Biomedical subjects

C A Yu

Publications and source records attributed to C A Yu.

At least 73 records · Page 4Linked to original sources

PubMed1992

Spin-label electron paramagnetic resonance and differential scanning calorimetry studies of the interaction between mitochondrial cytochrome c oxidase and adenosine triphosphate synthase complex.

The interaction between cytochrome c oxidase complex and adenosine triphosphate synthase (F1F0) complex in the purified, dispersed state and embedded in phospholipid vesicles was studied by differential scanning calorimetry and by spin-label electron paramagnetic resonance. The detergent-dispersed cytochrome oxidase and F1F0 complexes undergo endothermic thermodenaturation. However, when these complexes are embedded in phospholipid vesicles, they undergo exothermic thermodenaturation. The energy released is believed to result from the collapse of a strained interaction between unsaturated fatty acyl groups of phospholipids and an exposed area of the complex formed by the removal of interacting proteins. The exothermic enthalpy change of thermodenaturation of a protein-phospholipid exothermic enthalpy change of thermodenaturation of a protein-phospholipid vesicle containing both cytochrome oxidase complex and F1F0 was smaller than that of a mixture of protein-phospholipid vesicles formed from each individual electron transfer complex. This suggests specific interaction between cytochrome oxidase complex and F1F0 in the membrane. Further evidence for interaction between these two complexes is provided by saturation transfer EPR studies in which the rotational correlation time of spin-labeled cytochrome oxidase increases significantly when the complex is mixed with F1F0 prior to being embedded in phospholipid vesicles. From these results, it is concluded that at least a part of cytochrome oxidase and a part of F1F0 form a supermacromolecular complex in the inner mitochondrial membrane. No such supermacromolecular complex is detected between F1F0 and ubiquinol--cytochrome c reductase.

Animals↗

PubMed1992

Characterization of ubisemiquinone radicals in succinate-ubiquinone reductase.

A thenoyl trifluoroacetone-sensitive and antimycin-insensitive ubisemiquinone radical (Qs) is readily detected in purified succinate-cytochrome c reductase. When this reductase is resolved into succinate-Q and ubiquinol-cytochrome c reductases, Qs was not detected in either reductase. The difficulty in detecting such a radical in purified succinate-Q reductase has puzzled investigators for years. A deficiency of Q in the isolated complex is the reason for the failure to detect Qs. Upon addition of exogenous Q, a thenoyl trifluoroacetone-sensitive Q-radical is readily detectable in isolated succinate-Q reductase under a controlled redox potential. Maximum radical concentration is observed when 5 mol of exogenous Q, per mole of flavin, is added. The radical gives an EPR signal with a g-value of 2.005 and a line-width of 12 G. The Em of Qs is 84 mV at pH 7.4, with half-potentials of E1 = 40 mV and E2 = 128 mV. The Qs-radical does not show power saturation, even at 200 mW.

Antimycin A↗

PubMed1991

Protein-ubiquinone interaction in bovine heart mitochondrial succinate-cytochrome c reductase. Synthesis and biological properties of fluorine substituted ubiquinone derivatives.

To investigate the protein-ubiquinone interaction in the bovine heart mitochondrial succinate-cytochrome c reductase region of the respiratory chain, three fluorine substituted ubiquinone derivatives, 2,3-dimethoxy-6-(9'-fluorodecyl)-1,4-benzoquinone (9FQ), 2-methoxy-5-trifluoromethyl-6-decyl-1,4-benzoquinone (TFQ), and 2-methoxy-5-trifluoromethyl-6-(9'-fluorodecyl)-1,4-benzoquinone (9FTFQ), were synthesized. 9FQ was synthesized by radical coupling of Q0 and bis(10-fluoroundecanoyl)peroxide. The latter was prepared by fluorination of undecylenic acid followed by thionylchloride treatment and peroxidation. TFQ was synthesized from 2,2,2-trifluoro-p-cresol by methylation, nitration, reduction, acetylation, nitration, reduction, oxidation, and radical alkylation. 9FTFQ was prepared by the radical alkylation of 2-methoxy-5-trifluoromethyl-1,4-benzoquinone with bis(10-fluoroundecanoyl)peroxide. All three fluoro-Q derivatives are active (greater than 50% the activity of 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone) when used as electron acceptors for succinate-ubiquinone reductase. However, only 9FQ is active when used as an electron donor for ubiquinol-cytochrome c reductase or as an electron mediator for succinate-cytochrome c reductase. Both TFQ and 9FTFQ are competitive inhibitors for ubiquinol-cytochrome c reductase. A 19FNMR peak-broadening effect was observed for 9FQ when it was reconstituted with ubiquinone-depleted ubiquinol-cytochrome c reductase. A drastic up-field chemical shift was observed for TFQ when it was reconstituted with ubiquinone-depleted reductase. These results indicate that the binding environments of the benzoquinone ring and the alkyl side chain of the Q molecule are different. The strong up-field chemical shift for TFQ, and lack of significant chemical shift for 9FQ, suggest that the benzoquinone ring is bound near the paramagnetic cytochrome b heme.

