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S I Chan

Publications and source records attributed to S I Chan.

At least 73 records · Page 4Linked to original sources
PubMed1989

Analysis of membrane lipids by 500 MHz 1H NMR.

A nondestructive method has been developed for rapid analysis of lipid content of membrane extracts based on high field proton NMR spectroscopy. Lipid extraction is done by stepwise sonication of purified membranes into chloroform:methanol:water mixtures, and 1H spectra are recorded at 35 degrees C on final preparations consisting of at least 1 mg dried lipid solubilized in 2:1 CD3OD:CDCl3. Spectral peaks of lipid mixtures are assigned to lipid classes using a data base of standard lipid characteristic resonances derived from purified single membrane lipids and known mixtures of them. Peak intensities of characteristic peaks yield ratios of various lipids such as cholesterol:phospholipid and phosphatidylcholine:phosphatidylethanolamine, degree of unsaturation, average acyl chain length, total glycerol lipid content, and presence or absence of particular lipids, such as glycolipids or lysolipids. This procedure of membrane lipid analysis has been verified using known mixtures of purified standard lipids.

Glycerol↗
PubMed1988

The CO adduct of yeast cytochrome c oxidase. Mössbauer and photolysis studies.

Mössbauer spectra of 57Fe-enriched NADH-reduced yeast cytochrome c oxidase reveal two quadrupole doublets of unequal intensity; one (approximately 33%) is typical of high-spin ferrous heme with histidine coordination and is assigned to heme a3, while the other (approximately 67%) is typical of low-spin heme with two nitrogeneous axial ligands as expected from heme a. The excess intensity (approximately 17%) of the low-spin doublet must therefore be assigned to heme a3 in a modified environment. The Mössbauer spectra of the same sample exposed to CO show that 50% of the heme iron forms a CO adduct, consistent with heme a3 being inhibited by CO. While low-spin hem a has the same Mössbauer parameters as in the reduced sample, its intensity has dropped to 35%. A distinctly new high-spin species (approximately 15%) is observed and assigned to heme a in a modified environment. The comparable size of the unexpected high-spin heme a fraction in the CO adduct and the low-spin heme a3 fraction in the reduced enzyme suggest that they arise from the same material. This material is likely to be the inactive fraction that has been found in all preparations of resting yeast cytochrome c oxidase (Siedow, J.N., Miller, S., and Palmer, G. (1981) J. Bioenerg. Biomembr. 14, 171-179). The kinetics of CO recombination following photolysis of the CO complex further confirms the coexistence of two distinct fractions associated with active and inactive protein. The majority (approximately 74%), presumably active protein, recombines exponentially from 160 to 270 K following an Arrhenius law. The large activation enthalpy, delta H approximately 35 kJ/mol, is comparable to that found in the beef heart enzyme, suggesting that the flashed-off CO is bound by the nearby CuB as in the mammalian system (Fiamingo, F.G., Altschuld, R.A., Moh, P.P., and Alben, J.O. (1982) J. Biol. Chem. 250, 1639-1650). In the minority, presumably inactive, fraction the CO recombination has fast nonexponential kinetics with a distribution of activation enthalpies peaking near delta Hp = 13 kJ/mol reminiscent of CO binding to myoglobin. In this inactive fraction CuB is apparently not accessible to the flashed-off CO.

Algorithms↗
PubMed1988

Conversion of CuA to a type II copper in cytochrome c oxidase.

When cytochrome c oxidase is incubated at 43 degrees C for approximately 75 min in a solution containing the zwitterionic detergent sulfobetaine 12, the CuA site is converted into a type II copper as judged by changes in the 830-nm absorption band and the EPR spectrum of the enzyme. SDS-PAGE and sucrose gradient ultracentrifugation indicate concomitant loss of subunit III and monomerization of the enzyme during the heat treatment. Comparison of the optical and resonance Raman spectra of the heat-treated and native protein shows that the heme chromophores are not significantly perturbed; the resonance Raman data indicate that the small heme perturbations observed are limited to the cytochrome a3 site. Proton pumping measurements, conducted on the modified enzyme reconstituted into phospholipid vesicles, indicate that these vesicles are unusually permeable toward protons during turnover, as previously reported for the p-(hydroxymercuri)benzoate-modified oxidase and the modified enzyme obtained by heat treatment in lauryl maltoside. The sulfobetaine 12 modified enzyme is no longer capable of undergoing the recently reported conformational transition in which the tryptophan fluorescence changes upon reduction of the low-potential metal centers. Control studies on the monomeric and subunit III dissociated enzymes suggest that the disruption of this conformational change in the heat-treated oxidase is most likely associated with perturbation of the CuA site. These results lend support to the suggestion that the fluorescence-monitored conformational change of the native enzyme is initiated by reduction of the CuA site [Copeland et al. (1987) Biochemistry 26, 7311].

Animals↗
PubMed1988

Heat treatment of cytochrome c oxidase perturbs the CuA site and affects proton pumping behavior.

It has been previously reported that mild heat treatment (43 degrees C for ca. 60 min) abolishes the proton pumping activity of cytochrome c oxidase while leaving the oxidase activity and cytochromes a and a3 unperturbed [Sone, N., & Nicholls, P. (1984) Biochemistry 23, 6550-6554]. We herein describe the effects of this heat treatment on the electron paramagnetic resonance (EPR) and optical absorption signatures of the redox-active metal centers in the enzyme. We find that heat treatment of the oxidized enzyme causes a local structural perturbation at the CuA site. After heat treatment, the enzyme sample contains three subpopulations, each of which has a different structure at CuA. These include (i) native CuA, (ii) a type 2 copper species similar to the one produced by chemical modification by p-(hydroxymercuri)benzoate (pHMB) [Gelles, J., & Chan, S. I. (1985) Biochemistry 24, 3963-3972], and (iii) a novel type 1 copper species. In addition to changes at the CuA site, we find that heat treatment results in accelerated cyanide binding and the removal of subunit III. If the cytochrome c oxidase is heat treated while fully reduced, none of these changes are observed except for subunit III depletion. Furthermore, partial (CO mixed-valence derivative) reduction of the enzyme as well as ligand binding to cytochrome a3 also protects the enzyme against the heat-induced changes, indicating that the oxygen binding site plays a role in stabilizing the CuA site against structural perturbations.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1988

Heterogeneity in an isolated membrane protein. Has the 'authentic cytochrome oxidase' been identified?

The criteria of homogeneity or native state of a protein are prone to become ambiguous when applied to membrane proteins, such as cytochrome-c oxidase, which are purified by extraction with detergents. Properties of the purified material depend on the detergent used and on details of the purification protocol followed with any single batch of a preparation. We present arguments to show that the evidence presently available in published form does not justify the designation [(1987) J. Biol. Chem. 262, 3160-3164] of one type of preparation as being closer to the native state than others.

Cell Membrane↗
PubMed1988

NMR study of the interactions of polymyxin B, gramicidin S, and valinomycin with dimyristoyllecithin bilayers.

The interactions of three polypeptide antibiotics (polymyxin B, gramicidin S, and valinomycin) with artificial lecithin membranes were studied by nuclear magnetic resonance (NMR). Combination of 31P and 2H NMR allowed observation of perturbations of the bilayer membrane structure induced by each of the antibiotics in the regions of the polar headgroups and acyl side chains of the phospholipids. The comparative study of the effects of these membrane-active antibiotics and the lipid bilayer structure demonstrated distinct types of antibiotic-membrane interactions in each case. Thus, the results showed the absence of interaction of polymyxin B with the dimyristoyllecithin membranes. In contrast, gramicidin S exhibited strong interaction with the lipid above the gel to liquid-crystalline phase transition temperature: disordering of the acyl side chains was evident. Increasing the concentration of gramicidin S led to disintegration of the bilayer membrane structure. At a molar ratio of 1:16 of gramicidin S to lecithin, the results are consistent with coexistence of gel and liquid-crystalline phases of the phospholipids near the phase transition temperature. Valinomycin decreased the phase transition temperature of the lipids and increased the order parameters of the lipid side chains. Such behavior is consistent with penetration of the valinomycin molecule into the interior of the lipid bilayers.

