Biogenesis of mitochondrialc-type cytochromes

Cytochromesc andc ₁ are essential components of the mitochondrial respiratory chain. In both cytochromes the heme group is covalently linked to the polypeptide chain via thioether bridges. The location of the two cytochromes is in the intermembrane space; cytochromec is loosely attached to the surface of the inner mitochondrial membrane, whereas cytochromec ₁ is firmly anchored to the inner membrane. Both cytochromec andc ₁ are encoded by nuclear genes, translated on cytoplasmic ribosomes, and are transported into the mitochondria where they become covalently modified and assembled. Despite the many similarities, the import pathways of cytochromec andc ₁ are drastically different. Cytochromec ₁ is made as a precursor with a complex bipartite presequence. In a first step the precursor is directed across outer and inner membranes to the matrix compartment of the mitochondria where cleavage of the first part of the presequence takes place. In a following step the intermediate-size form is redirected across the inner membrane; heme addition then occurs on the surface of the inner membrane followed by the second processing reaction. The import pathway of cytochromec is exceptional in practically all aspects, in comparison with the general import pathway into mitochondria. Cytochromec is synthesized as apocytochromec without any additional sequence. It is translocated selectively across the outer membrane. Addition of the heme group, catalyzed by cytochromec heme lyase, is a requirement for transport. In summary, cytochromec ₁ import appears to follow a conservative pathway reflecting features of cytochromec ₁ sorting in prokaryotic cells. In contrast, cytochromec has invented a rather unique pathway which is essentially non-conservative.     

Characterization of the photosynthetic electron transport chain in normal and photobleachedAnabaena cylindrica by flash spectroscopy

Electron transport of normal and photobleachedAnabaena cylindrica was studied using spectral and kinetic analyses of absorbance transients induced by single turnover flashes. Between 500 and 600 nm two positive bands (540 and 566 nm) and two negative bands (515 and 554 nm) were found. Absorbance changes at 515 and 540 nm were partly characterized. None of these absorbance changes represent an electrochromic shift. Absorbance changes at 554 and 566 nm correspond to the oxidation of cytochromef and the reduction of cytochromeb ₅₆₃, respectively. We found a very slight 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) sensitivity of cytochromef in normal cells, while DCMU was completely ineffective for cytochromef reduction in photobleached cells. The absorbance change of cytochromeb ₅₆₃ increased, while the absorbance change of cytochromef was smaller than in normal cells. The increased O₂ evolution in photobleached cells and the negligible electron transport via cytochromef suggest the participation of other electron acceptor(s) in the electron-transport chain of photobleachedAnabaena cylindrica.     

Effect of dichlorophenolindophenol, dichlorophenolindophenol-sulfonate, and cytochromec on redox capacity and simultaneous net H+/K+ fluxes in aeroponically grown seedling roots of sunflower (Helianthus annuus L.): New evidence for a plasma membrane CN−-resistant redox chain

