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Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plastocyanin (Pc) is a soluble copper protein that transfers electrons from cytochrome b(6)f to photosystem I (PSI), two protein complexes that are localized in the thylakoid membranes in chloroplasts. The surface electrostatic potential distribution of Pc plays a key role in complex formation with the membrane-bound partners. It is practically identical for Pcs from plants and green algae, but is quite different for Pc from ferns. Here we report on a laser flash kinetic analysis of PSI reduction by Pc from various eukaryotic and prokaryotic organisms. The reaction of fern Pc with fern PSI fits a two-step kinetic model, consisting of complex formation and electron transfer, whereas other plant systems exhibit a mechanism that requires an additional intracomplex rearrangement step. The fern Pc interacts inefficiently with spinach PSI, showing no detectable complex formation. This can be explained by assuming that the unusual surface charge distribution of fern Pc impairs the interaction. Fern PSI behaves in a similar way as spinach PSI in reaction with other Pcs. The reactivity of fern Pc towards several soluble c-type cytochromes, including cytochrome f, has been analysed by flavin-photosensitized laser flash photolysis, demonstrating that the specific surface motifs for the interaction with cytochrome f are conserved in fern Pc.
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PMID:Functional characterization of the evolutionarily divergent fern plastocyanin. 1529 22

The structure of bovine liver cytochrome b(5), a soluble 93-residue proteolytic fragment of a 16 kDa membrane-bound hemoprotein, initially solved at 2.0 A resolution, has been refined at 1.5 A using data collected on a diffractometer. Refinement to 2.0 A resolution used the Hendrickson-Konnert procedure PROLSQ and was then extended to 1.5 A resolution using the program PROFFT. Only residues 3-87 could be identified in the model and these residues together with 93 water molecules gave an agreement factor of R = 0.161 for data in the resolution range 1.5-5 A. The structure was finally refined using the program X-PLOR, which enabled alternate conformers to be modelled for several surface side chains. Residues 1 and 2 at the amino terminus of the protein and residue 88 near the carboxyl terminus could be identified from these electron-density maps. However the remaining disordered carboxy-terminal residues could not successfully be included in the model. A total of 117 solvent molecules were included in the final refinement to give R = 0.164 for the data between 1.5 and 10 A.
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PMID:Refinement and structural analysis of bovine cytochrome b5 at 1.5 A resolution. 1529 27

Bacillus azotoformans is a Gram-positive denitrifying soil bacterium, which is capable of respiring nitrate, nitrite, nitric oxide, and nitrous oxide under anaerobic conditions. It contains a unique menaquinol-dependent nitric oxide reductase (qCu(A)NOR) with a Cu(A) center in its small subunit. The qCu(A)NOR exhibits menaquinol-dependent NO reductase activity, whereas reduced horse heart cytochrome c was inactive. Here we describe the purification of three membrane-bound c cytochromes from B. azotoformans. Their apparent molecular masses on SDS-PAGE are approximately 11 kDa. At neutral pH, these c cytochromes are negatively charged and the E(m) for all is close to 150 mV. Only one of these c cytochromes, which exhibits an alpha-band maximum at 551 nm, acts as a direct electron donor to qCu(A)NOR. Further investigation demonstrated that this cytochrome c(551) possesses two lipoyl moieties, which presumably function to anchor it to the membrane. Steady-state kinetic studies reveal that cytochrome c(551) is a noncompetitive inhibitor of NO reduction when menaquinol is used as an electron donor. This finding points to the presence of two different electron donation pathways in qCu(A)NOR. The ability of qCu(A)NOR to accept electrons from both menaquinol and cytochrome c(551) might be related to the regulation of the rate of NO reduction especially as a defense mechanism of B. azotoformans against the toxicity of NO. Growth experiments in batch culture indeed show that B. azotoformans is highly NO tolerant, in contrast to, for example, Paracoccus denitrificans that has a monofunctional cytochrome c-dependent NOR. We propose that the menaquinol pathway, which has a 4-fold greater maximal activity than the pathway via cytochrome c(551), is used for NO detoxification, whereas electron donation via the endogenous cytochrome c involves the cytochrome b(6)f complex serving the bioenergetic needs of the organism.
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PMID:NO reductase from Bacillus azotoformans is a bifunctional enzyme accepting electrons from menaquinol and a specific endogenous membrane-bound cytochrome c551. 1549 Nov 56

