Biomedical subjects

J E Draheim

Publications and source records attributed to J E Draheim.

9 recordsLinked to original sources

PubMed1992

Thermal denaturation of plastocyanin: the effect of oxidation state, reductants, and anaerobicity.

The thermal stability of plastocyanin (PC) was determined as a function of oxidation state of the copper center and the presence of oxidants, reductants, oxygen, and EDTA. It was found that the copper center and its ligands play a crucial role in maintaining the stability of PC. Thermal denaturation was monitored by using far-uv circular dichroism (CD) spectra to monitor changes in secondary structure, the near-uv CD ellipticity at 280 nm to monitor changes in tertiary structure, and the absorbance at 597 nm and the 255-nm CD transition to monitor changes in the copper center. Reduced PC (Tm = 71 degrees C) was found to be more stable than the oxidized form (Tm = 61 degrees C). The Tm was increased by addition of reductants, removal of oxygen, or addition of EDTA. Two distinct denatured forms (designated D1 and D2) were separated by anion exchange fast protein liquid chromatography. Neither form contained a native copper center. Form D2 retained the characteristic 280-nm CD band but showed an altered far-uv CD spectrum. Its formation was inhibited by the addition of reductants or the removal of oxygen. It could be refolded to form native, Cu-PC upon incubation with copper plus a reductant such as dithionite. These results suggest that its formation involves the reversible oxidation of a group on the PC molecule, possibly a ligand to the copper such as Cys 84 or Met 92. Form D1 occurred in the presence of ferricyanide or at high temperatures in the presence of oxygen. EDTA inhibited its formation. Form D1 lost the 280-nm CD transition and its far-uv CD spectrum was altered. No renaturation was observed suggesting that Form D1 is the product of an irreversible oxidation step possibly involving a histidine ligand to the copper. Forms D1 and D2 are not interconvertible and represent the endpoints of two different denaturation pathways.

Anaerobiosis↗

PubMed1991

Dramatic in situ conformational dynamics of the transmembrane protein bacteriorhodopsin.

The conformational dynamic capabilities of the in situ bacteriorhodopsin (bR) can be studied by determination of the changes of the bR net helical segmental tilt angle (the angle between the polypeptide segments and the membrane normal) induced by various perturbations of the purple membrane (PM). The analysis of the far-UV oriented circular dichroism (CD) of the PM provides one means of achieving this. Previous CD studies have indicated that the tilt angle can change from approximately 10 degrees to 39 degrees depending on the perturbants used with no changes in the secondary structure of the bR. A recent study has indicated that the bleaching-induced tilt angle can be enhanced from approximately 24 degrees to 39 degrees by cross-linkage and papain-digestion perturbations which by themselves do not alter the tilt angle. To add further credence, this study has been repeated using midinfrared (IR) linear dichroic spectral analysis. In contrast to the CD method, analysis by the IR method depends on the orientation of the amide plane of the helix assumed. Excellent consistency is achieved between the two methods only when it is assumed that the structural characteristics of the alpha-helices of the bR are equally alpha I and alpha II in nature. Furthermore, the analysis of the IR data becomes essentially independent of the three amide transitions utilized. The net tilt angle of segments completely randomized relative to the incident light must be 54.736 in view of helix symmetry. A value of 54.735 degrees +/- 0.001 degree was achieved by the IR method for the ethanol-treated PM film, establishing this kind of film as an ideal random state standard and demonstrating the accuracy potential of the IR method.

Bacteriorhodopsins↗

PubMed1989

Lipoxygenase isoenzymes: a spectroscopic and structural characterization of soybean seed enzymes.

Applying recent developments in protein purification techniques, a number of lipoxygenase isoenzymes have been isolated in satisfactory quantities for a detailed physical and structural characterization. Four seed isoenzymes from two soybean cultivars have been studied by peptide mapping, free thiol and iron content determinations, and C-terminal analysis as well as by uv-visible absorption and EPR spectroscopy. While differences between the type 1 enzyme and the other isoenzymes were readily detected using proteolytic peptide mapping, digestion with dilute hydrochloric acid and C-terminal analysis both revealed structural features which were similar in all of the isoenzymes. One clear difference between the lipoxygenases was in their free sulfhydryl group content. The uv-visible absorption spectrum of each native isoenzyme was consistent with expectations for the experimental aromatic amino acid content. All of the isoenzymes contained one non-heme iron atom per molecule of protein. The oxidation of each isoenzyme with product hydroperoxide resulted in the conversion of the iron from an EPR silent state into several forms with EPR signals characteristic of high spin iron(III). The EPR spectra of all isoenzymes were remarkably similar. A time course EPR and catalytic activity study revealed that the various EPR active states represent a complex equilibrium between iron atoms in different environments. The pH dependence for the EPR and absorption spectroscopy lends support to the hypothesis that acid/base chemistry represents an important aspect of lipoxygenase catalysis.

