Crystal structure of reduced thioredoxin reductase from Escherichia coli: Structural flexibility in the isoalloxazine ring of the flavin adenine dinucleotide cofactor

Catalysis by thioredoxin reductase (TrxR) from Escherichia coli requires al ternation between two domain arrangements. One of these conformations has b een 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 disu lfide on TrxR. As part of an investigation of conformational changes and in termediates 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. R eduction after crystallization resulted in significant local conformation c hanges. The isoalloxazine ring of the FAD cofactor, which is essentially pl anar in the oxidized enzyme, assumes a 34 degrees "butterfly" bend about th e N(5)-N(10) axis in reduced TrxR. Theoretical calculations reported by oth ers predict ring bending of 15-18 degrees for reduced isoalloxazines proton ated at N(1). The large bending in reduced TrxR is attributed in part to st eric interactions between the isoalloxazine ring and the sulfur of Cys138, formed by reduction of the active site disulfide, and is accompanied by cha nges 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 an y flavoprotein in the Protein Data Bank. Distributions of bending angles in published oxidized and reduced flavoenzyme structures are different from t hose found in studies of free flavins, indicating that the protein environm ent has a significant effect on bending.