Dimyristoylphosphatidylcholine↗
PubMed1988

On the nature of cysteine coordination to CuA in cytochrome c oxidase.

The resolution of new features in the 1H electron nuclear double resonance (ENDOR) spectrum of the oxidized CuA site in beef heart cytochrome c oxidase is presented. In a previous study, we assigned resonances in the CuA ENDOR spectrum to hyperfine interactions of methylene protons on one or two cysteine ligands to CuA (Stevens, T.H., Martin, C.T., Wang, H., Brudvig, G.W., Scholes, C.P., and Chan, S.I. (1982) J. Biol. Chem. 257, 12106-12113). In this work, a new 1H ENDOR resonance in the beef heart CuA ENDOR spectrum is reported and can be assigned to either anisotropy in a previously resolved cysteine methylene proton hyperfine interaction (Aiso = 12 MHz, Aaniso = 2.5 MHz) or to a third isotropic hyperfine coupling (A = 13.6 MHz) to a cysteine methylene proton of a second cysteine ligand to copper. In either case, the 1H ENDOR results require the delocalization of approximately 50% of the unpaired spin from copper onto either one or two cysteine ligands to CuA. To characterize further the CuA site, we have prepared yeast cytochrome c oxidase incorporating isotopically substituted [beta-13C]cysteine. The CuA ENDOR spectrum of this species shows only one clearly resolved 13C hyperfine interaction (A = 3.6 MHz). This result confirms the assignment of at least one strongly interacting cysteine ligand to CuA and suggests that if the assignment of two cysteine ligands to CuA is correct, the two cysteines interact with copper in a highly symmetric manner. A recent extended x-ray absorption fine structure study of native and modified forms of cytochrome c oxidase indicates the coordination of two sulfur ligands to CuA (Li, P.M., Gelles, J., Chan, S.I., Sullivan R.J., and Scott, R.A. (1987) Biochemistry 26, 2091-2095). In light of the new possibility of two symmetrically coordinated cysteine ligands to CuA, we propose a molecular orbital description of the oxidized CuA site which is characterized by a high degree of delocalization of unpaired spin away from copper and onto a pair of symmetrically coordinated cysteine sulfur ligands. We also present a protein model for the CuA site in which two cysteine ligands derived from subunit II lie on the face of an alpha-helix. This structure would allow the unprecedented stable coordination of two cysteine thiolate sulfurs to copper and may provide a mechanism for the redox-linked proton pumping by cytochrome c oxidase.

Copper↗
PubMed1988

pH dependence of the tryptophan fluorescence in cytochrome c oxidase: further evidence for a redox-linked conformational change associated with CuA.

When the low-potential metal centers of cytochrome c oxidase are reduced, the enzyme undergoes a conformational transition that shifts the fluorescence maximum of the emitting tryptophan residues from 329 to 345 nm. At pH 7.4, the change in this tryptophan fluorescence intensity is a nonlinear function of the electron equivalents added to the cyanide-inhibited enzyme. This nonlinear behavior is a result of the difference in redox potential between cytochrome a and CuA, which, at equilibrium, favors electron occupancy at cytochrome a. Studies on the cyanide-inhibited enzyme suggest that the conformational change is associated with reduction of CuA [Copeland, R. A., Smith, P. A., & Chan, S. I. (1987) Biochemistry 26, 7311-7316]. In this work we present tryptophan fluorescence data for the cyanide-inhibited enzyme at pH 8.9. Because of the pH dependence of the midpoint potential of cytochrome a in this form of the enzyme, the two low-potential centers become virtually isopotential at pH 8.9. The results obtained confirm our earlier conclusion that the observed conformational change is linked to the reduction of CuA only, rather than to the redox activity of both low-potential metal centers. We find that, in partially reduced cyanide-inhibited oxidase, raising the pH from 7.4 to 8.9 results in an intensification and red shift of the enzyme's tryptophan emission as the electron occupancy redistributes from cytochrome a to CuA. Moreover, when the fluorescence change is plotted as a function of the number of electrons added to the enzyme at pH 8.9, the data fit the nearly linear function expected for a conformational change triggered by reduction of CuA exclusively.

