochrome c was reoxidized in first-orderkinetics by this cytochrome b₅₆₁. The specific activity was0.7 s^–1 per mg protein at 20°C at the concentrationof 30 µMM cytochrome c. Activity was inhibited by KCN and NaN₃, but not by antimycin.The addition of a low concentration of KCN to the cytochromeb₅₆₁ produced a change in the absorption spectrum, evidencethat KCN interacts with the heme moiety of cytochrome b₅₆₁.Results of this and preceeding studies show that the cytochromeoxidase (cytochrome "o") described earlier (Sasaki et al. 1970)is cytochrome b₅₆₁. (Received May 16, 1983; Accepted September 8, 1983)     

Chromatic Regulation of Cytochrome Composition and Electron Transfer from Cytochrome b6-f Complex to Photosystem I in Anacystis nidulans (Tx 20)

Cytochrome composition of the cyanobacterial photosyntheticsystem was studied with Anacystis nidulans (Tx 20) in relationto the chromatic regulation of photosystem composition. Comparisonof cytochrome compositions in cells with a high PS I/II ratio(3.0, grown under weak orange light) and with a low ratio (1.6,grown under weak red light) indicated that cytochrome compositionwas also changed in the chromatic regulation of photosystemcomposition. Two types of cytochrome change were observed: 1)contents of cytochromes C₅₅₃ and c₅₄₈ were changed in parallelwith the changes in PS I content, and 2) cytochrome b₅₅₃ andcytochrome b₆-f complex were held at a constant molar ratioto PS II. The molar ratio, PS II : cytochrome b₅₅₉ : cytochromeb₆-f complex : cytochrome c₅₅₃ : PS I : cytochrome C₅₄₈, inthe red-grown cells was 1 : 2.5 : 1.3 : 0.17 : 1.6 : 0.67, andthe ratio in the orange-grown cells, 1:2.4:0.9:0.32:3.0:1.2.In both types of cells, almost all cytochrome f in the cytochromeb₆-f complex was rapidly oxidized after multiple flash activation,indicating that all cytochrome b₆-f complexes in cells of bothtypes are functionally connected to PS I, even when the molarratio to PS I is largely changed. The content of cytochromeC₅₅₃ was at most 0.14 of PS I, suggesting that the cytochrometurns over several times per one turnover of PS I. ¹Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Fukazawa 2-1-1, Setagaya, Tokyo158, Japan. (Received January 20, 1986; Accepted March 17, 1986)     

Changes in the Cytochrome c Oxidase Activity in Response to Light Regime for Photosynthesis Observed with the Cyanophyte Synechocystis PCC 6714

Changes in the activity of cytochrome c oxidase (EC 1.9.3.1 [EC] ,Cyt-oxidase) in response to growth conditions were studied withthe cyanophyte Synechocystis PCC 6714 in relation to changesin PSI abundance induced by light regime for photosynthesis.The activity was determined with the V_max of mammalian cytochromec oxidation by isolated membranes. The activity of glucose-6-phosphate(G-6-P):NADP⁺ oxidoreductase (EC 1.1.1.49 [EC] ) was also determinedsupplementarily. Cyt-oxidase activity was enhanced by glucoseadded to the medium even when cell growth maintained mainlyby oxygenic photosynthesis. G-6-P:NADP⁺ oxidoreductase was alsoactivated by glucose. The enhanced level of Cyt-oxidase washigher under PSII light, which causes high PSI abundance, thanthat under PSI light, which causes low PSI abundance. The levelwas intermediate under hetetrotrophic conditions. Although theactivity level was low in cells grown under autotrophic conditions,the level was again lower in cells grown under PSI light thanunder PSII light. The change of Cyt-oxidase activity in responseto light regime occurred in the same direction as that for thevariation of PSI abundance. Results suggest that in SynechocystisPCC 6714, the capacity of electron turnover at the two terminalcomponents of thylakoid electron transport system, Cyt-oxidaseand PSI, changes in parallel with each other in response tothe state of thylakoid electron transport system. ¹Present address: Institute of Botany, Academia Sinica, Beijing100044, China ²Present address: Department of Botany, Utkal University, Bhubaneswar,India 751004     

Studies on the change of the respiratory system in roots in relation to the growth of plants II. Activity of iron-containing oxidases in roots of cereal crops with the aging of plants