Animals↗

PubMed1991

Inhibitory effects of dicyclohexylcarbodiimide on spinach cytochrome b6-f complex.

The electron transfer activity of purified cytochrome b6-f complex of spinach chloroplast is inhibited by dicyclohexylcarbodiimide (DCCD) in a concentration and incubation time dependent manner. The maximum inhibition of 75% is observed when 300 mole of DCCD per mole of protein (based on cytochrome f) is incubated with cytochrome b6-f complex at room temperature for 40 min. The inhibition of the complex is not due to the formation of cross links between subunits but due to the modification of carboxyls. The amount of DCCD incorporation is directly proportional to the activity loss, suggesting that some carboxyl groups in the complex are directly or indirectly involved in the catalytic function. The incorporated DCCD is located mainly at cytochrome b6 protein. The partially inhibited complex shows the same H+/e-ratio as that of the intact complex when embedded in phospholipid vesicles.

Binding Sites↗

PubMed1991

Immunochemical study of subunit VI (Mr 13,400) of mitochondrial ubiquinol-cytochrome c reductase.

A preparation containing the Mr 13,400 protein (subunit VI), phospholipid, and ubiquinone was isolated from bovine heart mitochondrial ubiquinol-cytochrome c reductase by a procedure involving Triton X-100 and urea solubilization, calcium phosphate-cellulose column chromatography at different pHs, acetone precipitation, and decanoyl-N-methylglucamide-sodium cholate extraction. The protein in this preparation corresponds to subunit VI of ubiquinol-cytochrome c reductase resolved in the sodium dodecyl sulfate-polyacrylamidce gel electrophoresis system of Schägger et al. (1987, FEBS Lett. 21, 161-168) and has the same amino acid sequence as that of the Mr 13,400 protein reported by Wakabayashi et al. (1985, J. Biol. Chem. 260, 337-343). The phospholipid and ubiquinone present in the preparation copurify with but are not intrinsic components of, the Mr 13,400 protein. This preparation has a potency and behavior identical to that of a free phospholipid preparation in restoring activity to delipidated ubiquinol-cytochrome c reductase. Antibodies against Mr 13,400 react only with Mr 13,400 protein and complexes which contain it. They do not inhibit intact, lipid-sufficient ubiquinol-cytochrome c reductase. However, when delipidated ubiquinol-cytochrome c reductase is incubated with antibodies prior to reconstitution with phospholipid, a 55% decrease in the restoration activity is observed, indicating that the catalytic site-related epitopes of the Mr 13,400 protein are buried in the phospholipid environment. Antibodies against Mr 13,400 cause an increase of apparent Km for ubiquinol-2 in ubiquinol-cytochrome c reductase. When mitoplasts or submitochondrial particles are exposed to a horseradish peroxidase conjugate of the Fab' fragment of anti-Mr 13,400 antibodies, peroxidase activity is found mainly in the submitochondrial particles preparation; little activity is detected in mitoplasts. This suggests that the Mr 13,400 protein is extruded toward the matrix side of the membrane.

Amino Acid Sequence↗

PubMed1991

The Rhodospirillum rubrum cytochrome bc1 complex: redox properties, inhibitor sensitivity and proton pumping.

