Received: 13 September 2010 Revised: 15 October 2010 Accepted: 15 October 2010 Published online in Wiley Online Library: 00 Month 2010 Role of 2-oxo and 2-thioxo modifications on the fragmentation reactions of the histidine radical cation y Adrian K. Y. Lam 1,2,3 , Craig A. Hutton 1,2 and Richard A. J. O’Hair 1,2,3 * 1 School of Chemistry, The University of Melbourne, Victoria 3010, Australia 2 Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne, Victoria 3010, Australia 3 ARC Centre of Excellence for Free Radical Chemistry and Biotechnology, School of Chemistry, Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne, Victoria 3010, Australia The fragmentation reactions of the radical cations, M R , of histidine, 2-oxo-histidine and 2-thioxo-histidine were examined using a combination of experiments performed on a linear ion trap and density functional theory (DFT) calculations at the UB3-LYP/6-311RRG(d,p) level of theory. Low-energy collision-induced dissociation (CID) on [Cu II (terpy)(M)] 2R complexes, formed via electrospray ionisation, produced the radical cations in sufficient yield to examine their unimolecular chemistry via an additional stage of CID. The CID spectrum of the radical cation of histidine is dominated by loss of water with the next most abundant ion arising from the combined loss of H 2 O and CO. In contrast, the CID spectra of the radical cations of 2-oxo-histidine and 2-thioxo-histidine are dominated by the combined loss of CO 2 and NH – –CH 2 . The observed differences are rationalised via DFT calculations which reveal that the barrier associated with loss of CO 2 from the histidine radical cation is higher than that for loss of H 2 O. In contrast, the introduction of an oxygen or sulfur atom into the side chain of histidine results in a reversal of the order of these barrier heights, thus making CO 2 loss the preferred pathway. Copyright ß 2010 John Wiley & Sons, Ltd. Histidine residues play important roles in the gas-phase fragmentation reactions of amino acid and peptide ions. For example, due to the basic and nucleophilic nature of the imidazole ring on the side chain, histidine residues can promote the formation of b ions in the collision-induced dissociation (CID) spectra of protonated peptides. [1–6] Histidine residues also play a role in electron capture dissociation (ECD) and electron transfer dissociation (ETD) fragmentation reactions of peptides, with recent studies investigating the effects that these electron-ion interactions have on the histidine residues. In a series of articles, Turec ˇek and co-workers described the radical rearrangements and unimolecular dissociations that are brought about by electron–ion interactions, noting that peptides containing a histidine residue and a free carboxylic acid moiety were able to undergo a carboxyl-catalysed radical rearrangement reaction. [7–10] Srikanth et al. also showed that oxidised histidine residues, which can be misassigned under CID conditions, could be correctly located through the use of ETD. [11] Histidine is also only one of seven of the twenty commonly occurring a-amino acids to form radical cations, M þ , upon CID of metal complexes [Cu(L n )M] 2þ (the others being methionine, phenylalanine, tyrosine, tryptophan, lysine and arginine). [12–16] The removal of an electron from histidine to form the canonical/hydrogen-deficient histidine radical cation has been found to affect both the acidities and the basicities of the functional groups involved in the intramo- lecular hydrogen bonding that stabilises the ion. [17] Unlike aliphatic amino acids, in which the spin density is localised onto the amino group, the radical centre within this system occurs on the imidazole side chain, increasing the acidity of the N-H group and thereby favouring structural confor- mations in which this group functions as a proton donor. [18,19] Ke et al. noted that two different populations of radical cations of the basic amino acids, histidine, lysine and arginine, could be formed from the corresponding copper(II) complexes and that these depended on the binding mode of the amino acid to the copper. [13] These results have recently been confirmed by Song et al., with the generation of two types of histidine radical cations through the use of Cu(II)-peptide complexes containing the sterically con- strained 1,4,7-triazacyclononane ligand. [14] As illustrated for histidine in Scheme 1, the type 1 radical cations, His1, are formed from a ‘charge solvated’ metal complex in which the amino acid binds to the copper via the amino group and the imidazole side chain. [13,20] Type 2 radical cations, His2, arise from a ‘salt bridge’ metal complex in which the histidine binds to the copper through the amino and carboxylate groups. In contrast to the relatively stable type 1 radical cations, type 2 radical cations are unstable and fragment through loss of CO 2 . Once formed, these radical cations do not ‘communicate’ with each other via intramolecular hydrogen atom transfer. Density functional theory (DFT) calculations at the UB3-LYP/6-311þþG(d,p) level of theory were used to rationalise this different behaviour based on the calculated barriers associated with HAT reactions leading to the various histidine isomers shown in Scheme 2 versus the barriers for fragmentation. Thus, when canonical His1 (relative energy of 0.0 kcal mol 1 ) is subjected to CID it undergoes a hydrogen atom transfer via a barrier of 8.4 kcal mol 1 to form a high energy conformer of the Rapid Commun. Mass Spectrom. 2011, 25, 251–261 (wileyonlinelibrary.com) DOI: 10.1002/rcm.4830 Research Article * Correspondence to: R. A. J. O’Hair, School of Chemistry, The University of Melbourne, Victoria 3010, Australia. E-mail: rohair@unimelb.edu.au y Part 76 of the series ’Gas-Phase Ion Chemistry of Biomolecules’. Rapid Commun. Mass Spectrom. 2011, 25, 251–261 Copyright ß 2010 John Wiley & Sons, Ltd. 251