Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The structure of "membranous cytochrome oxidase" has been investigated by X-ray diffraction, optical polarization spectroscopy and EPR spectroscopy. These studies indicate that the cytochrome oxidase molecules are oriented symmetrically in the membrane profile with a significant portion of their mass occurring within the extravesicular surface of the membrane; the oxidase molecultes span the membrane profile; the distribution of the oxidase molecules over the plane of these membranes is non-crystalline; the oxidase molecules contain bundles of alpha-helical polypeptide chain segments where the average orientation of the helices is normal to the membrane plane; and the average heme orientation within the oxidase molecules is such that the normal to the heme plane lies in the plane of the membrane.
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PMID:The structure of a cytochrome oxidase-lipid model membrane. 20 14

Oxidation factor, a protein required for electron transfer from succinate to cytochrome c in the mitochondrial respiratory chain, has been purified from isolated succinate . cytochrome c reductase complex. Purification of the protein has been followed by a reconstitution assay in which restoration of ubiquinol . cytochrome c reductase activity is proportional to the amount of oxidation factor added back to depleted reductase complex. The purified protein is a homogeneous polypeptide on acrylamide gel electrophoresis in sodium dodecyl sulfate and migrates with an apparent Mr = 24,500. Purified oxidation factor restores succinate . cytochrome c reductase and ubiquinol . cytochrome c reductase activities to depleted reductase complex. It is not required for succinate dehydrogenase nor for succinate . ubiquinone reductase activities of the reconstituted reductase complex. Oxidation factor co-electrophoreses with the iron-sulfur protein polypeptide of ubiquinol . cytochrome c reductase complex. The purified protein contains 56 nmol of nonheme iron and 36 nmol of acid-labile sulfide/mg of protein and possesses an EPR spectrum with the characteristic "g = 1.90" signal identical to that of the iron-sulfur protein of the cytochrome b . c1 complex. In addition, the optimal conditions for extraction of oxidation factor, including reduction with hydrosulfite and treatment of the b . c1 complex with antimycin, are identical to those which facilitate extraction of the iron-sulfur protein from the b . c1 complex. These results indicate that oxidation factor is a reconstitutively active form of the iron-sulfur protein of the cytochrome b . c1 complex first discovered by Rieske and co-workers (Rieske, J.S., Maclennan, D.H., and Coleman, R. (1964) Biochem. Biophys. Res. Commun. 15, 338-344) and thus demonstrate that this iron-sulfur protein is required for electron transfer from ubiquinol to cytochrome c in the mitochondrial respiratory chain.
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PMID:Purification of a reconstitutively active iron-sulfur protein (oxidation factor) from succinate . cytochrome c reductase complex of bovine heart mitochondria. 22 62

1. A partially purified tetanolysin preparation lysed the sterol-requiring Mycoplasma capricolum cells but had no effect on M. capricolum cells adapted to grow with no or very little cholesterol. The sterol-non-requiring Acholeplasma laidlawii cells grown either in a cholesterol-rich or a cholesterol-poor medium were unaffected by the tetanolysin preparation. 2. The lysis of M. capricolum cells by the tetanolysin preparation was temperature dependent, inhibited by cholesterol, sublytic concentrations of lucensomycin, and Mg2+. The sensitivity to lysis was greatly affected by the age of the culture, being highest in cells from the early logarithmic phase of growth and declining sharply thereafter. 3. Isolated M. capricolum membranes were capable of binding large amounts of the tetanolysin activity (up to 30 hemolytic units per mug membrane protein), 20 times as much as membranes of the adapted strain. The binding of tetanolysin activity to membranes was almost the same at 4,22, or 37 degrees C, and was very little affected by the age of the culture. The binding capacity of the membranes was not affected by the removal of 60-70% of membrane proteins by pronase digestion but markedly decreased with the removal of membrane lipids. 4. Of the five polypeptide bands detected in electrophorograms of the partially purified tetanolysin preparation, two bands (mol. wt. 44 000 and 42 000) were found to bind to the cholesterol-containing mycoplasma membrane preparation. EPR spectrometry revealed that the freedom of motion of fatty acid spin labels in the tetanolysin-treated membranes was markedly higher than that in untreated membranes. 5. The concept that tetanolysin interacts specifically with membrane cholesterol resulting in the shielding of cholesterol from its interaction with membrane phospholipids is discussed.
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PMID:Interaction between tetanolysin and Mycoplasma cell membrane. 79 33

