Thioredoxin reductase from Escherichia coli: evidence of restriction to a single conformation upon formation of a crosslink between engineered cysteines.
Thioredoxin reductase is a flavoprotein which catalyzes the reduction of the small protein thioredoxin by NADPH. It contains a redox active disulfide and an FAD in each subunit of its dimeric structure. Each subunit is further divided into two domains, the FAD and the pyridine nucleotide binding domains. The orientation of the two domains determined from the crystal structure and the flow of electrons determined from mechanistic studies suggest that thioredoxin reductase requires a large conformational change to carry out catalysis (Williams CH Jr, 1995, FASEB J 9:1267-1276). The constituent amino acids of an ion pair, E48/R130, between the FAD and pyridine nucleotide binding domains, were mutagenized to cysteines to form E48C,R130C (CC mutant). Formation of a stable bridge between these cysteines was expected to restrict the enzyme largely in the conformation observed in the crystal structure. Crosslinking with the bifunctional reagent N,N,1,2 phenylenedimaleimide, spanning 4-9 A, resulted in a >95 % decrease in thioredoxin reductase and transhydrogenase activity. SDS-PAGE confirmed that the crosslink in the CC-mutant was intramolecular. Dithionite titration showed an uptake of electrons as in wild-type enzyme, but anaerobic reduction of the flavin with NADPH was found to be impaired. This indicates that the crosslinked enzyme is in the conformation where the flavin and the active site disulfide are in close proximity but the flavin and pyridinium rings are too far apart for effective electron transfer. The evidence is consistent with the hypothesis that thioredoxin reductase requires a conformational change to complete catalysis.
Veine
D M
DM
Department of Veterans Affairs Medical Center, Ann Arbor, Michigan 48105, USA.
Ohnishi
K
K
Williams
C H
CH
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry* / Q000235:genetics
D003432:Cross-Linking Reagents; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine