Archives of Biochemistry and Biophysics 432 (2004) 188–195 www.elsevier.com/locate/yabbi 0003-9861/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2004.09.012 Oxidation of methionine residues in the prion protein by hydrogen peroxide Jesús R. Requena a,¤,1 , Mariana N. Dimitrova a,2 , Giuseppe Legname b,c , Susana Teijeira e , Stanley B. Prusiner b,c,d , Rodney L. Levine a a Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, United States b Institute for Neurodegenerative Diseases, University of California, San Francisco, CA, United States c Department of Neurology, University of California, San Francisco, CA, United States d Department of Biochemistry and Biophysics, University of California, San Francisco, CA, United States e Department of Pathology, Hospital Meixoeiro, Vigo, Spain Received 20 July 2004, and in revised form 9 September 2004 Available online 22 October 2004 Abstract Reaction of H 2 O 2 with the recombinant SHa(29–231) prion protein resulted in rapid oxidation of multiple methionine residues. Susceptibility to oxidation of individual residues, assessed by mass spectrometry after digestion with CNBr and lysC, was in general a function of solvent exposure. Met 109 and Met 112, situated in the highly Xexible amino terminus, and key residues of the toxic peptide PrP (106–126), showed the greatest susceptibility. Met 129, a residue located in a polymorphic position in human PrP and modulating risk of prion disease, was also easily oxidized, as was Met 134. The structural eVect of H 2 O 2 -induced methionine oxida- tion on PrP was studied by CD spectroscopy. As opposed to copper catalyzed oxidation, which results in extensive aggregation of PrP, this reaction led only to a modest increase in -sheet structure. The high number of solvent exposed methionine residues in PrP suggests their possible role as protective endogenous antioxidants.  2004 Elsevier Inc. All rights reserved. Keywords: Prion; Methionine; Oxidation; Hydrogen peroxide; Mass spectrometry; Circular dichroism The disease causing form of the prion proteins (PrP), 3 designated PrP Sc , accumulates in the prion diseases also called transmissible spongiform encephalopathies. PrP Sc is the only known component of the infectious prion particles. Mutations in the PrP gene cause the genetic forms of prion disease. The pathogenicity of PrP Sc derives from an altered conformation, the physical prop- erties of which are strikingly diVerent from those of the normal isoform, PrP C . The latter is a soluble, mono- meric, glycosylated protein attached to the membrane of neurons, and other cells through a glycosylphosphat- idylinositol anchor. Its function remains unknown although a strong, speciWc aYnity for copper has been well established. NMR analysis of recombinant PrP has shown it to be composed by 3  helices, a small portion of  sheet, and a long unstructured stretch spanning from the amino terminus to around position 120. In con- trast, PrP Sc is typically isolated as an insoluble aggregate with a characteristic partial resistance to proteinase K and a higher content in  sheet as compared to PrP C . The basic tenet of the prion hypothesis states that under certain circumstances PrP C can undergo a structural * Corresponding author. Fax: +1 34 981 582642. E-mail address: requenaj@usc.es (J.R. Requena). 1 Present address. Prion Research Unit, Departments of Microbiol- ogy and Medicine, University of Santiago, Santiago, Spain. 2 Analytical Science Department, Amgen Inc., Thousand Oaks, CA, United States 3 Abbreviation used: PrP, prion proteins.