Crystal structures of oxidized and reduced mitochondrial thioredoxin reductase provide molecular details of the reaction mechanism

Abstract

Thioredoxin reductase (TrxR) is an essential enzyme required for the efficient maintenance of the cellular redox homeostasis, particularly in cancer cells that are sensitive to reactive oxygen species. In mammals, distinct isozymes function in the cytosol and mitochondria. Through an intricate mechanism, these enzymes transfer reducing equivalents from NADPH to bound FAD and subsequently to an active-site disulfide. In mammalian TrxRs, the dithiol then reduces a mobile C-terminal selenocysteine-containing tetrapeptide of the opposing subunit of the dimer. Once activated, the C-terminal redox center reduces a disulfide bond within thioredoxin. In this report, we present the structural data on a mitochondrial TrxR, TrxR2 (also known as TR3 and TxnRd2). Mouse TrxR2, in which the essential selenocysteine residue had been replaced with cysteine, was isolated as a FAD-containing holoenzyme and crystallized (2.6 Å; R ‫؍‬ 22.2%; Rfree ‫؍‬ 27.6%). The addition of NADPH to the TrxR2 crystals resulted in a color change, indicating reduction of the active-site disulfide and formation of a species presumed to be the flavin-thiolate charge transfer complex. Examination of the NADP(H)-bound model (3.0 Å; R ‫؍‬ 24.1%; R free ‫؍‬ 31.2%) indicates that an active-site tyrosine residue must rotate from its initial position to stack against the nicotinamide ring of NADPH, which is juxtaposed to the isoalloxazine ring of FAD to facilitate hydride transfer. Detailed analysis of the structural data in conjunction with a model of the unusual C-terminal selenenylsulfide suggests molecular details of the reaction mechanism and highlights evolutionary adaptations among reductases.hioredoxins are the major cellular protein disulfide reductases and are responsible for the regulation of numerous biochemical processes within the cell (1). These proteins are maintained in a reduced state by thioredoxin reductases (TrxR), homodimeric f lavoproteins that catalyze the NADPHdependent reduction of thioredoxins (2, 3).Two forms of TrxRs have evolved with related but distinct modes of catalysis (2-5). Low-M r TrxRs (M r Ϸ 35 kDa) are typically found in prokaryotes, archaea, plants, and lower eukaryotes, whereas high-M r TrxRs (M r Ϸ 55 kDa) are observed in higher eukaryotes. To date, only the green algae Chlamydomonas reinhardtii has been shown to contain both a low-and a high-M r TrxR (6).The general features of catalysis are retained in both low-and high-M r TrxR (2, 4). TrxR transfers reducing equivalents from NADPH to its bound FAD, ultimately leading to the reduction of an active-site disulfide. In low-M r TrxRs, the catalytic cycle requires a large conformational change after dithiol activation (4,7,8). In high-M r TrxR, the active-site dithiol reduces a third redox active center in the highly mobile C terminus of the opposing subunit. This third group is responsible for the reduction of the disulfide bond within thioredoxin. Its nature is species-specific and ranges from a C-X-X-X-X-C disulfide in Plasmodium falciparum (9) to a vicinal disulfid...

Mapping the Catalytic Cycle of Schistosoma mansoni Thioredoxin Glutathione Reductase by X-ray Crystallography

“…2, panel A) (26,38). The recognized role of Tyr 296 (39), acting as a "gate" for NADPH binding, is confirmed in our structure. In fact, the phenolic ring of Tyr 296 , which points toward the re-face of the isoalloxazine ring of FAD in a T-shape configuration (see Structure 1 and also Ref.…”

Effects of thioredoxin reductase-1 deletion on embryogenesis and transcriptome

“…The allele was designed to allow excision of these first two protein-coding exons (Fig. 1a), which encode conserved amino acids 1-30 that contact flavin-adenine dinucleotide (FAD), are an integral part of the Rossman fold common to NADPH-binding proteins [45,46], and are found in all Txnrd1 mRNA isoforms [40]. Furthermore, when exons 1 and 2 are excised, the first in-frame AUG in the resultant mRNA (Met 70 ) is in a poor context for translation initiation and any polypeptide initiated here would lack both N-terminal active site cysteines (Cys 59 and Cys 64 ) (see below).…”

Crystal Structure and Catalysis of the Selenoprotein Thioredoxin Reductase 1

“…The structure of the selenenylsulfide motif and its location in the enzyme has so far only been modeled. The prior structure determinations of Sec-to-Cys mutant forms of mammalian TrxR (32,36,37) furthermore revealed that the C-terminal tail seems to be highly flexible, with the configuration of the final C-terminal residues therefore difficult to model with any appreciable certainty.…”

“…In 2001, the first crystal structure of a Sec-to-Cys mutant of rat TrxR1 was published (32), followed by several structure determinations of similar Sec-substituted mutants, i.e. mutants of mouse TrxR2 (33)(34)(35)(36), human TrxR1 (37) (also as directly deposited PDB entry 2CFY), and the non-selenoprotein orthologue of Drosophila (34,35). These structures as well as additional modeling studies and several extensive biochemical analyses (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43) have generated a generally accepted model for the major features of the catalytic mechanism of mammalian TrxRs.…”