AhpF can be dissected into two functional units: tandem repeats of two thioredoxin-like folds in the N-terminus mediate electron transfer from the thioredoxin reductase-like C-terminus to AhpC.
AhpF, the flavin-containing component of the Salmonella typhimurium alkyl hydroperoxide reductase system, catalyzes the NADH-dependent reduction of an active-site disulfide bond in the other component, AhpC, which in turn reduces hydroperoxide substrates. The amino acid sequence of the C-terminus of AhpF is 35% identical to that of thioredoxin reductase (TrR) from Escherichia coli. AhpF contains an additional 200-residue N-terminal domain possessing a second redox-active disulfide center also required for AhpC reduction. Our studies indicate that this N-terminus contains a tandem repeat of two thioredoxin (Tr)-like folds, the second of which contains the disulfide redox center. Structural and catalytic properties of independently expressed fragments of AhpF corresponding to the TrR-like C-terminus (F[208-521]) and the 2Tr-like N-terminal domain (F[1-202]) have been addressed. Enzymatic assays, reductive titrations, and circular dichroism studies of the fragments indicate that each folds properly and retains many functional properties. Electron transfer between F[208-521] and F[1-202] is, however, relatively slow (4 x 10(4) M(-)(1) s(-)(1) at 25 degrees C) and nonsaturable up to 100 microM F[1-202]. TrR is nearly as efficient at F[1-202] reduction as is F[208-521], although neither the latter fragment, nor intact AhpF, can reduce Tr. An engineered mutant AhpC substrate with a fluorophore attached via a disulfide bond has been used to demonstrate that only F[1-202], and not F[208-521], is capable of electron transfer to AhpC, thereby establishing the direct role this N-terminal domain plays in mediating electron transfer between the TrR-like part of AhpF and AhpC.
Poole
L B
LB
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA. lbpoole@wfubmc.edu
Godzik
A
A
Nayeem
A
A
Schmitt
J D
JD
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D004579:Electron Transport; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics; D054464:Peroxiredoxins; D017433:Protein Structure, Secondary; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013053:Spectrophotometry; D020080:Tandem Repeat Sequences; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry* / Q000235:genetics; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D010544:Peroxidases; D054464:Peroxiredoxins; C515937:ahpC protein, E coli; C515938:ahpF protein, E coli; D013880:Thioredoxin-Disulfide Reductase