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Oxidized and synchrotron cleaved structures of the disulfide redox center in the N-terminal domain of Salmonella typhimurium AhpF

¹ Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331-7305, USA
² Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA

(RECEIVED March 14, 2005; FINAL REVISION June 9, 2005; ACCEPTED June 9, 2005)

The flavoprotein component (AhpF) of Salmonella typhimurium alkyl hydroperoxide reductase contains an N-terminal domain (NTD) with two contiguous thioredoxin folds but only one redox-active disulfide (within the sequence -Cys₁₂₉-His-Asn-Cys₁₃₂-). This active site is responsible for mediating the transfer of electrons from the thioredoxin reductase-like segment of AhpF to AhpC, the peroxiredoxin component of the two-protein peroxidase system. The previously reported crystal structure of AhpF possessed a reduced NTD active site, although fully oxidized protein was used for crystallization. To further investigate this active site, we crystallized an isolated recombinant NTD (rNTD); using diffraction data sets collected first at our in-house X-ray source and subsequently at a synchrotron, we showed that the active site disulfide bond (Cys129–Cys132) is oxidized in the native crystals but becomes reduced during synchrotron data collection. The NTD disulfide bond is apparently particularly sensitive to radiation cleavage compared with other protein disulfides. The two data sets provide the first view of an oxidized (disulfide) form of NTD and show that the changes in conformation upon reduction of the disulfide are localized and small. Furthermore, we report the apparent pK_a of the active site thiol to be ~5.1, a relatively low pK_a given its redox potential (~265 mV) compared with most members of the thioredoxin family.

Keywords: X-ray crystallography; thioredoxin; disulfide; peroxiredoxin; thiolate; alkyl hydroperoxide reductase; synchrotron radiation; pK_a; radiation damage

Abbreviations: AhpF, alkyl hydroperoxide reductase flavoprotein or NADH:peroxiredoxin oxidoreductase • NTD, N-terminal domain of AhpF • rNTD, recombinant NTD • DTT, 1,4-dithiothreitol • DTNB, 5,5'-dithio-bis-(2-nitrobenzoic acid) • TNB, 2-nitro-5-thiobenzoate.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051459705.

Reprint requests to: P. Andrew Karplus, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331-7305, USA; e-mail: karplusp{at}ucs.orst.edu; fax: (541) 737-0481.

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