Summary Excised roots from axenically grown sunflower seedlings reduced or oxidized exogenously added 2,6-dichlorophenolindophenol (DCIP), DCIP-sulfonate (DCIP-S), and cytochromec, and affected simultaneous H⁺/K⁺ net fluxes. Experiments were performed with nonpretreated living and CN^–-pretreated poisoned roots (control and CN^–-roots). CN^–-roots showed no H⁺/K⁺ net flux activity but still affected the redox state of the compounds tested. The hydrophobic electron acceptor DCIP decreased the rate of H⁺ efflux in control roots with extension of the maximum rate and optimal pH ranges, then the total net H⁺ efflux (H⁺) equalled that of the roots without DCIP. The simultaneously measured K⁺ influx rate was first inhibited, then inverted into efflux, and finally influx recovered to low rates. This effect could not be due to uptake of the negatively charged DCIP, but due to the lower H⁺ efflux and the transmembrane electron efflux caused by DCIP, which would depolarize the membrane and open outward K⁺ channels. The different H⁺ efflux kinetics characteristics, together with the small but significant DCIP reduction by CN^–-roots were taken as evidence that an alternative CN^–-resistant redox chain in the plasma membrane was involved in DCIP reduction. The hydrophilic electron acceptor DCIP-S enhanced both H⁺ and K⁺ flux rates by control roots. DCIP-S was not reduced, but slightly oxidized by control roots, after a lag, while CN^–-roots did not significantly oxidize or reduce DCIP-S. Perhaps the hydrophobic DCIP could have access to and drain electrons from an intermediate carrier deep inside the membrane, to which the hydrophilic DCIP-S could not penetrate. Also cytochromec enhanced H⁺ and K⁺, consistent with the involvement of the CN^–-resistant redox chain. Control roots did not reduce but oxidize cytochromec after a 15 min lag, and CN^–-roots doubled the rate of cytochromec oxidation without any lag. NADH in the medium spontaneously reduced cytochromec, but control or CN^–-roots oxidized cytochromec, despite of the presence of NADH. In this case CN^–-roots were less efficient, while control roots doubled the rate of cytochromec oxidation by CN^–-roots, after a 10 min lag in which cytochromec was reduced at the same rate as the medium plus NADH did. CN^–-roots seemed to have a fully activated CN^–-resistant branch. The described effects on K⁺ flux were consistent with the current hypothesis that redox compounds changed the electric membrane potential (de- or hyperpolarization), which induces the opening of voltage-gated in- or outward K⁺ channels.Abbreviations Cyt c cytochromec - DCIP 2,6-dichlorophenolindophenol - DCIP-S 2,6-dichlorophenolindophenol 3-sulfonate - HCF(III) hexacyanoferrate (III) - PM plasma membrane - SHAM salicylhydroxamic acid - VH⁺ and VK⁺ H⁺ efflux and K⁺ influx rates - H⁺ and K⁺ total H⁺ efflux and K⁺ influx at the end of the experiment - H⁺ and K⁺ buffering power of the titrated medium     

The effect of membrane potential on the redox state of cytochromeb 561 in antimycin-inhibited submitochondrial particles

The oxidation of cytochromeb ₅₆₁ by ATP was measured in submitochondrial particles inhibited by antimycin. The redox potential of the bulk (M phase) was controlled by the ratio of fumarate:succinate, and the oxidation of cytochromeb was calculated and expressed as a change in redox potential (E _h) measured in millivolts. The oxidation of cytochromeb ₅₆₁ is an energy-driven reaction affected only by the component of the proton motive force. The oxidation (measured in millivolts) is a function of the phosphate potential, reaching a maximal value of 40 mV at G_ATP<–12 kcal/mole. The maximal measured value of ATP-dependent was 100 mV. Thus only a fraction of the membrane potential effects the redox state of cytochromeb ₅₆₁. In contrast to the ATP-induced oxidation of cytochromeb ₅₆₁, cytochromeb ₅₆₆ is in redox equilibrium with fumarate succinate either in the presence or in the absence of ATP. The selective oxidation ofb ₅₆₁ is explained within the term of theQ cycle as a reflection of on the electron electrochemical potential. The positive electric potential of theC phase causes cytochromeb ₅₆₆ to act as oxidant with respect to cytochromeb ₅₆₁. In the presence of antimycin cytochromeb ₅₆₁ cannot equilibrate with the quinone and undergoes oxidation, while cytochromeb ₅₆₆ reequilibrates with the quinone and thus regains redox equilibrium with the fumarate succinate redox buffer.Abbreviations used: ETP_H, phosphorylating submitochondrial particles; TMPD,N ₁ N ₁ NN-tetramethyl-p-phenylenediamine; FCCP, carbonylcyanidep-trifluoromethoxyphenylhydrazone; Mes, 2-(N-morpholino) ethanesulfonic acid.     

