A DFT Study of the Mechanism of Ni Superoxide Dismutase (NiSOD): Role of the Active Site Cysteine-6 Residue in the Oxidative Half-Reaction RAJEEV PRABHAKAR, KEIJI MOROKUMA, DJAMALADDIN G. MUSAEV Cherry L. Emerson Center for Scientific Computation, and Department of Chemistry, Emory University, 1515 Dickey Dr., Atlanta, Georgia 30322 Received 12 December 2005; Accepted 21 February 2006 DOI 10.1002/jcc.20455 Published online in Wiley InterScience (www.interscience.wiley.com). Abstract: In the present DFT study, the catalytic mechanism of H 2 O 2 formation in the oxidative half-reaction of NiSOD, E-Ni(II) + O 2 À + 2H + ? E-Ni(III) + H 2 O 2 , has been investigated. The main objective of this study is to investigate the source of two protons required in this half-reaction. The proposed mechanism consists of two steps: superoxide coordination and H 2 O 2 formation. The effect of protonation of Cys6 and the proton donating roles of side chains (S) and backbones (B) of His1, Asp3, Cys6, and Tyr9 residues in these two steps have been studied in detail. For protonated Cys6, superoxide binding generates a Ni(III)–O 2 H species in a process that is exothermic by 17.4 kcal/mol (in protein environment using the continuum model). From the Ni(III)–O 2 H species, H 2 O 2 formation occurs through a proton donation by His1 via Tyr9, which relative to the resting position of the enzyme is exothermic by 4.9 kcal/mol. In this pathway, a proton donating role of His1 residue is proposed. However, for unprotonated Cys6, a Ni(II)–O 2 À species is generated in a process that is exothermic by 11.3 kcal/mol. From the Ni(II)–O 2 À species, the only feasible pathway for H 2 O 2 formation is through donation of protons by the Tyr9(S)–Asp3(S) pair. The results discussed in this study elucidate the role of the active site residues in the catalytic cycle and provide intricate details of the complex functioning of this enzyme. q 2006 Wiley Periodicals, Inc. J Comput Chem 27: 1438–1445, 2006 Key words: DFT study; Ni superoxide dusmutase; cysteine-6 residue Introduction Superoxide dismutases (SODs) are metalloenzymes that protect cells from oxidative damage by dismuting superoxide into hydrogen peroxide and oxygen through alternate oxidation and reduction of their catalytic metal ions. 1,2 In the literature, three different classes of SODs have been characterized: (1) MnSOD and FeSOD, (2) CuZnSOD, and (3) NiSOD. NiSOD is the most recent class of SOD, which is discovered in Streptomyces 3,4 and cyanobacteria. 5 On the basis of amino acid sequence, metal ligand environment, and spectroscopic properties, NiSOD is dis- tinct from other known SODs. 6,7 However, all SODs are known to have very similar catalytic rate constants, pH dependence, and catalytic functions. 8 Therefore, like its counterparts, the cat- alytic dismutation function of NiSOD occurs through the oxida- tive and the reductive half-reactions, which can be described by the following two equations: E-Ni(II) þ O À 2 þ 2H þ ! E-Ni(III) þ H 2 O 2 ð1Þ E-Ni(III) þ O À 2 ! E-Ni(II) þ O 2 ð2Þ where E-Ni(II) and E-Ni(III) represent the reduced and oxidized states of the metal center in the enzyme. In this study, the source of two protons required for H 2 O 2 formation in the oxida- tive half-reaction has been investigated in detail. The X-ray structures of NiSOD from Streptomyces seoulensis have been determined at 1.68 and 1.30 A ˚ resolutions. 8,9 In the reduced state of the enzyme the Ni(II) is tetra-coordinated with square planar geometry. In this state, as shown in Figure 1, the Ni(II) forms a NiÀÀS bond with Cys6, NiÀÀS and NiÀÀN bonds with the side chain and the amide backbone, respectively, of Cys2 and an NiÀÀN bond with the backbone of His1. The two active site water molecules form hydrogen bonding network between the backbone of Cys2 and the phenol group of Tyr9. Correspondence to: K. Morokuma; e-mail: morokuma@emory.edu Contract/grant sponsor: the National Science Foundation; contract/grant number: CHE-0209660 Contract/grant sponsor: AFOSR; contract/grant number: DURIP FA9550- 04-1-0321 (for support of the computer facilities) This article contains supplementary material available via the Internet at q 2006 Wiley Periodicals, Inc. http://www.interscience.wiley.com/jpages/0192-8651/suppmat.