Characterization of the thioredoxin system in the facultative phototroph Rhodobacter sphaeroides Y

Abstract

This paper reports the purification and characterization of a thioredoxin system (thioredoxin, thioredoxin reductase, NADPH) from the facultative phototroph Rhodobacter sphaeroides Y. R. sphaeroides 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. R. sphaeroides Y thioredoxin reductase was purified with the same assay using NADPH and E. coli thioredoxin. R. sphaeroides 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 intead of a Lys. R. sphaeroides Y thioredoxin contains two trytophan residues. The fluorescence intensity of the tryptophan residues is quenched in oxidized thioredoxin; on reduction, a much smaller increase is observed with R. sphaeroides Y thioredoxin than with the E. coli protein. However, the presence of 5 M guanidine .cntdot. HCl results in the complete exposure of the two tryptophan residues. R. sphaeroides 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 R. sphaeroides Y thioredoxin with a Km value of 3.3 .+-. 0.6 .mu.M. A kinetic study of the two thioredoxin systems shows that they have a high degree of cross-reactivity.