Structure of Intact AhpF Reveals a Mirrored Thioredoxin-like Active Site and Implies Large Domain Rotations during Catalysis

• Published in: Biochemistry (Easton) Vol. 40; no. 13; pp. 3900 - 3911
• Main Authors: Wood, Zachary A, Poole, Leslie B, Karplus, P. Andrew
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 03-04-2001
• Subjects: AhpF protein; Amino Acid Sequence; Binding Sites; Catalysis; Computer Simulation; Crystallization; Crystallography, X-Ray; Dimerization; Escherichia coli - enzymology; Escherichia coli Proteins; Models, Molecular; Molecular Sequence Data; NADH-peroxiredoxin oxidoreductase; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Peroxidases - chemistry; Peroxidases - metabolism; Peroxiredoxins; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Salmonella typhimurium; Salmonella typhimurium - enzymology; Sequence Homology, Amino Acid; Thermodynamics; Thioredoxin Reductase 1; Thioredoxin-Disulfide Reductase - chemistry; Thioredoxin-Disulfide Reductase - metabolism; Thioredoxins - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi002765p

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 antioxidant enzymes. The structure of AhpF from Salmonella typhimurium at 2.0 Å resolution, determined using multiwavelength anomalous dispersion, shows that the C-terminal portion of AhpF (residues 210−521) is structurally like Escherichia coli thioredoxin reductase. In addition, AhpF has an N-terminal domain (residues 1−196) formed from two contiguous thioredoxin folds, but containing just a single redox-active disulfide (Cys129−Cys132). A flexible linker (residues 197−209) connects the domains, consistent with experiments showing that the N-terminal domain acts as an appended substrate, first being reduced by the C-terminal portion of AhpF, and subsequently reducing AhpC. Modeling 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 Pyrococcus furiosis, we describe a new class of protein disulfide oxidoreductases based on a novel mirror-image active site arrangement, with a distinct carboxylate (Glu86) being functionally equivalent to the key acid (Asp26) of E. coli thioredoxin. A final fortuitous result is that the N-terminal redox center is reduced and provides a high-resolution view of the thiol−thiolate hydrogen bond that has been predicted to stabilize the attacking thiolate in thioredoxin-like proteins.