Distribution of iron-containing oxidases in aging nodal rootsof rice and wheat was studied. Activities of cytochrome c oxidase(1.9.3.1 [EC] , cytochrome c : O₂ oxidoreductase), catalase (1.11.1.6 [EC] ,H₂O₂: H₂O₂ oxidoreductase) and peroxidase (1.11.1.7 [EC] , donor:H₂O₂ oxidoreductase) in wheat roots were comparatively higherthan were those in rice roots at corresponding stages. Cytochromec oxidase in roots remained active throughout the lives of therice and wheat crops. In rice roots, catalase seemed to playa distinct role around the panicle formation stage. Decay ofcatalase activity took place earlier than did that of peroxidaseand cytochrome c oxidase activities. In wheat roots similarenzyme activity changes were not observed. Data may suggestthat the high activity of iron containing oxidases at the panicleformation stage (I) may be chiefly due to catalase activityin rice roots. ¹Paper presented at the 14th Annual Meeting of the Society ofthe Science of Soil and Manure, Japan (1968). (Received November 21, 1968; )     

Cytochrome b560 in chromatophores from Chromatium vinosum

A membrane-bound cytochrome of the B-type in Chromatium chromatophores,cytochrome b₅₆₀, was reduced both by flash light activationand continuous illumination in the presence of antimycin atcontrolled ambient redox potentials. The light-minus-dark differencespectra had peaks at 560 and 430 nm, and troughs at 445 and415 nm. The reduction was observed in the ambient redox potentialfrom 400 to about 200 mV. However, below 200 mV, a re-reductionof photooxidized C-type cytochrome superimposed the reductionof cytochrome b₅₆₀ In the absence of antimycin, the reductionwas not observed, suggesting that the reoxidation of cytochromeb₅₆₀ was faster than the reduction. Dark titrations at various pH values showed that E_m7 of thecytochrome b₅₆₀ was about 40 mV and the E_m value was pH-dependent(–60 mV/pH) from pH 6 to 9. Cytochrome b₅₆₀ had a pK ataround pH 9. The content and some properties of cytochrome b₅₆₀ were similarin chromatophores from either photoautotrophically or photoheterotrophicallygrown cells. The possibility of involvement of cytochrome b₅₆₀ in the photosyntheticelectron transfer is discussed. (Received April 19, 1980; )     

Comparison between Electron Transfers through Plastocyanin in Spinach Chloroplasts and Cytocbrome C2 in Rhodopseudomonas sphaeroides

Photosynthetic electron transfers through the water-solubleperipheral membrane proteins of plastocyanin and cytochromec₂, were studied in spinach chloroplasts and the photosyntheticbacterium Rhodopseudomonas sphaeroides. In spinach chloroplasts,the rate of flash-induced oxidation of cytochrome f was highlydependent on the salt concentration in the suspending medium.The maximum rate with a half time of 200 µs was observedin the presence of 50 mas KCl or 5 mM MgCl₂. The salt effectwas similar to that on the reaction rate between P700 in thylakoidfragments and externally added plastocyanin. On the other hand,in intact cells of R. sphaeroides, in which cytochrome c₂ islocated in the periplasmic space exposed to the outer ionicenvironment, the rate of cytochrome c₁ oxidation via cytochromec₂ was almost independent of salt concentration. This independencewas a contrast to the strong dependence on salt concentrationof reactions between isolated reaction centers and cytochromec₂ These results suggest that plastocyanin reacts collisionallywith the photosystem I reaction center and cytochrome b₆f complexin a manner that is controlled by the surface electrostaticpotential. Cytochrome c₂, on the other hand, reacts with thebacterial reaction center and cytochrome bc₁ complex probablyby forming a complex prior to activation of the reaction center. ¹ Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Fukazawa 2-1-1, Setagaya, Tokyo158, Japan.     

Cytochromes of Mitochondria from Rice Seedlings Germinated under Water and Their Changes during Air Adaptation

Rice seeds were germinated for up to 5 days under water (submerged)and some for another day in air (air-adapted). Control seedswere germinated for 6 days throughout in air. Low-temperaturedifference spectra of shoot mitochondria were compared amongthese three types of seedlings. All cytochromes found in theaerobic seedlings were present in the submerged seedlings. However,there were some differences in the cytochromes b₅₅₃ and c ofthese two types of seedlings. The cytochrome aa₃ peak heightand cytochrome oxidase activity per mitochondrial protein increased1.6- and 2.8-fold, respectively, during air adaptation. Slightlyhigher concentrations of the b-type cytochromes than found inair-adapted mitochondria were already present in submerged mitochondria.The computed difference between the dithionite-reduced differencespectra of mitochondria from submerged seedlings before andafter air adaptation, showed that cytochromes aa₃ and c hadincreased more than cytochrome b₅₅₇ during air adaptation. (Received November 16, 1987; Accepted March 16, 1988)     

The Molecular Basis of Genetic Diversity among Cytoplasms of Triticum and Aegilops. III. Chloroplast Genomes of the M and Modified M Genome-Carrying Species

