title: Essential role of the flexible linker on the conformational equilibrium of bacterial peroxiredoxin reductase for effective regeneration of peroxiredoxin.
abstract: Reactive oxygen species (ROS) can damage DNA, proteins, and lipids, so cells have antioxidant systems that regulate ROS. In many bacteria, a dedicated peroxiredoxin reductase, alkyl hydroperoxide reductase subunit F (AhpF), catalyzes the rapid reduction of the redox-active disulfide center of the antioxidant protein peroxiredoxin (AhpC) to detoxify ROS such as hydrogen peroxide, organic hydroperoxide, and peroxynitrite. AhpF is a flexible multidomain protein that enables a series of electron transfers among the redox centers by accepting reducing equivalents from NADH. A flexible linker connecting the N-terminal domain (NTD) and C-terminal domain (CTD) of AhpF suggests that the enzyme adopts a large-scale domain motion that alternates between the closed and open states to shuttle electrons from the CTD via the NTD to AhpC. Here, we conducted comprehensive mutational, biochemical, and biophysical analyses to gain insights into the role of the flexible linker and the residues critical for the domain motions ofEscherichia coliAhpF (EcAhpF) during electron transfer. Small-angle X-ray scattering studies of linker mutants revealed that a group of charged residues,200EKR202, is crucial for the swiveling motion of the NTD. Moreover, NADH binding significantly affectedEcAhpF flexibility and the movement of the NTD relative to the CTD. The mutants also exhibited a decrease in H2O2reduction by the AhpF-AhpC ensemble. We propose that a concerted movement involving the NTD, C-terminal NADH, and FAD domains, and the flexible linker between them is essential for optimal intra-domain cross-talk and for efficient electron transfer to the redox partner AhpC required for peroxidation.
authors.affiliation: From the Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), Singapore 138671.
authors.forename: Neelagandan
authors.initials: N
authors.lastname: Kamariah
authors.affiliation: From the Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), Singapore 138671.
authors.forename: Birgit
authors.initials: B
authors.lastname: Eisenhaber
authors.affiliation: From the Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), Singapore 138671.|the School of Computer Engineering, Nanyang Technological University, Singapore 637553, Republic of Singapore.
authors.forename: Frank
authors.initials: F
authors.lastname: Eisenhaber
authors.affiliation: From the Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), Singapore 138671, ggrueber@ntu.edu.sg.|the School of Biological Sciences, Nanyang Technological University, Singapore 637551, and.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Grüber
mesh_terms: D004252:DNA Mutational Analysis; D004220:Disulfides / Q000737:chemistry; D004583:Electrons; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry; D009154:Mutation; D009243:NAD / Q000737:chemistry; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D054464:Peroxiredoxins / Q000737:chemistry*; D011485:Protein Binding; D000072417:Protein Domains; D055503:Protein Multimerization; D017382:Reactive Oxygen Species / Q000737:chemistry; D012542:Scattering, Radiation
chemical_list: D004220:Disulfides; D029968:Escherichia coli Proteins; D017382:Reactive Oxygen Species; D009243:NAD; D006861:Hydrogen Peroxide; D054464:Peroxiredoxins; C515937:ahpC protein, E coli; C515938:ahpF protein, E coli; D009113:Muramidase; D010100:Oxygen
keywords: antioxidant; bioenergetics; electron transfer; enzyme mechanism; flavoprotein; protein structure; small-angle X-ray scattering (SAXS)