A detergent-solubilized, three-subunit-containing cytochrome bc1 complex, isolated from the photosynthetic bacterium R. rubrum, has been shown to be highly sensitive to stigmatellin, myxothiazol, antimycin A and UHDBT, four specific inhibitors of these complexes. Oxidation-reduction titrations have allowed the determination of Em values for all the electron-carrying prosthetic groups in the complex. Antimycin A has been shown to produce a red shift in the alpha-band absorbance maximum of one of the cytochrome b hemes in the complex and stigmatellin has been shown to alter both the Em and EPR g-values of the Rieske iron-sulfur protein in the complex. Western blots have revealed antigenic similarities between the cytochrome subunits of the R. rubrum complex and those of the related photosynthetic bacteria, Rb. capsulatus and Rb. sphaeroides. The R. rubrum complex has been incorporated into liposomes. These liposomes exhibit respiratory control and are able to couple electron transfer from quinol to cytochrome c to proton translocation across the liposome membrane in a manner consistent with a Q-cycle mechanism. It can thus be concluded that neither electron transport nor coupled proton translocation by the cytochrome bc1 complex requires more than three subunits in R. rubrum.

Antimycin A↗

PubMed1991

Essentiality of the molecular weight 15,000 protein (subunit IV) in the cytochrome b-c1 complex of Rhodobacter sphaeroides.

The cytochrome b-c1 complex from Rhodobacter sphaeroides was resolved into four protein subunits by a phenyl-Sepharose CL-4B column eluted with different detergents. Individual subunits were purified to homogeneity. Antibodies against subunit IV (Mr = 15,000) were raised and purified. These antibodies had a high titer with isolated subunit IV and with the b-c1 complex from R. sphaeroides. They inhibited 95% of the ubiquinol-cytochrome c reductase activity of the cytochrome b-c1 complex, indicating that subunit IV is essential for the catalytic function of this complex. When detergent-solubilized chromatopores were passed through an anti-subunit IV coupled Affi-Gel 10 column, no no ubiquinol-cytochrome c reductase activity was detected in the effluent, and four proteins, corresponding to the four subunits in the isolated complex, were adsorbed to the column. This indicated that subunit IV in an integral part of the cytochrome b-c1 complex. No change in the apparent Kms for Q2H2 and for cytochrome c was observed with anti-subunit IV treated complex. Antibodies against subunit IV had little effect on the stability of the ubisemiquinone radical in this complex, suggesting that they do not bind to the subunit near its ubiquinone-binding site.

Antibodies↗

PubMed1991

The catalytic role of subunit IV of the cytochrome b6-f complex from spinach chloroplast.

The catalytic role of subunit IV, the Mr 17,000 protein, in the chloroplast cytochrome b6-f complex was established through trypsinolysis of the complex under controlled conditions. When purified chloroplast cytochrome b6-f complex, 1 mg/ml, in 50 mM Tris-succinate buffer (pH 7.0) containing 1% sodium cholate and 10% glycerol is treated with 80 micrograms of trypsin at room temperature for various lengths of time, the activity of the cytochrome b6-f complex decreases as the incubation time increases. A maximal inactivation of 80% is reached at 7 min of incubation. The trypsin inactivation is accompanied by the destruction of the proton translocation activity of the complex. No alteration of absorption and EPR spectral properties was observed in the trypsin-inactivated complex. Subunit IV is the only subunit in the cytochrome b6-f complex that is digested by trypsin, and the degree of digestion correlates with the decrease of electron transfer activity. The binding of azido-Q to subunit IV of the complex decreases as the extent of inactivation of the cytochrome b6-f complex by trypsin increases. The residue molecular mass of trypsin cleaved subunit IV is about 14 kDa, suggesting that the cleavage site is at lysine 119 or arginine 125 or 126. When the thylakoid membrane was assayed for cytochrome b6-f complex activity, very little activity was observed; and the activity was not sensitive to trypsinolysis. Upon sonication, activity and sensitivity to trypsinolysis was greatly increased, suggesting that subunit IV protrudes from the lumen side of the membrane.

Affinity Labels↗

PubMed1991

Crystallization of mitochondrial ubiquinol-cytochrome c reductase.

Ubiquinol-cytochrome c reductase of beef heart mitochondria was crystallized in the presence of decanoyl-N-methylglucamide, heptanetriol, and sodium chloride with poly(ethylene glycol) as precipitant. The largest crystal has dimensions of 4 x 2 x 1 mm. The crystalline enzyme is composed of 10 subunits. It contains 2.5 nmol of ubiquinone, 8.4 nmol of cytochrome b, 4.2 nmol of cytochrome c1, 4.2 nmol of iron-sulfur cluster, and 140 nmol of phospholipid per milligram of protein. Of the last, 36% is with diphosphatidylglycerol. The crystals are very stable in the cold and show full enzymatic activity when redissolved in aqueous solution. Absorption spectra of the redissolved crystals show a Soret to UV ratio of 0.88 and 1.01 in the oxidized and the reduced forms, respectively.

Animals↗

PubMed1991

Electron spin echo envelope modulation spectroscopy supports the suggested coordination of two histidine ligands to the Rieske Fe-S centers of the cytochrome b6f complex of spinach and the cytochrome bc1 complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria.

Electron spin echo envelope modulation (ESEEM) experiments performed on the Rieske Fe-S clusters of the cytochrome b6f complex of spinach chloroplasts and of the cytochrome bc1 complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria show modulation components resulting from two distinct classes of 14N ligands. At the g = 1.92 region of the Rieske EPR spectrum of the cytochrome b6f complex, the measured hyperfine couplings for the two classes of coupled nitrogens are A1 = 4.6 MHz and A2 = 3.8 MHz. Similar couplings are observed for the Rieske centers in the three cytochrome bc1 complexes. These ESEEM results indicate a nitrogen coordination environment for these Rieske Fe-S centers that is similar to that of the Fe-S cluster of a bacterial dioxygenase enzyme with two coordinated histidine ligands [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871]. The Rieske Fe-S cluster lacks modulation components from a weakly coupled peptide nitrogen observed in water-soluble spinach ferredoxin. Treatment with the quinone analogue inhibitor DBMIB causes a shift in the Rieske EPR spectrum to g = 1.95 with no alteration in the magnetic coupling to the two nitrogen atoms. However, the ESEEM pattern of the DBMIB-altered Rieske EPR signal shows evidence of an additional weakly coupled nitrogen similar to that observed in the spinach ferredoxin ESEEM patterns.

Animals↗

PubMed1991

Involvement of a histidine residue in the interaction between membrane-anchoring protein (QPs) and succinate dehydrogenase in mitochondrial succinate-ubiquinone reductase.

The involvement of a histidine residue of the membrane-anchoring protein (QPs) fraction in reconstitution of succinate dehydrogenase to form succinate-ubiquinone reductase is studied by using a histidine-modifying reagent, diethylpyrocarbonate (DEPC). A maximum inactivation of 80% of reconstitutive activity is obtained when QPs is treated with 1 mM DEPC at 0 degrees C for 30 min in 50 mM Tris-HCl (pH 7.0). DEPC also inactivates about 85% of intact succinate-ubiquinone reductase. The inactivation of succinate-ubiquinone reductase by DEPC is a result of the modification of essential histidine residues of succinate dehydrogenase. The inactivation is not a result of the modification of the histidine residue in QPs which is essential for interaction with succinate dehydrogenase because the QPs dissociated from the inactivated succinate-ubiquinone reductase is active in reconstitution with active succinate-dehydrogenase. Apparently, the essential histidine in QPs is shielded by succinate dehydrogenase and thus inaccessible to DEPC modification in succinate-ubiquinone reductase. The involvement of a histidine residue of QPs in interaction with succinate dehydrogenase is further evident by the presence of 553 nm shoulder on the alpha-absorption peak of reduced cytochrome b-560 (a characteristic of physical association of QPs with succinate dehydrogenase) in the DEPC-inactivated succinate-ubiquinone reductase. This shoulder disappears from a mixture of succinate dehydrogenase and DEPC-treated QPs when reduced with dithionite. About one histidine residue per molecule of QPs is modified in the DEPC-treated sample, suggesting that only one histidine residue is essential for interaction with succinate dehydrogenase. This essential histidine group is located in the smaller subunit (Mr 13,000) of QPs.