Animals↗
PubMed1988

Chemical modification of the CuA site affects the proton pumping activity of cytochrome c oxidase.

Cytochrome c oxidase in which the CuA site has been perturbed by extensive modification of the enzyme with the thiol reagent p-(hydroxymercuri)benzoate has been reconstituted into phospholipid vesicles. The reconstituted vesicles lack respiratory control, and the orientation of the enzyme in the vesicles is similar to that of the native cytochrome c oxidase. In the proton translocation assay, the vesicles containing the modified enzyme behave as if they are unusually permeable to protons. When the modified and native proteins were coreconstituted, a substantial portion of the latter became uncoupled as revealed by low respiratory control and low overall proton pumping activity. These results suggest that the modified enzyme catalyzes a passive transport of protons across the membrane. When milder conditions were used for the chemical modification, a majority of the thiols reacted while the CuA site remained largely intact. Reconstitution of such a partially modified cytochrome c oxidase produced vesicles with respiratory control and proton translocating activity close to those of reconstituted native enzyme. It thus appears that the appearance of a proton leak is related to the perturbation of the CuA site. These observations suggest that the structure of CuA may be related to the role of this site in the proton pumping machinery of cytochrome c oxidase.

Animals↗
PubMed1988

A proposal for the site and mechanism of redox-linked proton translocation in cytochrome c oxidase.

In this paper, we present a detailed mechanistic model for the redox-linked proton pump of cytochrome c oxidase. Using the spectroscopic properties of CuA, we have constructed a molecular orbital model for the electronic structure of the CuA site. One prediction of this model is that a ligand rearrangement should occur upon reduction of CuA. We further describe a detailed mechanism for proton pumping which utilizes this redox-linked CuA ligand rearrangement as its central feature. The turnover cycle of this proton pump model is then discussed in terms of the theoretical thermodynamic and kinetic requirements of a redox-linked proton pump.

Binding Sites↗
PubMed1987

Cytochrome c oxidase exhibits a rapid conformational change upon reduction of CuA: a tryptophan fluorescence study.

When cytochrome c oxidase is reduced, it undergoes a conformational change that shifts its tryptophan fluorescence maximum from 329 to 345 nm. Studies of ligand-bound, mixed-valence forms of the enzyme show that this conformational change is dependent on the redox state of the low-potential metal centers, cytochrome a and CuA. The intrinsic fluorescence of oxidized cytochrome c oxidase is not effectively quenched by Cs+; however, marked quenching is observed for the reduced enzyme with a Stern-Volmer constant of 0.69. These observations, together with the significant red shift of the emission maximum, suggest that the emitting tryptophan residues are becoming more solvent accessible in the reduced enzyme. Stopped-flow spectra show that this conformational transition occurs rapidly upon reduction of the low-potential sites with a pseudo-first-order rate constant of 4.07 +/- 0.40 s-1. The conformational change monitored by tryptophan fluorescence is suggested to be related to the previously proposed "open-closed" transition of cytochrome c oxidase. Reductive titration of the cyanide-inhibited enzyme with ferrocytochrome c shows a nonlinear response of the fluorescence shift to added electron equivalents. A theoretical treatment of the reduction of the two interacting sites of the cyanide-inhibited enzyme has been developed that gives the population of each redox state as a function of the total number of electrons accepted by the enzyme. This treatment depends on two parameters: the difference in redox potential between the two metals and the redox interaction between the redox centers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Extended X-ray absorption fine structure of copper in CuA-depleted, p-(hydroxymercuri)benzoate-modified, and native cytochrome c oxidase.