Metallosphaera sedula is a thermoacidophilic Crenarchaeon which is capable of leaching metals from sulfidic ores. The authors have investigated the presence and expression of genes encoding respiratory complexes in this organism when grown heterotrophically or chemolithotrophically on either sulfur or pyrite. The presence of three gene clusters, encoding two terminal oxidase complexes, the quinol oxidase SoxABCD and the SoxM oxidase supercomplex, and a gene cluster encoding a high-potential cytochrome b and components of a bc(1) complex analogue (cbsBA-soxL2N gene cluster) was established. Expression studies showed that the soxM gene was expressed to high levels during heterotrophic growth of M. sedula on yeast extract, while the soxABCD mRNA was most abundant in cells grown on sulfur. Reduced-minus-oxidized difference spectra of cell membranes showed cytochrome-related peaks that correspond to published spectra of Sulfolobus-type terminal oxidase complexes. In pyrite-grown cells, expression levels of the two monitored oxidase gene clusters were reduced by a factor of 10-12 relative to maximal expression levels, although spectra of membranes clearly contained oxidase-associated haems, suggesting the presence of additional gene clusters encoding terminal oxidases in M. sedula. Pyrite- and sulfur-grown cells contained high levels of the cbsA transcript, which encodes a membrane-bound cytochrome b with a possible role in iron oxidation or chemolithotrophy. The cbsA gene is not co-transcribed with the soxL2N genes, and therefore does not appear to be an integral part of this bc(1) complex analogue. The data show for the first time the differential expression of the Sulfolobus-type terminal oxidase gene clusters in a Crenarchaeon in response to changing growth modes.
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PMID:Respiratory gene clusters of Metallosphaera sedula - differential expression and transcriptional organization. 1563 23

Gram+ bacteria are capable of complete denitrification just like Gram- (Gram-negative) bacteria. However, Gram+ (Gram-positive) bacteria have a very small periplasmic-like space. This leads to the question of whether those enzymes and electron carriers involved in denitrification, which are normally located in the periplasmic space in Gram- bacteria, are located in the periplasmic-like space in Gram+ bacteria or have been modified as membrane-bound proteins. Using Bacillus azotoformans as a Gram+ bacterial model, our study demonstrates that anaerobic denitrification is catalysed by four membrane-bound enzymes and that the electron carriers are membrane-bound c-type cytochromes and menaquinol. NADH dehydrogenase is coupled with the denitrification pathway providing menaquinol. In addition, the cytochrome b(6)f complex forms part of the denitrification pathway, oxidizing menaquinol and reducing at least three different membrane-bound c-type cytochromes. We determined that the NO reductase, qCu(A)NOR (where NOR stands for nitric oxide reductase), can accept electrons from two donors, a specific cytochrome c(551) and menaquinol. Similarly, nitrite reductase, a copper enzyme, and nitrous oxide reductase may be bifunctional enzymes. Regarding the bifunctionality of qCu(A)NOR, we propose that the menaquinol-linked pathway is involved in the detoxification of NO.
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PMID:Membrane-bound denitrification in the Gram-positive bacterium Bacillus azotoformans. 1566 84

An overexpression system for spinach apocytochrome b(6) as a fusion protein to a maltose-binding protein in Escherichia coli was established using the expression vector pMalp2. The fusion of the cytochrome b(6) to the periplasmic maltose-binding protein directs the cytochrome on the Sec-dependent pathway. The cytochrome b(6) has a native structure in the bacterial cytoplasmic membrane with both NH(2) and COOH termini on the same, periplasmic side of the membrane but has the opposite orientation compared to that in thylakoid. Our data also show that in the E. coli cytoplasmic membrane, apocytochrome b(6) and exogenic hemes added into a culture media spontaneously form a complex with similar spectroscopic properties to native cytochrome b(6). Reconstituted membrane-bound cytochrome b(6) contain two b hemes (alpha band, 563 nm; average E(m,7) = -61 +/- 0.84 and -171 +/- 1.27 mV).
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PMID:Integration of the thylakoid membrane protein cytochrome b6 in the cytoplasmic membrane of Escherichia coli. 1589

The photosynthetic purple bacteria such as Rb. sphaeroides possesses an intracytoplasmic membrane (ICM) and a variety of pigment-binding membrane proteins located in the ICM, acting as photoreceptor. Such photosynthetic apparatus is concentrated in the ICM. It is composed of three multimeric membrane-bound proteins; light-harvesting complexes (LH 1, LH 2), a reaction center (RC) and a cytochrome b/c1 complex. We have purified these membranes, which are called chromatophores, and characterized the structure and dynamics of the photosynthetic membrane-bound proteins by means of multi-nuclear solid state NMR. First, the isotropic chemical shift of carbonyl carbons in natural abundance and [1-(13)C] Phe labeled chromatophores indicates that the membrane-bound proteins take mainly the helical conformation. Second, the chemical shifts of side-chain resonances of uniformly (15)N-labeled chromatophores indicate the side-chain histidine residue is mainly hydrogen bonded, whereas structural heterogeneity of arginine and lysine side-chains are probed by those wide distribution of (15)N shifts. Thirdly, the [beta-(2)H(3)]Ala and [epsilon-(2)H(2)]Tyr labeling of the chromatophores are performed and dynamics of the [beta-(2)H]Ala and the [epsilon-(2)H(2)]Tyr labeled chromatophores are studied by means of (2)H solid state NMR. The dynamics of [beta-(2)H(3)]Ala is found to be a 10(8)Hz three-site jump motion with 10 degrees liberation along the Calpha-Cbeta bond axis. The (2)H-NMR powder pattern spectrum of [epsilon-(2)H(2)] Tyr labeled chromatophores was interpreted with an averaged correlation time of 5x10(5) Hz with 180 degrees two-fold flips, the result of the averaging of two kinds of split spectra in terms of motional time scale.
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PMID:Structure and dynamics of photosynthetic membrane-bound proteins in Rhodobacter Sphaeroides, studied with solid-state NMR spectroscopy. 1622 36