Diethyl Pyrocarbonate↗

PubMed1991

Hematoporphyrin-promoted photoinactivation of mitochondrial ubiquinol-cytochrome c reductase: selective destruction of the histidine ligands of the iron-sulfur cluster and protective effect of ubiquinone.

Purified ubiquinol-cytochrome c reductase of beef heart mitochondria is very stable in aqueous solution; it suffers little damage upon illumination with visible light under aerobic or anaerobic conditions. However, it is rapidly inactivated when the photosensitizer hematoporphyrin is present during illumination. The hematoporphyrin-promoted photoactivation is dependent on sensitizer dose, illumination time, and oxygen. Singlet oxygen is shown to be the destructive agent in this system. The photoinactivation of ubiquinol-cytochrome c reductase is prevented by excess exogenous ubiquinone, regardless of its redox state. This protective effect is not due to protein-ubiquinone interactions but to the singlet oxygen scavenger property of ubiquinone. Ubiquinone also protects against hematoporphyrin-promoted photoinactivation of succinate-ubiquinone reductase and cytochrome c oxidase. The photoinactivation site in ubiquinol-cytochrome c reductase is the iron-sulfur cluster of Rieske's protein. Two histidine residues, presumably serving as two ligands for the iron-sulfur cluster of Rieske's protein, are destroyed. No polypeptide bond cleavage is detected. Photoinactivation has little effect on the spectral properties of cytochromes b and c1 but alters their reduction rates substantially. this photoinactivation also causes the formation of proton-leaking channels in the complex. When the photoinactivated reductase is co-inlaid with intact ubiquinol-cytochrome c reductase or cytochrome c oxidase in a phospholipid vesicle, no proton ejection can be detected during the oxidation of their corresponding substrates.

Animals↗

PubMed1990

EPR characterization of the cytochrome b-c1 complex from Rhodobacter sphaeroides.

EPR characteristics of cytochrome c1, cytochromes b-565 and b-562, the iron-sulfur cluster, and an antimycin-sensitive ubisemiquinone radical of purified cytochrome b-c1 complex of Rhodobacter sphaeroides have been studied. The EPR specra of cytochrome c1 shows a signal at g = 3.36 flanked with shoulders. The oxidized form of cytochrome b-562 shows a broad EPR signal at g = 3.49, while oxidized cytochrome b-565 shows a signal at g = 3.76, similar to those of two b cytochromes in the mitochondrial complex. The distribution of cytochromes b-565 and b-562 in the isolated complex is 44 and 56%, respectively. Antimycin and 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone (DBMIB) have little effect on the g = 3.76 signal, but they cause a slight downfield and upfield shifts of the g = 3.49 signal, respectively. 5-Undecyl-6-hydroxyl-4,7-dioxobenzothiazole (UHDBT) shifts the g = 3.49 signal downfield to g = 3.56 and sharpens the g = 3.76 signal slightly. Myxothiazol causes an upfield shift of both g = 3.49 and g = 3.76 signals. EPR characteristics of the reduced iron-sulfur cluster in bacterial cytochrome b-c1 complex are: gx = 1.8 with a small shoulder at g = 1.76, gy = 1.89 and gz = 2.02, similar to those observed with the mitochondrial enzyme. The gx = 1.8 signal decreased and the shoulder increased concurrently as the redox potential decreased, indicating that the environment of the iron-sulfur cluster is sensitive to the redox state of the complex. UHDBT sharpens the gz and and shifts it downfield from g = 2.02 to 2.03, and shifts gx upfield from g = 1.80 to 1.78. UHDBT also causes an upfield shift of gy but to a much lesser extent compared to the other two signals. Addition of DBMIB causes a downfield shift of the gy from 1.89 to 1.94 and broadens the gx signal with an upfield to g = 1.75. Myxothiazol and antimycin show little effect on the gy and gz signals, but they broaden and shift the gx signal upfield to g = 1.74. However, the myxothiazol effect is partially reversed by UHDBT. An antimycin-sensitive ubisemiquinone radical was detected in the cytochrome b-c1 complex. At pH 8.4, the antimycin-sensitive ubisemiquinone radical has a maximal concentration of 0.66 mol per mol complex at 100 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Antimycin A↗

PubMed1990

The small molecular mass ubiquinone-binding protein (QPc-9.5 kDa) in mitochondrial ubiquinol-cytochrome c reductase: isolation, ubiquinone-binding domain, and immunoinhibition.