Pyruvate formate-lyase (acetyl-CoA:formate C-acetyltransferase, EC 2.3.1.54) from anaerobic Escherichia coli cells converts pyruvate to acetyl-CoA and formate by a unique homolytic mechanism that involves a free radical harbored in the protein structure. By EPR spectroscopy of selectively 13C-labeled enzyme, the radical (g = 2.0037) has been assigned to carbon-2 of a glycine residue. Estimated hyperfine coupling constants to the central 13C nucleus (A parallel = 4.9 mT and A perpendicular = 0.1 mT) and to 13C nuclei in alpha and beta positions agree with literature data for glycine radical models. N-coupling was verified through uniform 15N-labeling. The large 1H hyperfine splitting (1.5 mT) dominating the EPR spectrum was assigned to the alpha proton, which in the enzyme radical is readily solvent-exchangeable. Oxygen destruction of the radical produced two unique fragments (82 and 3 kDa) of the constituent polypeptide chain. The N-terminal block on the small fragment was identified by mass spectrometry as an oxalyl residue that derives from Gly-734, thus assigning the primary structural glycyl radical position. The carbon-centered radical is probably resonance-stabilized through the adjacent carboxamide groups in the polypeptide main chain and could be comparable energetically with other known protein radicals carrying the unpaired electron in tyrosine or tryptophan residues.
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PMID:The free radical in pyruvate formate-lyase is located on glycine-734. 131 May 45

We present here a simple and rapid method which allows relatively large quantities of oxygen-evolving photosystem II- (PS-II-) enriched particles to be obtained from wild-type and mutants of the cyanobacterium Synechocystis 6803. This method is based on that of Burnap et al. [Burnap, R., Koike, H., Sotiropoulou, G., Sherman, L. A., & Inoue, Y. (1989) Photosynth. Res. 22, 123-130] but is modified so that the whole preparation, from cells to PS-II particles, is achieved in 10 h and involves only one purification step. The purified preparation exhibits a 5-6-fold increase of O2-evolution activity on a chlorophyll basis over the thylakoids. The ratio of PS-I to PS-II is about 0.14:1 in the preparation. The secondary quinone electron acceptor, QB, is present in this preparation as demonstrated by thermoluminescence studies. These PS-II particles are well-suited to spectroscopic studies as demonstrated by the range of EPR signals arising from components of PS-II that are easily detectable. Among the EPR signals presented are those from a formal S3-state, attributed to an oxidized amino acid interacting magnetically with the Mn complex in Ca(2+)-deficient PS-II particles, and from S2 modified by the replacement of Ca2+ by Sr2+. Neither of these signals has been previously reported in cyanobacteria. Their detection under these conditions indicates a similar lesion caused by Ca2+ depletion in both plants and cyanobacteria. The protocol has also been applied to mutants which have site-specific changes in PS-II. Data are presented on mutants having changes on the electron donor (Y160F) and electron acceptor (G215W) side of the D2 polypeptide.
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PMID:Oxygen-evolving photosystem II preparation from wild type and photosystem II mutants of Synechocystis sp. PCC 6803. 131 Dec 5

Site-directed mutants of Escherichia coli fumarate reductase in which FrdB Cys204, Cys210, and Cys214 were individually replaced by Ser and in which Val207 was replaced by Cys were constructed and overexpressed in a strain of E. coli lacking a wild-type copy of fumarate reductase and succinate dehydrogenase. The consequences of these mutations on bacterial growth, enzymatic activity, and the EPR properties of the constituent iron-sulfur clusters were investigated. The FrdB Cys204Ser, Cys210Ser, and Cys214Ser mutations result in enzymes with negligible activity that have dissociated from the membrane and consequently are incapable of supporting cell growth under conditions requiring a functional fumarate reductase. EPR studies indicate that these effects are associated with loss of both the [3Fe-4S] and [4Fe-4S] clusters, centers 3 and 2, respectively. In contrast, the FrdB Val207Cys mutation results in a functional membrane-bound enzyme that is able to support growth under anaerobic and aerobic conditions. However, EPR studies indicate that the indigenous [3Fe-4S]+,0 cluster (Em = -70 mV), center 3, has been replaced by a much lower potential [4Fe-4S]2+,+ cluster (Em = -350 mV), indicating that the primary sequence of the polypeptide determines the type of clusters assembled. The results of these studies afford new insights into the role of centers 2 and 3 in mediating electron transfer from menaquinol, the residues that ligate these clusters, and the intercluster magnetic interactions in the wild-type enzyme.
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PMID:[3Fe-4S] to [4Fe-4S] cluster conversion in Escherichia coli fumarate reductase by site-directed mutagenesis. 131 45

