Structure and mechanism of mammalian thioredoxin reductase: The active site is a redox-active selenolthiol/selenenylsulfide formed from the conservedcysteine-selenocysteine sequence
Lw. Zhong et al., Structure and mechanism of mammalian thioredoxin reductase: The active site is a redox-active selenolthiol/selenenylsulfide formed from the conservedcysteine-selenocysteine sequence, P NAS US, 97(11), 2000, pp. 5854-5859
Citations number
38
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Mammalian thioredoxin reductases (TrxR) are homodimers, homologous to gluta
thione reductase (CR), with an essential selenocysteine (SeCys) residue in
an extension containing the conserved C-terminal sequence -Gly-Cys-SeCys-Gl
y, In the oxidized enzyme, we demonstrated two nonflavin redox centers by c
hemical modification and peptide sequencing: one was a disulfide within the
sequence -Cys(59)-Val-Asn-Val-Gly-Cys(64), identical to the active site of
CR; the other was a selenenylsulfide formed from Cys(497)-SeCys(498) and c
onfirmed by mass spectrometry. In the NADPH reduced enzyme, these centers w
ere present as a dithiol and a selenolthiol, respectively. Based on the str
ucture of GR, we propose that in TrxR, the C-terminal Cys(497)-SeCys(498) r
esidues Of one monomer are adjacent to the Cys(59) and Cys(64) residues of
the second monomer, The reductive half-reaction of TrxR is similar to that
of GR followed by exchange from the nascent Cys59 and Cys64 dithiol to the
selenenylsulfide of the other subunit to generate the active-site selenolth
iol. Characterization of recombinant mutant rat TrxR with SeCys(498) replac
ed by Cys having a 100-fold lower k(cat) for Trx reduction revealed the C-t
erminal redox center was present as a dithiol when the Cys(59)-Cys(64) was
a disulfide, demonstrating that the selenium atom with its larger radius is
critical for formation of the unique selenenylsulfide. Spectroscopic redox
titrations with dithionite or NADPH were consistent with the structure mod
el. Mechanisms of TrxR in reduction of Trx and hydroperoxides have been pos
tulated and are compatible with known enzyme activities and the effects of
inhibitors, like goldthioglucose and 1-chloro-2,4-dinitrobenzene.