The small molecular mass ubiquinone-binding protein (QPc-9.5 kDa) was purified to homogeneity from 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinol+ ++- labeled bovine heart mitochondrial ubiquinol-cytochrome c reductase. The N-terminal amino acid sequence of the isolated protein is Gly-Arg-Gln-Phe-Gly-His-Leu-Thr-Arg-Val-Arg-His-, which is identical with that of a Mr = 9500 protein in the reductase [Borchart et al. (1986) FEBS Lett. 200, 81-86]. A ubiquinone-binding peptide was prepared from [3H]azidoubiquinol-labeled QPc-9.5 kDa protein by trypsin digestion followed by HPLC separation. The partial N-terminal amino acid sequence of this peptide, Val-Ala-Pro-Pro-Phe-Val-Ala-Phe-Tyr-Leu-, corresponds to amino acid residues 48-57 in the reported Mr = 9500 protein. According to the proposed structural model for the Mr = 9500 protein, the azido-Q-labeled peptide is located in the membrane on the matrix side. These results confirm our previous assessment that the Mr = 13,400 subunit is not the small molecular weight Q-binding protein. Purified antibodies against QPc-9.5 kDa have a high titer with isolated QPc-9.5 kDa protein and complexes that contain it. Although antibodies against QPc-9.5 kDa do not inhibit intact succinate- and ubiquinol-cytochrome c reductases, a decrease of 85% and 20% in restoration of succinate- and ubiquinol-cytochrome c reductases, respectively, is observed when delipidated succinate- or ubiquinol-cytochrome reductases are incubated with antibodies prior to reconstitution with ubiquinone and phospholipid, indicating that epitopes at the catalytic site of QPc-9.5 kDa are buried in the phospholipid environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

Cloning and sequencing of a cDNA encoding the Rieske iron-sulfur protein of bovine heart mitochondrial ubiquinol-cytochrome c reductase.

Two cDNA clones encoding bovine heart mitochondrial Rieske iron-sulfur protein were obtained by immunological screening of a bovine heart cDNA expression library in lambda gt11 with antiserum directed against Rieske iron-sulfur protein isolated from bovine heart mitochondrial ubiquinol-cytochrome c reductase. The cDNA inserts were 1005 and 1100 base pairs with an open reading frame of 807 base pairs which encoded a 196-amino acid mature Rieske iron-sulfur protein and a 73-amino acid presequence. The amino acid sequence of Rieske iron-sulfur protein deduced from nucleotide sequencing is the same as that obtained from protein sequencing except at residues #73 and #191 which are Ser and Asp instead of Ala and Gly, respectively.

Amino Acid Sequence↗

PubMed1990

Effect of substituents of the benzoquinone ring on electron-transfer activities of ubiquinone derivatives.

