In thioredoxin reductase (TrxR) from Escherichia coli, cycles of reduction and reoxidation of the flavin adenine dinucleotide (FAD) cofactor depend on rate-limiting rearrangements of the FAD and NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) domains. We describe the structure of the flavin-reducing conformation of E. coli TrxR at a resolution of 3.0 angstroms. The orientation of the two domains permits reduction of FAD by NADPH and oxidation of the enzyme dithiol by the protein substrate, thioredoxin. The alternate conformation, described by Kuriyan and co-workers, permits internal transfer of reducing equivalents from reduced FAD to the active-site disulfide. Comparison of these structures demonstrates that switching between the two conformations involves a "ball-and-socket" motion in which the pyridine nucleotide-binding domain rotates by 67 degrees.

Thioredoxin reductase (EC 1.6.4.5) is a widely distributed flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin. Thioredoxin plays several key roles in maintaining the redox environment of the cell. Like all members of the enzyme family that includes lipoamide dehydrogenase, glutathione reductase and mercuric reductase, thioredoxin reductase contains a redox active disulfide adjacent to the flavin ring. Evolution has produced two forms of thioredoxin reductase, a protein in prokaryotes, archaea and lower eukaryotes having a M r of 35 000, and a protein in higher eukaryotes having a M r of 55 000. Reducing equivalents are transferred from the apolar flavin binding site to the protein substrate by distinct mechanisms in the two forms of thioredoxin reductase. In the low M r enzyme, interconversion between two conformations occurs twice in each catalytic cycle. After reduction of the disulfide by the flavin, the pyridine nucleotide domain must rotate with respect to the flavin domain in order to expose the nascent dithiol for reaction with thioredoxin; this motion repositions the pyridine ring adjacent to the flavin ring. In the high M r enzyme, a third redox active group shuttles the reducing equivalent from the apolar active site to the protein surface. This group is a second redox active disulfide in thioredoxin reductase from Plasmodium falciparum and a selenenylsulfide in the mammalian enzyme. P. falciparum is the major causative agent of malaria and it is hoped that the chemical difference between the two high M r forms may be exploited for drug design.Keywords: flavoprotein; thioredoxin; thioredoxin reductase; selenium, disulfide; dithiol; selenenylsulfide; redox active; ribonucleotide reductase; transcription factor activation; drug design.With most enzymes, the structure, and the mechanism that is associated with it, are essentially the same regardless of the enzyme source, whether that be prokaryote, archaea or eukaryote, i.e. evolution has decided on one way to effect catalysis. However, there are enzymes where the same reaction is catalyzed by more than one structure and mechanism (e.g. methionine synthase [1]), and thioredoxin reductase is another such enzyme [2,3]. Thioredoxin reductase is a flavoprotein that catalyzes the reduction of thioredoxin by NADPH [4,5]. The substrate thioredoxin is a small protein of M r 12 000 which in its dithiol state plays a key role in maintaining the redox environment of the cell [6]. Important functions of reduced thioredoxin include the reduction of nucleotides to deoxynucleotides and the modulation of transcription factors such as NF-kB in eukaryotes [6±8]. C H A R A C T E R I S T I C S O F A N E N Z Y M E F A M I L YThioredoxin reductase is a member of the family of dimeric flavoenzymes that catalyze the transfer of electrons between pyridine nucleotides and disulfide/dithiol compounds and promote catalysis via FAD and a redox active disulfide (Table 1) [9±13]. The family includes lipoamide dehydrogenase, glutathione reductase and mercuric reduc...

B(12)-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that uses bound cobalamin as an intermediate methyl carrier. Major domain rearrangements have been postulated to explain how cobalamin reacts with three different substrates: homocysteine, methyltetrahydrofolate and S-adenosylmethionine (AdoMet). Here we describe the 3.0 A structure of a 65 kDa C-terminal fragment of MetH that spans the cobalamin- and AdoMet-binding domains, arranged in a conformation suitable for the methyl transfer from AdoMet to cobalamin that occurs during activation. In the conversion to the activation conformation, a helical domain that capped the cofactor moves 26 A and rotates by 63 degrees, allowing formation of a new interface between cobalamin and the AdoMet-binding (activation) domain. Interactions with the MetH activation domain drive the cobalamin away from its binding domain in a way that requires dissociation of the axial cobalt ligand and, thereby, provide a mechanism for control of the distribution of enzyme conformations.

Catalysis by thioredoxin reductase (TrxR) from Escherichia coli requires alternation between two domain arrangements. One of these conformations has been observed by X-ray crystallography (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800-816). This form of TrxR, denoted FO, permits the reaction of enzyme-bound reduced FAD with a redox-active disulfide on TrxR. As part of an investigation of conformational changes and intermediates in catalysis by TrxR, an X-ray structure of the FO form of TrxR with both the FAD and active site disulfide reduced has been determined. Reduction after crystallization resulted in significant local conformation changes. The isoalloxazine ring of the FAD cofactor, which is essentially planar in the oxidized enzyme, assumes a 34 degree "butterfly" bend about the N(5)-N(10) axis in reduced TrxR. Theoretical calculations reported by others predict ring bending of 15-28 degrees for reduced isoalloxazines protonated at N(1). The large bending in reduced TrxR is attributed in part to steric interactions between the isoalloxazine ring and the sulfur of Cys138, formed by reduction of the active site disulfide, and is accompanied by changes in the positions and interactions of several of the ribityl side-chain atoms of FAD. The bending angle in reduced TrxR is larger than that for any flavoprotein in the Protein Data Bank. Distributions of bending angles in published oxidized and reduced flavoenzyme structures are different from those found in studies of free flavins, indicating that the protein environment has a significant effect on bending.

Thioredoxin reductase is a homodimeric flavoenzyme containing a flavin adenine dinucleotide (FAD) and a redox-active disulfide in each subunit. Structural work on the enzyme from Escherichia coli suggests that thioredoxin reductase exists in two conformations, both of which are necessary for catalysis [Waksman, G., Krishna, T. S. R., Williams, C. H., Jr., & Kuriyan, J. (1994) J. Mol. Biol. 236, 800-816]. These factors make it likely that the mechanism of this enzyme is complex. The rapid reaction of enzyme with nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) (the reductive half-reaction), proceeds in three phases. The first phase represents the formation of an NADPH-FAD charge transfer complex. The second phase involves FAD reduction, with loss of the NADPH-FAD charge transfer band. The third phase shows a slower decrease in absorbance at 456 nm and the formation of a reduced flavin-NADP+ charge transfer band. These and other results indicate that NADP+ and NADPH compete for the single binding site on oxidized and fully reduced enzyme and that NADP+ release does not limit the third phase of reduction. Experiments that include examination of the reductive half-reactions of active-site mutants, having the active-site disulfide removed by mutating one or both of the active-site cysteines, indicate that the third phase does not represent reduction by a second equivalent of NADPH. Comparison of the rate constants and temperature dependence of the reductive half-reaction with those of turnover show that the reductive half-reaction is not solely rate-limiting in catalysis. The results suggest that wild type and each altered enzyme exists in a unique equilibrium of conformers. It is proposed that the third phase of the reductive half-reaction represents a flavin reduction event largely limited by the conformational change proposed in the structural work.