Cytochrome c oxidase contains four redox-active metal centers: two heme irons, cytochromes a and a3, and two copper ions, CuA and CuB. Due to the paucity of spectroscopic signatures for both copper sites in cytochrome c oxidase, the ligands and structures for these sites have remained ambiguous. The specific depletion of CuA from the p-(hydroxymercuri)benzoate- (pHMB-) modified cytochrome c oxidase recently reported [Gelles, J., & Chan, S. I. (1985) Biochemistry 24, 3963-3972] is herein described. Characterization of this enzyme shows that the structures of the remaining metal centers are essentially unperturbed by the CuA modification and depletion (P. M. Li, J. Gelles, and S. I. Chan, unpublished results). Copper extended X-ray absorption fine structure (EXAFS) measurements on the CuA-depleted cytochrome c oxidase reveal coordination of three (N, O) ligands and one (S, Cl) ligand at the CuB site. Comparison of EXAFS results obtained for the CuA-depleted, pHMB-modified, and "unmodified control" enzymes has allowed the deconvolution of the EXAFS in terms of the inner coordination spheres for CuA as well as CuB. On the basis of these data, it is found that the structure for the CuA site is consistent with two (N, O) ligands and two S ligands.

Copper↗
PubMed1987

Evidence for a ferryl Fea3 in oxygenated cytochrome c oxidase.

Evidence is reported which shows that a reactive ferryl Fea3/cupric CuB binuclear couple is present at the dioxygen reduction site in "oxygenated" cytochrome c oxidase; when the fully reduced enzyme is reoxidized at low temperatures; and when partially reduced cytochrome c oxidase is reoxidized with dioxygen at room temperature.

Animals↗
PubMed1987

Effect of chlorophyll a on the phase behavior of hydrated monogalactosyldiacylglycerols.

We have studied the effect of chlorophyll a (chl a) on the X-ray diffraction patterns and the appearance of freeze-fracture electron micrographs of aqueous dispersions of monogalactosyldiacylglycerols (MGDG), the most abundant lipid in the thylakoid membrane. In MGDG systems containing 0-18 mol% of chl a, the diffraction patterns indicate the presence of a well-ordered reverse hexagonal phase. When 30 mol% of chl a was incorporated into the MGDG, the low-angle X-ray diffraction lines of the hexagonal lattice were slightly broadened and were accompanied by additional weak lines. With higher mol percents of chl a, the low-angle lines could no longer be indexed on a hexagonal or lamellar lattice. The freeze-fracture electron micrographs of similar samples showed that the patterns characteristic of the reverse hexagonal phase of an aqueous dispersion of pure MGDG were replaced by large liposomes, the fracture pattern of which is circular. We conclude that chl a in excess of 20 mol% destabilized the orderly reverse hexagonal phase of aqueous MGDG dispersions and disturbed the long-range order of the lipid array. These results are summarized in a temperature-composition isobaric phase diagram over a temperature range of -60 degrees C to 60 degrees C.

Chlorophyll↗
PubMed1986

Molecular mechanisms of band 3 inhibitors. 1. Transport site inhibitors.

The band 3 protein of red cells is a transmembrane ion transport protein that catalyzes the one-for-one exchange of anions across the cell membrane. 35Cl NMR studies of Cl- binding to the transport sites of band 3 show that inhibitors of anion transport can be grouped into three classes: (1) transport site inhibitors (examined in this paper), (2) channel-blocking inhibitors (examined in the second of three papers in this issue), and (3) translocation inhibitors (examined in the third of three papers in this issue). Transport site inhibitors fully or partially reduce the affinity of Cl- for the transport site. The dianion 4,4'-di-nitrostilbene-2,2'-disulfonate (DNDS) and the arginine-specific reagent phenylglyoxal (PG) each completely eliminate the transport site 35Cl NMR line broadening, and each compete with Cl- for binding. These results indicate that DNDS and PG share a common inhibitory mechanism involving occupation of the transport site: one of the DNDS negative charges occupies the site, while PG covalently modifies one or more essential positive charges in the site. In contrast, 35Cl NMR line broadening experiments suggest that 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) leaves the transport site partially intact so that the affinity of Cl- for the site is reduced but not destroyed. This result is consistent with a picture in which DIDS binds near the transport site and partially occupies the site.

Anion Exchange Protein 1, Erythrocyte↗