ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
O7. Crystal Structure of PerR: Characterization of the Regulation Site in
the Active Protein and Unambiguous Identification of 2-oxo-histidine in the
Oxidized Form
V. Duarte a , D. Traoré a , A. El Ghazouani a , L. Jacquamet b , F. Borel b , J.-L. Ferrer b ,
D. Lascoux c , J.-L. Ravanat d , G. Blondin a , C. Caux-Thang a , J.-M. Latour a
a
iRTSV - LCBM, CEA, 17 av. des Martyrs, 38054, Grenoble, France
e-mail: victor.duarte@cea.fr
b
IBS - LCCP, CEA, 41 rue Jules Horowitz, 38027, Grenoble, France
c
IBS - LSMP, CEA, 41 rue Jules Horowitz, 38027, Grenoble, France
d
iNAC - SCIB - LAN, CEA, 17 av. des Martyrs, 38054, Grenoble, France
Oxidative stress is generated by exposure to elevated levels of Reactive Oxygen Species (ROS). To avoid the
harmful effects of ROS, cells constitutively express proteins to protect themselves. The expression of these
proteins is under control of specific regulators. In Bacillus subtilis, the PerR protein is a metal-dependent sensor
of H2O2. PerR is a dimeric zinc protein with a regulatory metal-binding site that coordinates either Fe 2+ (PerR-
Zn-Fe) or Mn 2+ (PerR-Zn-Mn). While most of the peroxide sensors use redox-active cysteines to detect H2O2, it
has been shown that reaction of PerR-Zn-Fe with H2O2 leads to the oxidation of one histidine (H) residue that
binds the Fe 2+ ion. This metal-catalyzed oxidation of PerR leads to the incorporation of one oxygen atom into
either H37 or H91. However the exact position of the added oxygen is still unknown. The present study reports
the crystal structure of the active PerR-Zn-Mn protein, which reveals the nature of the regulatory metal binding
site. We also present the x-ray structure of the oxidized PerR protein (PerR-Zn-ox) that clearly shows a 2-oxohistidine
residue in position 37. 2-oxo-histidine formation is also demonstrated and quantified by HPLC-
MS/MS. EPR experiments indicate that PerR-Zn-ox shows a significant affinity for the regulatory metal, albeit
lower than that of the wild-type protein. However, due to the predominant oxidation of H37, the oxidized PerR
protein shows a drastically reduced affinity for the DNA.
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