Original Contribution The thermodynamics of thiol sulfenylation Lionel Billiet a, b, c, d , Paul Geerlings a , Joris Messens b, c, d, ⁎, Goedele Roos a, b, c, d, ⁎ a General Chemistry, VUB, Vrije Universiteit Brussel, B-1050 Brussels, Belgium b Department of Structural Biology, VIB, Vrije Universiteit Brussel, B-1050 Brussels, Belgium c Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium d Brussels Center for Redox Biology, Brussels, Belgium abstract article info Article history: Received 23 June 2011 Revised 27 December 2011 Accepted 28 December 2011 Available online 28 January 2012 Keywords: Sulfenylation Peroxiredoxin Redox potential Density functional theory Free radicals Protein sulfenic acids are essential cysteine oxidations in cellular signaling pathways. The thermodynamics that drive protein sulfenylation are not entirely clear. Experimentally, sulfenic acid reduction potentials are hard to measure, because of their highly reactive nature. We designed a calculation method, the reduction potentials from electronic energies (REE) method, to give for the first time insight into the thermodynamic aspects of protein sulfenylation. The REE method is based on the correlation between reaction path- independent reaction energies and free energies of a series of analogous reactions. For human peroxiredoxin (Tpx-B), an antioxidant enzyme that forms a sulfenic acid on one of its active-site cysteines during reactive oxygen scavenging, we found that the reduction potential depends on the composition of the active site and on the protonation state of the cysteine. Interaction with polar residues directs the RSO - /RS - reduction to a lower potential than the RSOH/RSH reduction. A conserved arginine that thermodynamically favors the sulfenylation reaction might be a good candidate to favor the reaction kinetics. The REE method is not limited to thiol sulfenylation, but can be broadly applied to understand protein redox biology in general. © 2012 Elsevier Inc. All rights reserved. In an oxygen-dependent environment, oxidative stress is always lying in wait. When oxygen is incompletely reduced, reactive oxygen species (ROS) such as peroxide and oxide radicals can arise as natural by-products of oxygen metabolism or from exposure to external agents such as light, ionizing radiation, or redox drugs [1,2]. In partic- ular, the electron-rich, polarizable element sulfur is very sensitive to oxidation, making the cysteine and methionine residues the most oxidation-sensitive targets in proteins. The cysteine thiol (–SH) can easily be oxidized to a sulfenic acid (–SOH) by two-electron oxidants such as peroxides, haloamines, hypohalides, and peroxynitrites [3]. Sulfenic acid formation, also called sulfenylation, has long been regarded as a harmful cysteine modification, but is nowadays known as an essential intermediate in intramolecular disulfide bond formation and as a messenger in signaling pathways [2]. It is essential to know which protein structural elements drive sulfenylation. For this study, we focus on thioredoxin peroxidase B (Tpx-B) from human erythrocytes. Peroxiredoxins (Prx's) have evolved to protect the cellular machinery from the potentially harmful consequences of oxidation [4]. The Prx enzymes are ubiquitous thiol-dependent thioredoxin peroxidases. Basically, all Prx's use similar reaction mech- anisms [5]. In the first step, the active-site cysteine (often referred to as peroxidatic cysteine, Cys P ) is oxidized to sulfenic acid (Cys P –OH). In the subsequent steps, Cys P –OH is regenerated to Cys p –SH via a free thiol from the resolving cysteine (Cys R ), leading to the formation of the Cys P –Cys R disulfide [6,7]. Tpx-B is an obligate homodimer hav- ing Cys P (Cys51) and Cys R (Cys172) on different subunits (Fig. 1A) [8]. A third cysteine residue (Cys70) is not involved during catalysis. Highly conserved polar residues Tyr43, Thr48, Glu54, Arg127, and Arg150 and nonpolar residues Pro44, Val50, and Trp86 constitute the active site around Cys51 (Figs. 1B and C) [8,9]. The conserved res- idues surrounding Cys172 and Cys70 are less polar than those of Cys51 (Fig. 1D). The thiolate (S - ) form of Cys51 has a larger reactivity than the thiol (SH) form toward oxidation [3]. Although a wealth of kinetic data is available for the reaction of Prx's with various oxidants such as H 2 O 2 , peroxynitrite, and organic hydroperoxides [10–14], the driving force behind this oxidation, i.e., the reduction potential, is not known. Experimentally determined enzymatic catalyzed and noncata- lyzed RSOH/RSH reduction potentials are not available. This is pre- sumably due to the highly reactive nature of sulfenic acids [15] and high reaction rates between thiols and oxidants [3]. For example, rate constants on the order of ~10 8 M -1 s -1 (pH 7.4, 37 °C) are reported for the oxidation of Prx2 with H 2 O 2 [16], but they vary depending on the studied Prx and oxidant [2]. Computational chem- istry might be helpful in unraveling the sulfenylation thermodynam- ics. Reduction potentials can be calculated in good accordance with experiments for small and medium-sized systems. High-level, com- putationally expensive free energy calculations are performed in a thermodynamic cycle linking the process in the gas phase with that in solvent [17–19]. Alternatively, strategies based on free energy Free Radical Biology & Medicine 52 (2012) 1473–1485 ⁎ Corresponding authors. Fax: + 32 2 6291963. E-mail addresses: joris.messens@vib-vub.be (J. Messens), groos@vub.ac.be (G. Roos). 0891-5849/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2011.12.029 Contents lists available at SciVerse ScienceDirect Free Radical Biology & Medicine journal homepage: www.elsevier.com/locate/freeradbiomed