Spirulina-acyl-lipid desaturases are membrane-bound enzymes found in thylakoid and plasma membranes. These enzymes carry out the fatty acid desaturation process of Spirulina to yield gamma-linolenic acid (GLA) as the final desaturation product. In this study, Spirulina-Delta(6) desaturase encoded by the desD gene was heterologously expressed and characterized in Saccharomyces cerevisiae. We then conducted site-directed mutagenesis of the histidine residues in the three histidine boxes to determine the role of these amino acid residues in the enzyme function. Our results showed that while four mutants showed complete loss of Delta(6)-desaturase activity and two mutants showed only trace of the activity, the enzyme activity could be partially restored by chemical rescue using exogenously provided imidazole. This study reveals that the histidine residues (which have imidazole as their functional group) in the conserved clusters play a critical role in Delta(6)-desaturase activity, possibly by providing a di-iron catalytic center. In our previous study, this enzyme was expressed in Escherichia coli. The results reveal that the enzyme can function only in the presence of an exogenous cofactor, ferredoxin, provided in vitro. This evidence suggests that baker's yeast has a cofactor that can complement ferredoxin, thought to act as an electron donor for the Delta(6) desaturation in cyanobacteria, including Spirulina. The electron donor of the Spirulina-Delta(6) desaturation in yeast is more likely to be cytochrome b(5), which is absent in E. coli. This means that the enzyme expressed in S. cerevisiae can catalyze the biosynthesis of the product, GLA, in vivo.
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PMID:Functional expression of Spirulina-Delta6 desaturase gene in yeast, Saccharomyces cerevisiae. 1632 83

Although cytochrome b-559 has long been known as a membrane-bound redox component closely linked to the reaction center of the oxygen-generating photosystem (PSII), its role in photosynthesis has remained obscure. This paper reports evidence and outlines a hypothesis in support of a "b-559 cycle"-i.e., a light-induced, cytochrome b-559-dependent, cyclic electron transport pathway around PSII that promotes translocation of protons from plastoquinol into the aqueous domain (lumen) of photosynthetic membranes (thylakoids). Light-induced proton transport coupled to light-induced electron transport is an essential aspect of energy transduction in photosynthesis because it generates an electrochemical proton gradient that drives ATP synthesis by the process of photosynthetic phosphorylation. The principal carrier of electrons and protons in thylakoids is the plastoquinone/plastoquinol couple. We propose that the b-559 cycle functions as a redox-linked proton pump that may operate jointly with the Rieske iron-sulfur pathway in oxidizing plastoquinol. The overall effect of such concerted oxidation of plastoquinol would be the translocation into the thylakoid lumen of two protons for each electron transferred from water to plastocyanin via plastoquinone.
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PMID:Cytochrome b-559 and proton conductance in oxygenic photosynthesis. 1659 7

Reactions of photosynthetic electron transport and photophosphorylation were studied in preparations from the blue-green alga, Phormidium luridum. Osmotic lysis of protoplasts proved to be a superior technique for the production of cell-free preparations with high enzymatic activity. Such lysed protoplasts sustain high rates of photophosphorylation coupled to the photo-reduction of NADP(+) or ferricyanide. P/2e(-) ratios close to unity were routinely observed. The same preparations, and also those prepared by grinding the cells in solutions containing sucrose or ethylene glycol, are active in cyclic photophosphorylation mediated by phenazine methosulfate or dichloro-phenolindophenol. The particles prepared by grinding the cells are, however, inactive in non-cyclic photophosphorylation.Extensive washing of the membranes with solutions containing sucrose removes the majority of the residual soluble fraction of the algal cell which includes cytochromes C(554) and C(549) and phycocyanin. Cyclic photophosphorylation activity is unimpaired by this treatment, but is abolished when the membranes are washed with very dilute buffers. This activity is restored by the addition of a soluble protein which is not a known redox constituent such as cytochrome C(554) or plastocyanin, and may be a coupling factor.Analysis of the well-washed membranes by low temperature (77 degrees K) difference spectrophotometry reveals the presence of cytochrome b(6) and a bound form of cytochrome C(554) in proportions similar to that found in higher plant chloroplasts. The concentration of the membrane-bound cytochrome C(554), relative to cytochrome b(6) is not altered by extensive washing, sonication or treatment with 1% digitonin. This indicates that this cytochrome is an integral component of the cytoplasmic lamellae and we suggest that it is of functional significance. The soluble form of cytochrome C(554), which is present in concentrations about 3-fold higher than the bound form, depending upon growth conditions, is not essential for cyclic photophosphorylation. The concentration of cytochrome b(6): chlorophyll a was found to be 1:500.Under the conditions employed, we were unable to detect a bound form of the low potential cytochrome C(549).
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PMID:Photosynthetic Reactions by Lysed Protoplasts and Particle Preparations from the Blue-Green Alga, Phormidium luridum. 1665 76


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