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The reduction of Escherichia coli thioredoxin by thioredoxin reductase was studied by stopped-flow spectrophotometry. The reaction showed no dependence on thioredoxin concentration, indicating that complex formation was rapid and occurred during the dead time of the instrument. The kobs for the reaction of approximately 20 s-1 probably reflects the rate of electron transfer from thioredoxin reductase to thioredoxin and agrees with the kcat observed by steady-state kinetics. The reaction rate was unaffected by increasing the ionic strength, suggesting a lack of electrostatic stabilization in the interaction of the two proteins. A mutant thioredoxin in which a positively charged lysine in the active-site region was changed to a glutamic acid residue resulted in an electrostatic destabilization. Thioredoxin K36E was still a substrate for the reductase, but binding was impaired so that the rate could be measured by stopped-flow techniques as reflected by a dependence on protein concentration. Raising the ionic strength in this reaction served to shield the negative charge and increased the rate of binding to the reductase.
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