Interactions of cytochromec with phospholipid membranes

Summary Cytochromec added during the formation of lecithin-cardiolipin liquid crystals in 0.015m KCl is readily bound. After successive washings with 0.15m KCl, only about 50% of this bound cytochromec is removed. The remaining cytochromec is resistant to further salt extraction, and the amount of this cytochromec that is bound varies with the concentration of added cytochromec to a maximum binding ratio of 170, mole ratio cytochromec to phospholipid. This binding appears to be electrostatic; it is competitively inhibited by increasing the initial molarity of KCl from 0.015 to 0.10m. Binding of cytochromec is insignificant in the absence of cardiolipin, and is affected by varying the pH. Electron microscope studies of osmium tetroxide-stained thin sections show that the liquid crystals consist of vesicles, each of which contains a large number of concentric, alternating light and dense lines. The dense lines have been identified by other workers with the polar head groups of the phospholipids on the surface of a bilayer, and the light area represents the hydrophobic interior. The addition of cytochromec causes an average decrease in the number of lines per vesicle. It increases the center-to-center distance between two neighboring light or dense lines and the width of the dense lines. On the basis of this evidence and electrostatic binding, it is concluded that cytochromec is binding on the polar surfaces of the phospholipid bilayers comprising the liquid crystalline vesicles.     

Influence of 8-azido-ATP and other anions on the activity of cytochromec oxidase

The effect of ATP and other anions on the kinetics of cytochromec oxidation by reconstituted bovine heart cytochromec oxidase was investigated. The following results were obtained: (1) ATP and other polyvalent anions increase theK _m for cytochromec and theV _max (if assayed by the photometric method). The magnitude of the effect is proportional to the charge of the anion as follows from the series of increasing effectiveness: P_iii. (2) The kinetic effects are obtained in the millimolar physiological concentration range. (3) The kinetic changes are not saturated at high concentrations. (4) A specific interaction site for ATP at the cytosolic domain of the enzyme is concluded from the increase ofK _m for cytochromec after photolabelling of proteoliposomes with 8-azido-[-³²P]-ATP, which is protected by ATP but not by ADP. (5) No specific binding site for ATP could be identified by photolabelling with 8-azido-[-³²P]-ATP. The labelling is only partly protected by ATP or ADP.Abbreviations CCP carbonylcyanide-m-chlorophenylhydrazone - TMPD N,N,N,N-tetramethyl-1,4-phenylenediamine dihydrochloride - 8-N₃-ATP 8-azido-adenosine-5-triphosphate Dedicated to Professor Dr. Friedhelm Schneider on the occasion of his 60th birthday.     

The reactions of the oxidase and reductases ofParacoccus denitrificans with cytochromesc

Electron transport in theParacoccus denitrificans respiratory chain system is considerably more rapid when it includes the membrane-bound cytochromec ₅₅₂ than with either solubleParacoccus c ₅₅₀ or bovine cytochromec; a pool function for cytochromec is not necessary. Low concentrations ofParacoccus or bovine cytochromec stimulate the oxidase activity. This observation could explain the multiphasic Scatchard plots which are obtained. A negatively charged area on the back side ofParacoccus c which is not present in mitochondrialc could be a control mechanism forParacoccus reactions.Paracoccus oxidase and reductase reactions with bovinec show the same properties as mammalian systems; and this is true ofParacoccus oxidase reactions with its own soluble cytochromec if added polycation masks the negatively charged area. Evidence for different oxidase and reductase reaction sites on cytochromec include: (1) stimulation of the oxidase but not reductase by a polycation; (2) differences in the inhibition of the oxidase and reductases by monoclonal antibodies toParacoccus cytochromec; and (3) reaction of another bacterial cytochromec withParacoccus reductases but not oxidase. Rapid electron transport occurs in cytochromec-less mutants ofParacoccus, suggesting that the reactions result from collision of diffusing complexes.     

C1 metabolism inParacoccus denitrificans: Genetics ofParacoccus denitrificans

Paracoccus denitrificans is able to grow on the C₁ compounds methanol and methylamine. These compounds are oxidized to formaldehyde which is subsequently oxidized via formate to carbon dioxide. Biomass is produced by carbon dioxide fixation via the ribulose biphosphate pathway. The first oxidation reaction is catalyzed by the enzymes methanol dehydrogenase and methylamine dehydrogenase, respectively. Both enzymes contain two different subunits in an ₂₂ configuration. The genes encoding the subunits of methanol dehydrogenase (moxF andmoxI) have been isolated and sequenced. They are located in one operon together with two other genes (moxJ andmoxG) in the gene ordermoxFJGI. The function of themoxJ gene product is not yet known.MoxG codes for a cytochromec _551i , which functions as the electron acceptor of methanol dehydrogenase. Both methanol dehydrogenase and methylamine dehydrogenase contain PQQ as a cofactor. These so-called quinoproteins are able to catalyze redox reactions by one-electron steps. The reaction mechanism of this oxidation will be described. Electrons from the oxidation reaction are donated to the electron transport chain at the level of cytochromec. P. denitrificans is able to synthesize at least 10 differentc-type cytochromes. Five could be detected in the periplasm and five have been found in the cytoplasmic membrane. The membrane-bound cytochromec ₁ and cytochromec ₅₅₂ and the periplasmic-located cytochromec ₅₅₀ are present under all tested growth conditions. The cytochromesc _551i andc _553i , present in the periplasm, are only induced in cells grown on methanol, methylamine, or choline. The otherc-type cytochromes are mainly detected either under oxygen limited conditions or under anaerobic conditions with nitrate as electron acceptor or under both conditions. An overview including the induction pattern of allP. denitrificans c-type cytochromes will be given. The genes encoding cytochromec ₁, cytochromec ₅₅₀, cytochromec _551i , and cytochromec _553i have been isolated and sequenced. By using site-directed mutagenesis these genes were mutated in the genome. The mutants thus obtained were used to study electron transport during growth on C₁ compounds. This electron transport has also been studied by determining electron transfer rates inin vitro experiments. The exact pathways, however, are not yet fully understood. Electrons from methanol dehydrogenase are donated to cytochromec _551i . Further electron transport is either via cytochromec ₅₅₀ or cytochromec _553i to cytochromeaa ₃. However, direct electron transport from cytochromec _551i to the terminal oxidase might be possible as well. Electrons from methylamine dehydrogenase are donated to amicyanin and then via cytochromec ₅₅₀ to cytochromeaa ₃, but other routes are used also.P. denitrificans is studied by several groups by using a genetic approach. Several genes have already been cloned and sequenced and a lot of mutants have been isolated. The development of a host/vector system and several techniques for mutation induction that are used inP. denitrificans genetics will be described.     

Interaction of cytochrome c L with methanol dehydrogenase from Methylophaga marina 42:

The reaction of methanol dehydrogenase with cytochrome c _L from Methylophaga marina and the reactions of the non-physiological substrates, Wurster's blue and ascorbic acid, with both proteins were studied as a function of temperature (4–32 °C), pressure (1–2000 bar) and ionic strength using the optical high pressure stopped-flow method. The thermodynamic parameters H, S and V were determined for all reactions where electron transfers are involved. These data allowed the determination of the Maxwell relationships which proved the internal thermodynamic consistency of the system under study. A conformational change on the cytochrome c _L level was deduced from both breaks in the Arrhenius plots and the variation of the V with temperature.Abbreviations MOPS 4-morpholinepropanesulfonic acid - CHES 2-(cyclohexylamino)ethanesulfonic acid - MDH methanol dehydrogenase - EDTA ethylenedinitrilotetraacetic acid disodium salt - BTB bromothymol blue (3,3-dibromothymolsulfoneph-thalein) - PQQ 2,7,9-tricarboxy-lH-pyrrolo-[2,3f]quinoline-4,5-dione - cytochrome c _HH mammalian horse heart cytochrome c     

Organization and function of cytochromeb and ubiquinone in the cristae membrane of beef heart mitochondria

