Requirement for the Two AhpF Cystine Disulfide Centers in Catalysis of Peroxide Reduction by Alkyl Hydroperoxide Reductase

Abstract

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 (Cys135Cys138), while the other two (Cys129Cys132) 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 (Ser129Ser132) and three which perturb the TrR-like disulfide center (Ser345Ser348, Ser345Cys348, and Cys345Ser348). Fluorescence, absorbance, and circular dichroism spectra show relatively small perturbations for mutations at the disulfide center proximal to the flavin (Cys345Cys348) and no changes for the Ser129Ser132 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 Cys345Cys348, 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 Cys129Cys132 in mediating electron transfer between Cys345Cys348 and the AhpC active-site disulfide.

Attachment of the N-Terminal Domain of Salmonella typhimurium AhpF to Escherichia coli Thioredoxin Reductase Confers AhpC Reductase Activity but Does Not Affect Thioredoxin Reductase Activity

“…This result, along with the thiol content of reduced Nt-TrR C342,345S of about 4 per subunit, indicates that electron transfer from the reduced flavin to the N-terminal disulfide center can occur in the absence of the Cys342-Cys345 center. This observation is in accordance with the results of similar titrations with the corresponding mutant of AhpF, AhpF C345,348S (8). Titrations of both of these mutants require relatively long equilibration times after each addition, indicating that the normal pathway of electron transfer has been disrupted.…”

“…For example, assays of crude bacterial extracts for AhpC activity using excess flavoprotein are much more straightforward and sensitive using Nt-TrR than using AhpF or its counterpart from Streptococcus mutans, Nox-1, which has an even higher oxidase activity (30,50). It is notable that the flavoproteinassociated properties of Nt-TrR (e.g., specificity for NADPH rather than NADH as reductant, low oxidase and transhydrogenase activities, and high flavin fluorescence) closely follow those characteristics of the parental TrR protein, while the new activities observed for Nt-TrR (AhpC-dependent peroxidase and DTNB reductase activities) are characteristic of intact AhpF with a functional N-terminal disulfide center (2,3,8). In all AhpF mutants characterized to date, DTNB reductase activities have paralleled AhpC-linked peroxidase activities, indicating a direct role for the N-terminal domain in both activities.…”

“…The additional stretch of about 200 amino acids at the N-terminus of AhpF contains an extra redox-active disulfide center required for catalysis of AhpC reduction (8). Our working hypothesis has been that the C-terminal part of AhpF undergoes electron transfer from NADH to the TrR-like Cys345-Cys348 redox center via the flavin in a manner quite analogous to those transfers in TrR ( Figure 1B,C).…”

Purification and Characterization of a Chimeric Enzyme fromHaemophilus influenzae Rd That Exhibits Glutathione-dependent Peroxidase Activity

“…Studies with the separate regions of the PGdx enzyme will provide new insights into the catalytic mechanism and the interaction between its two regions. (15) for a Grx-reducible poplar Prx but adapted here for PGdx; see "Discussion" for a detailed discussion of each reaction (1)(2)(3)(4)(5). The top black part of the letter E corresponds to the N-terminal Prx region, whereas the bottom gray part of the letter represents the C-terminal Grx region.…”

Crystallization and preliminary X-ray analysis of the catalytic core of the alkylhydroperoxide reductase component AhpF from Escherichia coli

“…The AhpF component reduces an internal redox-active disul®de bridge between Cys345 and Cys348 by electron transfer from reduced pyridine nucleotides via the cofactor¯avin. The reduction of this disul®de bridge is followed by a cascade of disul®de-exchange reactions, ®rst intramolecularly to a disul®de in the N-terminal region of AhpF, then intermolecularly to the disul®de Cys46±Cys165 of the AhpC component (Ellis & Poole, 1997;Li Calzi & Poole, 1997). The Ahp reductase system has a profound in¯uence on the activity of certain antimycobacterial drugs, as the expression of the ahpC gene is strongly enhanced in isonicotinic acid hydrazide (INH) resistant strains of Mycobacterium tuberculosis (Zhang et al, 1996).…”