Characterization of the thioredoxin system in the facultative phototroph Rhodobacter sphaeroides Y.
This paper reports the purification and characterization of a thioredoxin system (thioredoxin, thioredoxin reductase, NADPH) from the facultative phototroph Rhodobacter sphaeroides Y. Rhodobacter sph. Y thioredoxin was purified to homogeneity with an assay based on the reduction of 5,5'-dithiobis(2-nitrobenzoic acid) by NADPH and Escherichia coli thioredoxin reductase. Rhodobacter sph. Y thioredoxin reductase was purified with the same assay using NADPH and E. coli thioredoxin. Rhodobacter sph. Y thioredoxin contained 102 amino acid residues and had a single intrachain disulfide bond. The two half-cystine residues are part of the active site made up of the sequence -Ala-Glu-Trp-Cys-Gly-Pro-Cys-Arg- which is identical to that of E. coli thioredoxin except for the presence of an Arg instead of a Lys. Rhodobacter sph. Y thioredoxin contains two tryptophan residues. The fluorescence intensity of the tryptophan residues is quenched in oxidized thioredoxin; on reduction, a much smaller increase is observed with Rhodobacter sph. Y thioredoxin than with the E. coli protein. However, the presence of 5 M guanidine X HCl results in the complete exposure of the two tryptophan residues. Rhodobacter sph. Y thioredoxin reductase has structural and functional similarities to E. coli thioredoxin reductase: it has a molecular mass of 68 kDa, and consists of two, probably identical, subunits. Each subunit has one bound FAD molecule. The enzyme is highly specific for NADPH; it is also highly specific for Rhodobacter sph. Y thioredoxin with a Km value of 3.3 +/- 0.6 microM. A kinetic study of the two thioredoxin systems shows that they have a high degree of cross-reactivity.
D000596:Amino Acids / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D009249:NADP / Q000302:isolation & purification; D012242:Rhodobacter sphaeroides / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D013880:Thioredoxin-Disulfide Reductase / Q000302:isolation & purification; D013879:Thioredoxins / Q000378:metabolism*
D000596:Amino Acids; D001426:Bacterial Proteins; D013879:Thioredoxins; D009249:NADP; D013880:Thioredoxin-Disulfide Reductase