The arrangement and function of the redox centers of the mammalianbc ₁ complex is described on the basis of structural data derived from amino acid sequence studies and secondary structure predictions and on the basis of functional studies (i.e., EPR data, inhibitor studies, and kinetic experiments). Two ubiquinone reaction centers do exist—a QH₂ oxidation center situated at the outer, cytosolic surface of the cristae membrane (Q₀ center), and a Q reduction center (Q _i center) situated more to the inner surface of the cristae membrane. The Q₀ center is formed by theb-566 domain of cytochromeb, the FeS protein, and maybe an additional small subunit, whereas the Q _i center is formed by theb-562 domain of cytochromeb and presumably the 13.4kDa protein (QP-C). The Q binding proteins are proposed to be protein subunits of the Q reaction centers of various multiprotein complexes. The path of electron flow branches at the Q₀ center, half of the electrons flowing via the high-potential cytochrome chain to oxygen and half of the electrons cycling back into the Q pool via the cytochromeb path connecting the two Q reaction centers. During oxidation of QH₂, 2H⁺ are released to the cytosolic space and during reduction of Q, 2H⁺ are taken up from the matrix side, resulting in a net transport across the membrane of 2H⁺ per e^– flown from QH₂ to cytochromec, the H⁺ being transported across the membrane as H (H⁺ + e^–) by the mobile carrier Q. The authors correct their earlier view of cytochromeb functioning as a H⁺ pump, proposing that the redox-linkedpK changes of the acidic groups of cytochromeb are involved in the protonation/deprotonation processes taking place during the reduction and oxidation of Q. The reviewers stress that cytochromeb is in equilibrium with the Q pool via the Q _i center, but not via the Q₀ center. Their view of the mechanisms taking place at the reductase is a Q cycle linked to a Q-pool where cytochromeb is acting as an electron pump.     

Spectrophotometric titrations of ferricytochrome C with ferrohexacyanide in the pH range 5 to 7

Spectrophotometric titrations were conducted on the system horse heart ferricytochromec plus ferrohexacyanide in the pH range 5 to 7 and at temperatures 8, 18, 22 and 28°C. A difference extinction coefficient for reducedvs. oxidized cytochromec at 550 nm of 21 mmol^–1cm^–1 was used in part of the evaluations. On the assumption that only one electron-transferlinked proton dissociation is effective for both ferro- and ferricytochromec in this pH range, various possible models are developed with only three conforming with the experimental pH dependence of the spectrophotometric equilibrium constant. The data conform best to a model with protonic dissociation constants between pH 5 and 7 such that the reduced cytochromec species is at least a factor of 3 more acidic than the one for oxidized cytochromec (with pK_H 6). This interpretation holds least for the data at 22°C, which points to a structural rearrangement at about this temperature (Czerlinski and Bracokova, 1973; Zabinski and Czerlinski, 1974; Zabinski, et al., 1974). While the extinction coefficient of ferrocytochromec shows no significant change with pH and temperature, the one for ferricytochromec does: it is about 5% larger at pH 5 than at pH 7 (550 nm). Graphs for the absorption change of ferricytochromec (pH 7 as reference) document the details over the wavelength range 500 to 750 nm.     

Differential exposure of components of cytochromeb−c 1 region in beef heart mitochondria and electron transport particles

The reduction of cyctochromesc +c ₁ by durohydroquinone and ferrocyanide in electron transport particles (ETP) and intact cytochromec-depleted beef heart mitochondria has been studied. At least 94% of the ETP are in an inverted orientation. Durohydroquinone reduces 80% ofc +c ₁ in ETP but less than 20% in mitochondria; sonication of mitochondria allows reduction of cytochromesc +c ₁ (80%). Addition of ferrocyanide (effective redox potential +245 mV) to electron transport particles results in 30% reduction of cytochromesc +c ₁. Addition of ferrocyanide to intact cytochromec-depleted mitochondria does not reduce cytochromec ₁; treatment withN,N,N,N-tetramethylphenylenediamine, Triton X-100, or sonic oscillation results in 30% reduction of cytochromesc +c ₁. TheK _m value of ferrocyanide oxidase for K-ferrocyanide is pH-dependent in ETP only, increasing with increasing pH. The extent of reduction of cytochromec ₁ is also pH-dependent in ETP only, the extent of reduction increasing with decreasing pH. On the basis of these data cytochromec ₁ is exposed to the matrix face and cytochromec is exposed to the cytoplasmic face. No redox center other than cytochromec in the segment between the antimycin site and cytochromec is exposed on the C-side.Abbreviations Used: MES, 2(N-morpholino)-ethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; TMPD,N,N,N,N-tetramethylphenylenediamine; ETP, electron transport particles; NAD-NADH, nicotinamide adenine dinucleotide; PMS, phenazine methosulfate.     