The restriction fragment patterns of chloroplast DNAs of all M or modified M genome-carrying Aegilops species, and those of common wheat (Triticum aestivum), Ae. umbellulata and Ae. squarrosa as referants, have been analyzed using eight restriction endonucleases, BamHI, EcoRI, HindIII, KpnI, PstI, SalI, SmaI and XhoI. Nine distinctly different chloroplast genomes are evident, and the mutual relatedness among them is estimated based on the number of different restriction fragments. The results lead to the following conclusions. (1) Chloroplast genomes of three Comopyrum species, Ae. comosa, Ae. heldreichii and Ae. uniaristata, are more closely related with each other and are greatly different from those of the Amblyopyrum species, Ae. mutica, and of Ae. umbellulata and Ae. squarrosa. (2) Ae. crassa's chloroplast genome lies at the center of chloroplast genome diversification, whereas those of common wheat, Ae. squarrosa and Ae. uniaristata are three extreme forms lying far from the center. (3) Chloroplast genomes of three 4x species, Ae. biuncialis, Ae. columnaris and Ae. triaristata, arose from Ae. umbellulata, and that of a fourth 4x species, Ae. ventricosa , arose from Ae. squarrosa. The chloroplast origins of two other 4x species, Ae. ovata and Ae. crassa, remain unsolved. (4) The chloroplast genomes of two Ae. mutica strains are identical, even though their cytoplasms exert quite different effects on male fertility, heading date and growth vigor of common wheat.     

Contents of Cytochromes, Quinones and Reaction Centers of Photosystems I and II in a Cyanobacterium Synechococcus sp.

The contents of photosystem I and photosystem II reaction centers,cytochrome c-553, cytochrome c-550, cytochrome f, cytochromeb-559, cytochrome b-563, plastoquinone and vitamin K₁ in thecyanobacterium Synechococcus sp. were determined. About threephotosystem I reaction centers were present for each photosystemII reaction center. The amounts of cytochromes functioning betweenthe two photosystems were approximately half those of the photosystemI reaction center. Plastocyanin was not detected, while plastoquinoneand vitamin K₁ were present in excess of other electron carriersand reaction centers. The results indicate the importance ofplastoquinone and cytochrome c-553 for cooperation of the tworeaction centers through electron transport. ¹Present address: Toray Basic Research Laboratory, 1111 Tebiro,Kamakura, Kanagawa 248, Japan. (Received June 17, 1982; Accepted January 17, 1983)     

The Isolation and Characterization of a Cytochrome b6f Complex from the Cyanobacterium Spirulina sp.

A cytochrome b₆f complex was isolated and purified from Spirulinasp. The complex was solubilized with n-heptyl ß-D-thioglucosideand chromatographed on a DEAE-Toyopearl 650M column. The purifiedcomplex contained a small amount of chlorophyll and carotenoid.At least four polypeptides were present in the complex: cytochromef (29 kDa), cytochrome b₆(23 kDa), iron-sulfur protein (ISP,23 kDa), and a 17 kDa polypeptide. Each polypeptide was separatedfrom the complex treated with 2-mercaptoethanol or urea. Theabsorption spectra of cytochrome b₆ and cytochrome f were similarto those of Anabaena and spinach as expected. The complex wasactive in supporting ubiquinol-cytochrome c oxidoreductase activity.Fifty percent inhibition of the activity was accomplished by1 µM dibromothymoquinone (DBMIB). The K_m values for ubiquinol-2and cytochrome c (horse heart) were 5.7 µM and 7.4 µM,respectively. (Received August 15, 1988; Accepted November 14, 1988)     

Isolation and Characterization of the Cytochrome bf Complex from Whole Thylakoids, Grana, and Stroma Lamellae Vesicles from Spinach Chloroplasts

The cytochrome bf complex was isolated from spinach thylakoids,and also from separated grana and stroma lamellae vesicles,by a procedure involving NaBr washing, detergent treatment andcentrifugation in sucrose gradients. The resulting complex fromall three types of membranes, were almost completely devoidof chlorophyll and carotenoids. The complexes have kinase activitytowards histone III-S and contain a 64 kDa protein claimed tobe a kinase. Electrophoretic analyses indicate that the complexesare in dimeric form and composed of six polypeptides with molecularmasses of 34/33, 23, 20, 17, 12 and 4 kDa. The complexes containtwo moles cytochrome b₆ per mole cytochrome f and one mole RieskeFeS. The 17 kDa and 4 kDa polypeptides are the so called subunit4 and 5 respectively. The 12 kDa protein was identified as plastocyaninby immunoblotting. Plastocyanin and the 4 kDa protein were presentin the cytochrome bf complex even after a second repeated sucrosedensity gradient centrifugation. The sucrose gradient sedimentation pattern was different forthe grana and stroma lamellae complexes. The complex from thestroma lamellae arrives at a higher density than the grana complex.Furthermore, the gradient centrifugation diagram of the stromalamellae consists of one main peak while the diagram of thegrana complex shows two peaks. There is significantly more plastocyaninand 4 kDa protein in the bf complex isolated from stroma lamellaethan from grana. In addition there is a 15 kDa protein in thecomplex isolated from the grana vesicles. Immunoblot analysisafter crosslinking indicated that the 4 kDa protein and theplastocyanin are associated in the cytochrome bf complex. Theoxidoreductase activity is higher (about 50%) in the cytochromebf complex from the grana than from the stroma lamellae fraction.We suggest that a difference in composition of the cytochromebf complex between the two membranes might be important in theregulation of cyclic and non cyclic electron flow. ¹Present address: Department of Plant Physiology II, Universityof Warsaw, 00 927 Warsaw, Poland     

