REQUIREMENT FOR THE 2 AHPF CYSTINE DISULFIDE CENTERS IN CATALYSIS OF PEROXIDE REDUCTION BY ALKYL HYDROPEROXIDE REDUCTASE

AhpF, the alkyl hydroperoxide reductase component which transfers elec trons from pyridine nucleotides to the peroxidase protein, AhpC, posse sses two redox-active disulfide centers in addition to one FAD per sub unit; the primary goal of these studies has been to test for the requi rement of one or both of these disulfide centers in catalysis. Two hal f-cystine residues of one center (CYS345CYS348) align with those of th e homologous Escherichia coli thioredoxin reductase (TrR) sequence (Cy s(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 cen ter (Ser(345)Ser(348),Ser(345)Cys(348), and Cys(345)Ser(348)) Fluoresc ence, absorbance, and circular dichroism spectra show relatively small perturbations for mutations at the disulfide center proximal to the f lavin (Cys(345)Cys(348)) and no changes for the Ser(129)Ser(132) mutan t; identical circular dichroism spectra in the ultraviolet region indi cate unchanged secondary structures in all mutants studied. Oxidase an d 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 muta tions, dropping to less than 5% for DTNB reductase activity and to les s than 2% for peroxidase activity in the presence of AhpC. Reductive t itrations 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 b e reduced, although only slowly, These results emphasize the necessity for both redox-active disulfide centers in AhpF for catalysis of disu lfide 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.