Requirement for the two AhpF cystine disulfide centers in catalysis of peroxide reduction by alkyl hydroperoxide reductase

AhpF, the alkyl hydroperoxide reductase component which transfers electrons from pyridine nucleotides to the peroxidase protein, AhpC, possesses two redox-active disulfide centers in addition to one FAD per subunit; the primary goal of these studies has been to test for the requirement of one or both of these disulfide centers in catalysis. Two half-cystine residues of one center (CYS345CYS348) align with those of the homologous Escherichia coli thioredoxin reductase (TrR) sequence (Cys(135)Cys(138)), while the other two (Cys(129)Cys(132)) reside in the additional N-terminal region of AhpF which has no counterpart in TrR, We have employed site-directed mutagenesis techniques to generate four mutants of AhpF, including one which removes the N-terminal disulfide (Ser(129)Ser(132)) and three which perturb the TrR-like disulfide center (Ser(345)Ser(348),Ser(345)Cys(348), and Cys(345)Ser(348)) Fluorescence, absorbance, and circular dichroism spectra show relatively small perturbations for mutations at the disulfide center proximal to the flavin (Cys(345)Cys(348)) and no changes for the Ser(129)Ser(132) mutant; identical circular dichroism spectra in the ultraviolet region indicate unchanged secondary structures in all mutants studied. Oxidase and transhydrogenase activities are preserved in all mutants, indicating no role for cystine redox centers in these activities, Both DTNB and AhpC reduction by AhpF are dramatically affected by each of these mutations, dropping to less than 5% for DTNB reductase activity and to less than 2% for peroxidase activity in the presence of AhpC. Reductive titrations confirm the absence of one redox center in each mutant; even in the absence of Cys(345)Cys(348), the N-terminal redox center can be reduced, although only slowly, These results emphasize the necessity for both redox-active disulfide centers in AhpF for catalysis of disulfide reductase activity and support a direct role for Cys(129)Cys(132) in mediating electron transfer between Cys(345)Cys(348) and the AhpC active-site disulfide.