Effects of axial methionine coordination on the in-plane asymmetry of the heme electronic structure of cytochrome<Emphasis Type="Italic"> c</Emphasis>

The paramagnetic susceptibility () tensors of the oxidized forms of thermophile Hydrogenobacter thermophilus cytochrome c₅₅₂ (Ht cyt c₅₅₂) and a quintuple mutant (F7A/V13 M/F34Y/E43Y/V78I; qm) of mesophile Pseudomonas aeruginosa cytochrome c₅₅₁ (Pa cyt c₅₅₁) have been determined on the basis of the redox-dependent ¹H NMR shift changes of the main-chain NH and CH proton resonances of non-coordinated amino acid residues and the NMR structures of the reduced forms of the corresponding proteins (J. Hasegawa, T. Yoshida, T. Yamazaki, Y. Sambongi, Y. Yu, Y. Igarashi, T. Kodama, K. Yamazaki, Y. Kyogoku, Y. Kobayashi (1998) Biochemistry 37:9641–9649; J. Hasegawa, S. Uchiyama, Y. Tanimoto, M. Mizutani, Y. Kobayashi, Y. Sambongi,Y. Igarashi (2000) J Biol Chem 275:37824–37828). From the tensors determined, we obtained the contact shifts for heme methyl proton resonances, which provided the heme electronic structures of the oxidized forms of Ht cyt c₅₅₂ and qm. We also characterized the heme electronic structure of the cyanide adducts of the proteins, where the axial Met was replaced by an exogenous cyanide ion, through the analysis of ¹H NMR spectra. The results indicated that the heme electronic structures of both the proteins in their oxidized forms with axial His and Met coordination are largely different to each other, while those in their cyanide adducts are similar to each other. These results demonstrated that the orientation of the axial Met sulfur lone pair, with respect to heme, predominantly contributes to the spin delocalization into the porphyrin- system of heme in the oxidized proteins with axial His and Met coordination.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations COSY correlation spectroscopy - DQF-COSY double quantum filtered COSY - TOCSY total correlation spectroscopy - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect correlated spectroscopy - Cyt c cytochrome c - Pa cyt c₅₅₁ Pseudomonas aeruginosa cytochrome c₅₅₁ - Ht cyt c₅₅₂ Hydrogenobacter thermophilus cytochrome c₅₅₂ - _obs observed shift - _para paramagnetic shift - _dia diamagnetic shift - _con contact shift - _pc pseudo-contact shift     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

Initial purification study of the cytochromeb 561 of bean hypocotyl plasma membrane

Summary The plasma membrane (PM) of higher plants contains a major ascorbate-reducible, high-potentialb-type cytochrome, named cytochromeb ₅₆₁ (cytb ₅₆₁). In this paper a rapid purification protocol for the cytb ₅₆₁ of bean hypocotyls PM is described. An almost 200-fold increase of cytb ₅₆₁ specific concentration was achieved with respect to the PM fraction, which contained about 0.2 nmol of ascorbate-reducible heme per mg protein. The procedure can be performed in one day starting from purified PMs obtained by the phase-partitioning procedure. However, cytb ₅₆₁ proved to be unstable during chromatographic purification and the amount of protein finally recovered was low. Purified cytb ₅₆₁ eluted as a 130,000 Da protein-detergent complex from gel-filtration columns. It was completely reduced by ascorbate and reduced-minus-oxidized spectra showed -, - and -bands at 561, 530, and 429 nm respectively, not unlike the spectra of whole PMs. This work represents an initial approach to the biochemical characterization of the cytb ₅₆₁ of higher plants, formerly suggested to be related to cytb ₅₆₁ of animal chromaffin granules.Abbreviations cytb ₅₆₁ cytochromeb ₅₆₁ - PM plasma membrane - UPV upper-phase vesicles - GSII glucan synthase II - CCR NADH-dependent cytochromec reductase - CCO cytochromec oxidase - TX-100R reduced Triton X-100     