The effect of substituents on the 1,4-benzoquinone ring of ubiquinone on its electron-transfer activity in the bovine heart mitochondrial succinate-cytochrome c reductase region is studied by using synthetic ubiquinone derivatives that have a decyl (or geranyl) side-chain at the 6-position and various arrangements of methyl, methoxy and hydrogen in the 2, 3 and 5 positions of the benzoquinone ring. The reduction of quinone derivatives by succinate is measured with succinate-ubiquinone reductase and with succinate-cytochrome c reductase. Oxidation of quinol derivatives is measured with ubiquinol-cytochrome c reductase. The electron-transfer efficacy of quinone derivatives is compared to that of 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone. When quinone derivatives are used as the electron acceptor for succinate-ubiquinone reductase, the methyl group at the 5-position is less important than are the methoxy groups at the 2- and 3-positions. Replacing the 5-methyl group with hydrogen causes a slight increase in activity. However, replacing one or both of 2- and 3-methoxy groups with a methyl completely abolishes electron-acceptor activity. Replacing the 3-methoxy group with hydrogen results in a complete loss of electron-acceptor activity, while replacing the 2-methoxy with hydrogen results in an activity decrease by 70%, suggesting that the methoxy group at the 3-position is more specific than that at the 2-position. The structural requirements for quinol derivatives to be oxidized by ubiquinol-cytochrome c reductase are less strict. All 1,4-benzoquinol derivatives examined show partial activity when used as electron donors for ubiquinol-cytochrome c reductase. Derivatives that possess one unsubstituted position at 2, 3 or 5, with a decyl group at the 6-position, show substrate inhibition at high concentrations. Such substrate inhibition is not observed when fully substituted derivatives are used. The structural requirements for quinone derivatives to be reduced by succinate-cytochrome c reductase are less specific than those for succinate-ubiquinone reductase. Replacing one or both of the 2- and 3-methoxy groups with a methyl and keeping the 5-position unsubstituted (plastoquinone derivatives) yields derivatives with no acceptor activity for succinate-Q reductase. However, these derivatives are reducible by succinate in the presence of succinate-cytochrome c reductase. This reduction is antimycin-sensitive and requires endogenous ubiquinone, suggesting that these (plastoquinone) derivatives can only accept electrons from the ubisemiquinone radical at the Qi site of ubiquinol-cytochrome c reductase, and cannot accept electrons from the QPs of succinate-ubiquinone reductase.

Benzoquinones↗

PubMed1989

Purification and properties of isopenicillin N epimerase from Streptomyces clavuligerus.

Isopenicillin N epimerase, which catalyzes conversion of isopenicillin N to penicillin N, has been purified to electrophoretic homogeneity from the cell-free extract of Streptomyces clavuligerus by a procedure involving ammonium sulfate fractionation and chromatographies with DE-52, DEAE Affi-gel blue, Sephadex G-200, calcium phosphate-cellulose, and Mono Q. The purified epimerase is monomeric with a molecular weight of 47,000 or 50,000 as estimated by SDS-polyacrylamide gel electrophoresis or gel filtration, respectively. The enzyme contains 1 mol of pyridoxal 5'-phosphate per mol of protein, and shows absorption maxima at 280 and 420 nm. The epimerase catalyzes the complete 'racemization' on both the L-alpha-aminoadipyl side-chain of isopenicillin N and the D-alpha-aminoadipyl side-chain of penicillin N, so that an approximately equimolar mixture of the two penicillins is produced. The mixture is not truly racemic, since these penicillins are diastereomers rather than optical isomers. The chemical modification of primary amino groups of the epimerase by fluorescamine results in a great loss of the enzyme activity. The activity of purified enzyme is partially stimulated by the addition of sulfhydryl compounds. The activity is strongly inhibited by sulfhydryl group modifiers such as p-chloromercuribenzoate and N-ethylmaleimide.

Amino Acid Isomerases↗

PubMed1989

The Rhodospirillum rubrum cytochrome bc1 complex: peptide composition, prosthetic group content and quinone binding.

A cytochrome bc1 complex, essentially free of bacteriochlorophyll, has been purified from the photosynthetic purple non-sulfur bacterium Rhodospirillum rubrum. The complex catalyzes electron flow from quinol to cytochrome c (turnover number = 75 s-1) that is inhibited by low concentrations of antimycin A and myxothiazol. The complex contains only three peptide subunits: cytochrome b (Mr = 35,000); cytochrome c1 (Mr = 31,000) and the Rieske iron-sulfur protein (Mr = 22,400). Em values (pH 7.4) were measured for cytochrome c1 (+320 mV) and the two hemes of cytochrome b (-33 and -90 mV). Electron flow from quinol to cytochrome c is inhibited when the complex is pre-illuminated in the presence of a ubiquinone photoaffinity analog (azido-Q). During illumination, the azido-Q becomes covalently attached to the cytochrome b peptide and, to a lesser extent, to cytochrome c1.

Affinity Labels↗