Exploring the conformational equilbrium of E. coli thioredoxin reductase: Characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy.

P.A.W. van den Berg, S.B. Mulrooney, B. Gobets, I.H.M. van Stokkum, A. van Hoek, C.H. Williams Jr, A.J.W.G. Visser

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

The conformational dynamics of wild-type Escherichia coli thioredoxin reductase (TrxR) and the mutant enzyme C138S were studied by ultrafast time-resolved fluorescence of the flavin cofactor in combination with circular dichroism (both in the flavin fingerprint and far-UV regions) and steady-state fluorescence and absorption spectroscopy. The spectroscopic data show two conformational states of the enzyme (named FO and FR), of which the physical characteristics differ considerably. Ultrafast fluorescence lifetime measurements make it possible to distinguish between the two different populations: Dominant picosecond lifetimes of ∼1 ps (contribution 75%) and 7 ps (8%) are associated with the FO species in TrxR C138S. Long-lived fluorescence with two time constants in the range of 0.2 - 1 ns (total contribution 17%) originates from enzyme molecules in the FR conformation. The near absence of fast lifetime components in oxidized wild-type TrxR supports the idea of this enzyme being predominantly in the FR conformation. The emission spectrum of the FO conformation is blue-shifted with respect to that of the FR conformation. Because of the large difference in fluorescence characteristics, fluorescence measurements on time scales longer than 100 ps are fully determined by the fraction of enzyme molecules in the FR conformation. Binding of the thiol reagent phenyl mercuric acetate to wild-type enzyme and TrxR C138S stabilizes the enzymes in the FR conformation. Specific binding of the NADPH-analog, AADP
Original languageEnglish
Pages (from-to)2037-2049
JournalProtein Science
Volume10
DOIs
Publication statusPublished - 2001

Fingerprint

Dive into the research topics of 'Exploring the conformational equilbrium of E. coli thioredoxin reductase: Characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy.'. Together they form a unique fingerprint.

Cite this