Cytochromes,rubredoxin, and sulfur metabolism of the non-thiosulfate-utilizing green sulfur bacterium Pelodictyon luteolum

Two soluble c-type cytochromes (c-553 and c-555) and the nonheme iron-containing protein rubredoxin of the non-thiosulfate-utilizing green sulfur bacterium Pelodictyon luteolum were highly purified by ion exchange column chromatography, gel filtration and ammonium sulfate fractionation. Both cytochrome are small and basic hemoproteins, while rubredoxin is an acidic small nonheme iron protein. Cytochrome c-553 has a molecular weight of 13,000 determined by Sephacryl S-200 chromatography and of 10,700 by electrophoresis on SDS acrylamide gel, an isoelectric point at pH 10.2, a redox-potential of +220 mV. It shows maxima at 413 nm in the oxidized form, and the characteristic three maxima in the reduced state (-band at 553 nm, -band at 523 nm, -band at 417 nm). The best purity index (A ₂₈₀/A ₄₁₇) obtained was 0.18. Cytochrome c-555 (best purity index obtained: A ₂₈₀/A ₄₁₈=0.17) has an isoelectric point at pH 10.5, a molecular weight of 9,500 (by electrophoresis on SDS acrylamide gel) and a redox-potential of +160mV. The reduced form of this cytochrome shows the typical bands of c-type cytochromes at 555 (551) nm (-band), 523 nm (-band) and 418 nm (-band), while the oxidized form has the -band at 413 nm.Rubredoxin (best purity index obtained: A ₂₈₀/A ₄₉₀=3.5) is an acidic small protein. Its molecular weight estimated by gel filtration and SDS acrylamide gel electrophoresis is 27,000 and 6,300 respectively. The monomer of this protein contains one iron atom per molecule. Rubredoxin has an isoelectric point at pH 2.8 and shows maxima at 570 nm, 490 nm and 370 nm in the oxidized form.During anaerobic sulfide oxidation of a growing culture of Pelodictyon luteolum elemental sulfur is the first main product, which appears in the medium. Elemental sulfur is further oxidized to sulfate, after the available sulfide is completely consumed by the cells.Non-common abbreviations C Chlorobium - P Pelodictyon - SDS sodium dodecylsulfate - HIPIP high-potential-iron-sulfur-protein Offprint requests to: U. Fischer     

Studies on the sites expressing evolutionary changes in the structure of eukaryotic 5S ribosomal RNA

Summary We have determined the complete sequences of 5S rRNAs from a lamprey (Lampetra reissneri), a lancelet (Branchiostoma belcheri), silkworms (Philosamia cynthia ricini, Bombyx mori, Antheraea pernyi), and a silkworm hybrid (artificially fertilized hybrid species ofPhilosamia cynthia ricini x Bombyx mori ), as well as those of cotton seeds (Gossypium hirsutum L.). Having compared more than 170 eukaryotic 5S rRNAs of which seven sequences have been determined by our group as mentioned above, we have found that the evolutionary sites that exist at special locations in these structures are closely related to the evolution of eukaryotes. The changes proceed step by step in an orderly way, i.e., the change in nucleotide residues of the evolutionary sites depends on the order of the evolution of the species and shows group-specific patterns.     

Plastocyanin: Structural and functional analysis

Plastocyanin is one of the best characterized of the photosynthetic electron transfer proteins. Since the determination of the structure of poplar plastocyanin in 1978, the structure of algal (Scenedesmus, Enteromorpha, Chlamydomonas) and plant (French bean) plastocyanins has been determined either by crystallographic or NMR methods, and the poplar structure has been refined to 1.33 Å resolution. Despite the sequence divergence among plastocyanins of algae and vascular plants (e.g., 62% sequence identity between theChlamydomonas and poplar proteins), the three-dimensional structures are remarkably conserved (e.g., 0.76 Å rms deviation in the C positions between theChlamydomonas and poplar proteins). Structural features include a distorted tetrahedral copper binding site at one end of an eight-stranded antiparallel -barrel, a pronounced negative patch, and a flat hydrophobic surface. The copper site is optimized for its electron transfer function, and the negative and hydrophobic patches are proposed to be involved in recognition of physiological reaction partners. Chemical modification, cross-linking, and site-directed mutagenesis experiments have confirmed the importance of the negative and hydrophobic patches in binding interactions with cytochromef and Photosystem I, and validated the model of two functionally significant electron transfer paths in plastocyanin. One putative electron transfer path is relatively short (4 Å) and involves the solvent-exposed copper ligand His-87 in the hydrophobic patch, while the other is more lengthy (12–15 Å) and involves the nearly conserved residue Tyr-83 in the negative patch.     

The role ofc-type cytochromes in the terminal respiratory chain of the methylotrophic bacteriumMethylophilus methylotrophus

Whole cells of the methylotrophic bacteriumMethylophilus methylotrophus cultured under methanol-limited conditions contain approximately equal amounts of two majorc-type cytochromes,c _H andc _L. Virtually all of the cytochromec _H, and over one-third of the cytochromec _L, are loosely attached to the periplasmic surface of the respiratory membrane whence they can be released by sonication or by washing cells in ethylenediaminetetraacetate (EDTA). The latter causes inhibition of methanol oxidase activity and stimulation of ascorbate-N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) oxidase activity, neither of which effects are reversible by divalent metal ions. Kinetic analyses indicate that ascorbate-TMPD is oxidised via two routes, viz. a slow low-affinity pathway involving loosely membrane-boundc-type cytochromes plus cytochrome oxidaseaa ₃, and a faster higher-affinity pathway involving the firmly membrane-bound cytochrome oxidasec _L o complex; the former route predominates in the presence of divalent metal ions, and the latter route after exposure to EDTA. These and other results are discussed in terms of the spatial organisation of the terminal respiratory chain, and of the role ofc-type cytochromes in the oxidation of methanol and ascorbate-TMPD.Abbreviations EDTA Enthylenediaminetetraacetate - PMS Phenazinemethosulphate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - SDS Sodium dodecylsulphate - I₅₀ Concentration of inhibitor required to give 50% inhibition of enzyme activity - PQQ Pyrroloquinoline quinone     

Simultaneous measurements of optical and electrical properties of artificial membranes composed of mitochondrial lipids and their interaction with cytochromec

Summary A newly constructed cell, which allows simultaneous measurements of optical and electrical properties, was used to study bimolecular black membranes composed of beef heart mitochondrial lipids and their interaction with cytochromec.The results show that these highly charged membranes are stable only in relatively limited ranges of boundary conditions. In 0.1n KCl their maximum direct current (dc) resistance is 7×10⁸ Ohm cm²±10%; the series capacity at 1kHz is 0.43 F/cm²±3%; the entire thickness, determined by optical reflectivity, is 5.8±0.2 nm.The interaction between oxidized cytochromec and these lipid membranes is primarily of electrostatic nature, and dependent on the presence of highly charged phospholipids, such as diphosphatidyl glycerol (cardiolipin) and phosphatidyl ethanolamine. The attachment of cytochromec maximally causes a 2.5-fold increase in reflectivity, without any noticeable change in the capacity. This leads to a subsequent instability of the membrane (i.e., rupture) preceded by a rapid increase of the dc conductivity. This behavior is far less pronounced with reduced cytochromec.