Phenotypic and genetic diversity of leaf rust resistance in wheat wild relatives

Puccinia triticina (Pt), the causal agent of leaf rust evolves through forming new pathotypes that adversely affect the growth and yield of wheat cultivars. Therefore, continued production of resistant varieties through exploring novel sources of resistance in wild relatives which are abundantly found in Iran and the neighbouring regions is a major task in wheat breeding programs. The aim of the present study was to explore 60 wild wheat genotypes selected from the species Triticum monococcum, Aegilops tauschii, Ae. neglecta, Ae. cylindrica, Ae. triuncialis, Ae. umbellulata, Ae. speltoides, Ae. columnaris, Ae. crassa and Ae. ventricosa for resistance to leaf rust. The cultivar ‘Boolani’ and Thatcher near-isogenic lines were used as controls. Two-week-old seedlings were inoculated using 10 Pt pathotypes, and the infection types were recorded. The genotypes were also analysed for polymorphism using six sequence-tagged sites (STS) and sequence characterized amplified region (SCAR) markers. Forty-eight genotypes produced high infection types (3⁺) for two pathotypes, but the remaining genotypes produced low infection types of ‘0; ^=’ to ‘1⁺CN’ to all pathotypes. The latter included three accessions of Ae. tauschii, two accessions of each Ae. umbellulata, Ae. columnaris and Triticum monococcum, and one accession from each Ae. triuncialis, Ae. ventricosa and Ae. neglecta. Analysis for STS and SCAR markers suggested several genotypes could carry the genes Lr9, Lr10, Lr19, Lr24, Lr26 and Lr37 or their potential orthologs in addition to unknown resistance genes. In conclusion, the identified resistant genotypes could be further characterized and used in wheat breeding programs for leaf rust resistance.     

Oxygenated cytochrome o (Vitreoscilla) formed by treating oxidized cytochrome with superoxide anion

Oxygenated cytochrome o can be formed experimentally in twoways, i) by reaction of reduced cytochrome o with molecularoxygen, or ii) by reaction of oxidized cytochrome o with superoxideanion generated by the action of the xanthine oxidase system.It is thermodynamically feasible for oxidized cytochrome o plusO₂–, and reduced cytochrome o plus O₂ to appear as intermediatesin reactions i) and ii), respectively. Superoxide dismutase completely inhibits the xanthine oxidase-catalyzedconversion of oxidized cytochrome o into the oxygenated formbut it has relatively little effect on the oxygenated cytochromeo formation in the reaction system consisting of NADH, NADH-cytochromeo reductase, and cytochrome o. Thus, if superoxide anion doesplay a significant role in the latter system it must be efficientlycoupled to react with cytochrome o and inaccessible to superoxidedismutase. Direct electron transfer from the reductase to thecytochrome without the involvement of superoxide anion is analternative mechanism. (Received December 16, 1976; )     

N-Terminal Amino Acid Sequence and Processing Site of Sweet Potato Cytochrome c Oxidase Subunit II

The N-terminal amino acid sequence of sweet potato cytochromec oxidase subunit II polypeptide was determined. Comparisonsbetween the sequence and amino acid sequences deduced from thenucleotide sequences of other higher plant subunit II genesindicate a post-translational clevage of N-terminal extensionpart. ¹Present address: Institute of Low Temperature Science, HokkaidoUniversity, Sapporo, 060 Japan. (Received June 13, 1989; Accepted September 8, 1989)     

Cytochromes in the Cultured Mycobiont of a Lichen, Cladonia vulcani Sav

Cytochromes in a cultured cells of the mycobiont of Cladoniavulcani Sav. were studied, b-and c-type cytochromes and aa₃-typecytochrome c oxidase were found in the membrane fraction, whileb- and c-type cytochromes were found in the soluble fraction.Soluble cytochrome c-549.5 was purified, and some of its molecularproperties were determined. (Received June 27, 1994; Accepted October 3, 1994)