Structure of intact AhpF reveals a mirrored thioredoxin-like active site and implies large domain rotations during catalysis

AhpF, a homodimer of 57 kDa subunits, is a flavoenzyme which catalyzes the NADH-dependent reduction of redox-active disulfide bonds in the peroxidase AhpC, a member of the recently identified peroxiredoxin class of antioxidan t enzymes. The structure of AhpF from Salmonella typhimurium at 2.0 resolut ion, determined using multiwavelength anomalous dispersion, shows that the C-terminal portion of AhpF (residues 210-521) is structurally like Escheric hia coli thioredoxin reductase. In addition, AhpF has an N-terminal domain (residues 1-196) formed from two contiguous thioredoxin folds, but containi ng just a single redox-active disulfide (Cys129-Cys132). A flexible linker (residues 197-209) connects the domains, consistent with experiments showin g that the N-terminal domain acts as an appended substrate, first being red uced by the C-terminal portion of AhpF, and subsequently reducing AhpC. Mod eling studies imply that an intrasubunit electron transfer accounts for the reduction of the N-terminal domain in dimeric AhpF. Furthermore, comparing the N-terminal domain with protein disulfide oxidoreductase from Pyrococcu s furiosis, we describe a new class of protein disulfide oxidoreductases ba sed on a novel mirror-image active site arrangement, with a distinct carbox ylate (Glu86) being functionally-equivalent to the key acid (Asp26) of E. c oli thioredoxin. A final fortuitous result is that the N-terminal redox cen ter is reduced and provides a high-resolution view of the thiol-thiolate hy drogen bond that has been predicted to stabilize the attacking thiolate in thioredoxin-like proteins.