FLAVIN-DEPENDENT ALKYL HYDROPEROXIDE REDUCTASE FROM SALMONELLA-TYPHIMURIUM .2. CYSTINE DISULFIDES INVOLVED IN CATALYSIS OF PEROXIDE REDUCTION

The two-component alkyl hydroperoxide reductase enzyme system from Sal monella typhimurium catalyzes the pyridine nucleotide-dependent reduct ion of alkyl hydroperoxide and hydrogen peroxide substrates. This syst em is composed of a flavoenzyme, AhpF, which is related to the disulfi de-reducing enzyme thioredoxin reductase, and a smaller protein, AhpC, which lacks a chromophoric cofactor. We have demonstrated that NADH-l inked reduction of AhpF under anaerobic conditions converts two cystin e disulfide centers to their dithiol forms. The AhpC cystine disulfide center, shown to exist as an intersubunit disulfide bond, is stoichio metrically reducible by NADH in the presence of a catalytic amount of AhpF and can be reoxidized by ethyl hydroperoxide. Disulfide bridges w ithin oxidized AhpF form between Cys129 and Cys132 and between Cys345 and Cys348; the two C-terminal half-cystine residues, Cys476 and Cys48 9, exist as free thiol groups in oxidized AhpF and play no role in cat alysis. Removal of the N-terminal 202-amino acid segment containing th e Cys129-Cys132 disulfide center obliterates the ability of AhpF to tr ansfer electrons to 5,5'-dthiobis(2-nitrobenzoic acid) (DTNB) and AhpC , NADH added anaerobically to AhpF causes spectral changes consistent with preferential reduction of both disulfides relative to flavin redu ction; the reduction potentials of the disulfide centers are thus appr opriately poised for electron transfer from NADH and flavin to disulfi de-containing substrates (AhpC or DTNB), and ultimately to peroxides f rom AhpC, Blue, neutral flavin semiquinone is also generated in high y ields during reductive titrations (91% yield during dithionite titrati ons), although the relatively slow formation of this species indicates its catalytic incompetence, A long wavelength absorbance band beyond 900 nm attributable to an FADH(2) --> NAD(+) charge transfer interacti on is generated during NADH, but not dithionite, titrations and may be indicative of a species directly involved the catalytic cycle. A cata lytic mechanism including the transient formation of cysteine sulfenic acid within AhpC is proposed.