The ubiquinone complement of Rhodobacter capsulatus chromatophore membranes has been characterized by its isooctane solvent extractability and electrochemistry; we find that the main ubiquinone pool (Qpool) amounts to about 80% of the total ubiquinone and has an Em7 value close to 90 mV. To investigate the interactions of ubiquinone with the cyt bc1 complex, we have examined the distinctive EPR line shapes of the [2Fe-2S] cluster of the cyt bc1 complex when the Qpool-cyt bc1 complex interactions are modulated by changing the numbers of Q or QH2 present (by solvent extraction and reconstitution), by the exposure of the [2Fe-2S] to the Qpool in different redox states, by the presence of inhibitors specific for the Qo site (myxothiazol and stigmatellin) and Qi site (antimycin), and by site-specific mutations of side chains of the cyt b polypeptide (mutants F144L and F144G) previously identified as important for Qo site structure. Evidence suggests that the Qo site can accommodate two ubiquinone molecules. One (designated Qos) is bound relatively strongly and is second only to the ubiquinone of the QA site of the reaction center in its resistance to solvent extraction. In this strong interaction, the Qo site binds Q and QH2 with approximately equal affinities. Their bound states are distinguished by their effects on the [2Fe-2S] cluster spectral feature at gx at 1.783 (Q) and gx at 1.777 (QH2); titration of the line-shape change reveals an Em7 value of approximately 95 mV. The other molecule (Qow) is bound more weakly, in the same range as the ubiquinone of the QB site of the reaction center. Again, the affinities of the Q form (gx at 1.800) and QH2 form (gx at 1.777) are nearly equal, and the Em7 value measured is approximately 80 mV. These results are discussed in terms of earlier EPR analyses of the cyt bc1 complexes of other systems. A Qo site double-occupancy model is considered that builds on the previous model based on Qo site mutants [Robertson, D. E., Daldal, F.,& Dutton, P. L. (1990) Biochemistry 29, 11249-11260] and includes the recent suggestion that two of the [2F3-2S] cluster ligands of the R. capsulatus cyt bc1 complex are histidines [Gurbiel, R. J. Ohnishi, T., Robertson, D. E. Daldal, F., & Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. We speculate that the cyt bc1 complex complexes a full enzymatic turnover without necessary exchange of ubiquinone with the Qpool.
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PMID:Cytochrome bc1 complex [2Fe-2S] cluster and its interaction with ubiquinone and ubihydroquinone at the Qo site: a double-occupancy Qo site model. 131 87

A novel iron-sulfur protein has been isolated from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough). It is a stable monomeric protein, which has a molecular mass of 52 kDa, as determined by sedimentation-equilibrium centrifugation. Analysis of the metal and acid-labile sulfur content of the protein revealed the presence of 6.3 +/- 0.4 Fe/polypeptide and 6.2 +/- 0.7 S2-/polypeptide. Non-iron transition metals, heme, flavin and selenium were absent. Combining these data with the observation of a very anisotropic S = 1/2 [6Fe-6S]3+ prismane-like EPR signal in the dithionite-reduced protein, we believe that we have encountered the first example of a prismane-cluster-containing protein. The prismane protein has a slightly acidic amino acid composition and isoelectric point (pI = 4.9). The ultraviolet/visible spectrum is relatively featureless (epsilon 280 = 81 mM-1.cm-1, epsilon 400 = 25 mM-1.cm-1, epsilon 400,red = 14 mM-1.cm-1). The shape of the protein is approximately globular (S20.w = 4.18 S). The N-terminal amino acid sequence is MFS/CFQS/C QETAKNTG. Polyclonal antibodies against the protein were raised. Cytoplasmic localization was inferred from subcellular fractionation studies. Cross-reactivity of antibodies against this protein indicated the occurrence of a similar protein in D. vulgaris (Monticello) and Desulfovibrio desulfuricans (ATCC 27774). We have not yet identified a physiological function for the prismane protein despite trials for some relevant enzyme activities.
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PMID:Purification and biochemical characterization of a putative [6Fe-6S] prismane-cluster-containing protein from Desulfovibrio vulgaris (Hildenborough). 131 32

