title: NMR studies reveal a novel grab and release mechanism for efficient catalysis of the bacterial 2-Cys peroxiredoxin machinery.
abstract: In bacteria, an ensemble of alkyl hydroperoxide reductase subunits C (AhpC) and F (AhpF) is responsible for scavenging H2O2. AhpC donates electrons for the reduction of H2O2, which are provided after NADH oxidation by AhpF. The latter contains an N-terminal domain (NTD), catalyzing the electron transfer from NADH via a FAD of the C-terminal domain (CTD) into AhpC. The NADH-bound Escherichia coli AhpF structure revealed that NADH binding brings the substrate to the re-face of the FAD, making the Cys-Cys center of the CTD accessible to the NTD disulfide center for electron transfer (Kamariah et al. (2015) Biochim Biophys Acta 1847, 1139-1152). So far insight into the epitope and mechanism of AhpF and AhpC interaction as well as the electron transfer from the NTD to AhpC have been lacking. Here using NMR spectroscopy, we glean insight into the interaction of the NTD of AhpF with AhpC from E. coli. A coordinated disappearance of EcAhpF NTD peaks was observed in the presence of full length EcAhpC, indicating a long-lived AhpC-AhpF complex. C-terminal truncated EcAhpC resulted in a more dynamic interaction, revealing specific residue chemical shift perturbation and hence the binding epitope of the complex. Combined with docking studies, we have suggested that the C terminus of AhpC binds to the backside groove of the NTD. In addition, AhpC-AhpF formation is abolished under reducing conditions. We propose for the first time a binding mechanism in which the C terminus of AhpC wraps around the NTD, slowing the dissociation rate for an efficient electron transfer process, and a release mechanism mediated by the conformational change of the C terminus of AhpC upon reduction.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore City, Singapore.
authors.forename: Wilson
authors.initials: W
authors.lastname: Nartey
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore City, Singapore.
authors.forename: Sandip
authors.initials: S
authors.lastname: Basak
authors.affiliation: Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), Singapore City, Singapore.
authors.forename: Neelagandan
authors.initials: N
authors.lastname: Kamariah
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore City, Singapore.
authors.forename: Malathy S S
authors.initials: MS
authors.lastname: Manimekalai
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Robson
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Wagner
authors.affiliation: Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), Singapore City, Singapore.
authors.forename: Birgit
authors.initials: B
authors.lastname: Eisenhaber
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore City, Singapore.|School of Computer Engineering, Nanyang Technological University (NTU), Singapore City, Singapore.|Department of Biological Sciences, National University of Singapore, Singapore.
authors.forename: Frank
authors.initials: F
authors.lastname: Eisenhaber
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore City, Singapore.|Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), Singapore City, Singapore.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Grüber
mesh_terms: D055162:Biocatalysis; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D054464:Peroxiredoxins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D004151:Dipeptides; C046557:cysteinylcysteine; D054464:Peroxiredoxins
keywords: NMR spectroscopy; alkyl hydroperoxide reductase; bioenergetics; electron transport; reactive oxygen species