Catalysis↗

PubMed1988

Analysis of the near-ultraviolet absorption and circular dichroic spectra of parsley plastocyanin for the effects of pH and copper center conformation changes.

The absorption and circular dichroic (CD) spectra of parsley plastocyanin (PC) were measured in order to determine the effects of changes in primary amino acid sequence on both the copper center and protein components of the PC molecule. The near-ultraviolet (uv) absorption and CD spectra of parsley PC were found to be qualitatively similar to those of spinach, poplar, and lettuce PC, except for the near-uv CD spectrum of the reduced form at low pH (ca. pH 5.0). The CD spectrum of reduced parsley PC in the 250-265 nm wavelength region changes from positive to negative ellipticity upon reduction of pH, and is characterized by a pKa value of 5.7. This pKa value is the same as that for the protonation of the histidine 87 copper ligand, observed by NMR, and the change in conformation of the copper center. Similar processes are believed to occur in the other PC species at lower pH values. Thus, the pH-dependent perturbations of the near-uv CD spectra of reduced PC are interpreted as due to transitions in the reduced copper center. The increase in the near-uv absorption spectrum of reduced PC can be divided into pH-independent and pH-dependent portions. The pH-independent portion resembles the absorption spectrum of tetrahedral Cu(I) metallothionein, suggesting the presence of Cu(I)-Cys 84 and/or Cu(I)-Met 92 charge transfer transitions in the near-uv absorption spectra of reduced PC. The pH dependence of the absorption spectrum changes and the pH difference absorption spectrum indicate that tyrosine residues may contribute to at least a part of the pH-dependent portion of the absorption increase of reduced PC.

Chlorophyll↗

PubMed1986

Plastocyanin conformation. An analysis of its near ultraviolet absorption and circular dichroic spectra.

The near-ultraviolet absorption and circular dichroic spectra of plastocyanin are dependent upon the redox state, solution pH, and ammonium sulfate concentration. This dependency was observed in plastocyanin isolated from spinach, poplar, and lettuce. Removal of the copper atom also perturbed the near-ultraviolet spectra. Upon reduction there are increases in both extinction and ellipticity at 252 nm. Further increases at 252 nm were observed upon formation of apo plastocyanin eliminating charge transfer transitions as the cause. The spectral changes in the near-ultraviolet imply a flexible tertiary conformation for plastocyanin. There are at least two charge transfer transitions at approximately 295-340 nm. One of these transitions is sensitive to low pH's and is attributed to the His 87 copper ligand. The redox state dependent changes observed in the near-ultraviolet spectra of plastocyanin are attenuated either by decreasing the pH to 5 or by increasing the ammonium sulfate concentration to 2.7 M. This attenuation cannot be easily explained by simple charge screening. Hydrophobic interactions probably play an important role in this phenomenon. The pH and redox state dependent conformational changes may play an important role in regulating electron transport.

Circular Dichroism↗

PubMed1985

Plastocyanin conformation: the effect of the oxidation state on the pKa of nitrotyrosine-83.

Plastocyanin treated with tetranitromethane was nitrated at a single location, Tyr-83. Tyr-83 and its neighboring negative charges have been implicated as a binding site for positively charged redox agents (Chapman, S.K., Watson, A.D. and Sykes, A.G. (1983) J. Chem. Soc. Dalton Trans. 1983, 2543-2548). No effect was observed on either the plastocyanin midpoint redox potential or its reaction kinetics with P-700+ and cytochrome f. This makes nitration an ideal spectroscopic probe for monitoring changes in the environment of Tyr-83. The pKa of the nitrotyrosine was 8.6 and 8.3 for reduced and oxidized plastocyanin, respectively, indicating that the charge on the copper atom is 'felt' at Tyr-83. The high pKa value for both forms indicates that Tyr-83 is in a negatively charged environment, near residues Nos. 42-45 and Nos. 59-61. The extinction of the nitrotyrosine chromophore at 360 nm was not affected by a change in redox state. However, the ellipticity of this transition was greater for the oxidized form, indicating that environment of Tyr-83 is dependent upon the charge on the copper atom. This suggests an electrostatically driven conformational change at Tyr-83. A conformational change at Tyr-83 could regulate the binding of plastocyanin with its reaction partners in order to promote smooth electron transport.