To identify amino acid residues that influence the assembly or stability of the manganese cluster in photosystem II, we have generated site-directed mutations in the D1 polypeptide of the cyanobacterium, Synechocystis sp. PCC 6803. Indirect evidence has suggested that the D1 polypeptide provides some of the ligands that are required for metal binding. Mutations at position 170 of D1 were selected for characterization, since an aspartate to asparagine mutation (DN170D1) at this position completely abolishes photoautotrophic growth, while retention of a carboxylic acid at this position (aspartate to glutamate, DE170D1) supports photoautotrophic growth. Photosystem II particles were purified from control, DE170D1, and DN170D1 cells by a procedure that retains high rates of oxygen evolution activity in control particles [Noren, G.H., Boerner, R.J., & Barry, B.A. (1991) Biochemistry 30, 3943-3950]. Spectroscopic analysis shows that the tyrosine radical, Z+, which normally oxidizes the manganese cluster, is rapidly reduced in the DE170D1 mutant, but not in the DN170D1 mutant. A possible explanation of this block or dramatic decrease in the rate of electron transfer between the manganese cluster and tyrosine Z is an alteration in the properties of the metal center. Quantitation of manganese in these particles is consistent with aspartate 170 influencing the stability or assembly of the manganese cluster, since the aspartate to asparagine mutation results in a decrease in the manganese content per reaction center. Photosystem II particles from DN170D1 show a 60% decrease in the amount of specifically bound manganese per reaction center, when compared to control particles. Also, we observe a 70% decrease in the amount of specifically bound manganese per reaction center in partially purified DN170D1 particles and at least an 80% decrease in the amount of hydroxylamine-reducible manganese in DN170D1 thylakoid membranes. Single-turnover fluorescence assays and steady-state EPR measurements demonstrate that the remaining, endogenous manganese does not rapidly reduce tyrosine Z+ in the DN170D1 mutant. Additional evidence that aspartate 170 influences the assembly or stability of the metal site comes from analysis of the DE170D1 mutant. Although this mutant assembles a functional manganese cluster, as assessed by oxygen evolution and spectroscopic assays, the properties of the manganese site are perturbed.
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PMID:Evidence from directed mutagenesis that aspartate 170 of the D1 polypeptide influences the assembly and/or stability of the manganese cluster in the photosynthetic water-splitting complex. 132 68

Succinate:menaquinone-7 oxidoreductase (complex II) of the Gram-positive bacterium Bacillus subtilis consists of equimolar amounts of three polypeptides; a 65-kDa FAD-containing polypeptide, a 28-kDa iron-sulfur cluster containing polypeptide, and a 23-kDa membrane-spanning cytochrome b558 polypeptide. The enzyme complex was overproduced 2-3-fold in membranes of B. subtilis cells containing the sdhCAB operon on a low copy number plasmid and was purified in the presence of detergent. The cytochrome b558 subunit alone was similarly overexpressed in a complex II deficient mutant and partially purified. Isolated complex II catalyzed the reduction of various quinones and also quinol oxidation. Both activities were efficiently albeit not completely blocked by 2-n-heptyl-4-hydroxyquinoline N-oxide. Chemical analysis demonstrated two protoheme IX per complex II. One heme component was found to have an Em,7.4 of +65 mV and an EPR gmax signal at 3.68, to be fully reducible by succinate, and showed a symmetrical alpha-band absorption peak at 555 nm at 77 K. The other heme component was found to have an Em,7.4 of -95 mV and an EPR gmax signal at 3.42, was not reducible by succinate under steady-state conditions, and showed in the reduced state an apparent split alpha-band absorption peak with maxima at 553 and 558 nm at 77 K. Potentiometric titrations of partially purified cytochrome b558 subunit demonstrated that the isolated cytochrome b558 also contains two hemes. Some of the properties, i.e., the alpha-band light absorption peak at 77 K, the line shapes of the EPR gmax signals, and reactivity with carbon monoxide were observed to be different in B. subtilis cytochrome b558 isolated and in complex II. This suggests that the bound flavoprotein and iron-sulfur protein subunits protect or affect the heme environment in the assembled complex.
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PMID:Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase (complex II). 132 13


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