Chlorophyll↗

PubMed1985

Plastocyanin conformation. The effect of nitrotyrosine modification and pH.

Plastocyanin isolated from several species including spinach, poplar, and lettuce showed conformational changes both upon reduction and upon lowering the pH as determined by near-ultraviolet absorption and fluorescence measurements. The fluorescence excitation maximum was at 278 nm for all species of plastocyanin measured. In the case of spinach, the emission maximum was at 310-312 nm, similar to a tyrosine residue in solution. The fluorescence intensity increased 22% upon reduction of plastocyanin at pH 7.0. In poplar plastocyanin, the emission maximum was shifted to 335 nm and increased only 10% upon reduction. The 335 nm emission peak observed in poplar plastocyanin is attributed to Tyr 80 which is hydrogen bonded to a carbonyl group on the protein backbone. Tyr 83 was also shown to undergo fluorescence changes upon reduction since the redox state-dependent fluorescence changes decreased for a nitrotyrosine (nitrotyrosine-plastocyanin) derivative of this residue. These results show that the east face of the molecule, which contains both Tyr 80 and 83 as well as a possible binding site, undergoes conformational changes upon reduction. These conformational changes may be involved in promoting smooth electron transport between plastocyanin and its reaction partners. Both the absorption and fluorescence were found to be pH dependent. The quantum yield for fluorescence increased sharply below pH 6 for both oxidized and reduced spinach plastocyanin. This may be related to the appearance of a redox-inactive form of reduced plastocyanin. The conformational changes observed at low pH may provide a mechanism for control of electron transport by the proton gradient. Low concentrations of CaCl2 (10 mM) had no effect on plastocyanin fluorescence. However, addition of 2.7 M (NH4)2SO4 eliminated the redox-dependent fluorescence changes.

Chloroplasts↗

PubMed1985

Conformational changes in plastocyanin.

The visible and near-uv absorption and circular dichroic spectra were determined for spinach and poplar plastocyanin under a variety of conditions. The visible spectra showed that the copper center was invariant to changes in species, chemical modification with ethylenediamine, and addition of high concentrations of salt [2.7 M (NH4)2SO4]. In contrast, the near-uv spectra were sensitive to these conditions. Reduction of plastocyanin also altered its near-uv absorption and circular dichroic spectra. It is unlikely that these spectral changes were due to charge transfer bands since the near-uv CD spectrum of apo-plastocyanin was almost identical to that of reduced plastocyanin. There were no corresponding changes in the far-uv spectra which monitor protein secondary structure. The most likely explanation is that the protein has a flexible tertiary conformation. Conformational changes may be important in regulating electron transport. If plastocyanin is a mobile electron carrier, differential binding of the oxidized and reduced forms of plastocyanin to its reaction partners cytochrome f and P700 could facilitate electron transport.

Apoproteins↗

PubMed1984

Variable fluorescence of photosystem I particles and its application to the study of the structure and function of photosystem I.

Chlorophyll a fluorescence in Photosystem I (PSI) particles isolated according to the method of Bengis and Nelson [J. Biol. Chem. 252, 4564-4569 (1977)] was found to be dependent on the redox state of both P700 and X (an acceptor on the reducing side of PSI). Addition of dithionite plus neutral red to PSI caused an increase in fluorescence intensity and a shift of the main fluorescence peak from 689 to 674 nm. Addition of electron acceptors such as ferredoxin and methyl viologen decreased the fluorescence yield when added to PSI incubated under anaerobic conditions in the presence of excess dichlorophenol indophenol (DCIPH2). The Km for ferredoxin agreed with that determined from direct measurements of ferredoxin reduction, showing that X is a quencher of fluorescence. P700 was also found to be a quencher of fluorescence, since electron donors such as DCIPH2, TMPD, and plastocyanin decreased fluorescence with Km's nearly identical to those observed for P700+ reduction. Chemical modification of PSI (with ethylene diamine + a water-soluble carbodiimide) to make it positively charged increased the fluorescence yield and shifted the 689-nm peak to 674 nm. The Km's for DCIPH2 and ferredoxin were decreased. In contrast, modification of PSI with succinic anhydride, which increased the net negative charge, increased the Km for ferredoxin. Salts affected the interaction of methyl viologen with PSI. Both anion and cation selectivity were observed. Limited proteolysis increased the Km for both methyl viologen and ferredoxin, indicating that their binding site on PSI was altered. These results suggest that the binding site for ferredoxin is on either the 70- or the 20-kDa subunit of PSI.

Chlorophyll↗