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Journal of Molecular Structure: THEOCHEM 772 (2006) 89–92

DFT study of the uncatalyzed dioxygenation of acireductone

Tomasz Borowski a,*, Arianna Bassan b, Per E.M. Siegbahn b

a Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, ul. Niezapominajek 8, 30-239 Krakow, Polandb Department of Physics, Stockholm Center for Physics, Astronomy and Biotechnology, Stockholm University, S-106 91 Stockholm, Sweden

Received 5 June 2006; received in revised form 13 June 2006; accepted 21 June 2006Available online 27 June 2006

Abstract

Results of DFT calculations suggest that the uncatalyzed reaction between acireductone and dioxygen has a radical mechanism. In aninitiation step, calculated to be thermo-neutral, acireductone is one-electron oxidized by dioxygen to a radical species. This radical inter-mediate reacts with triplet dioxygen forming a ketoacid radical and formate. In a spontaneous propagation step acireductone reduces theketoacid radical to the ketoacid anion.� 2006 Elsevier B.V. All rights reserved.

Keywords: DFT; Reaction mechanism; Acireductone; Dioxygen

Acireductone (ACR; 1,2-dihydroxy-3-keto-5-(methyl-thio)pentene; see Fig. 1) is an intermediate formed in acommon metabolic cycle called the methionine salvagepathway [1,2]. Two enzymes that catalyze the oxidationof ACR by dioxygen were identified, and interestingly, theyshare a polypeptide chain and differ only in the identity of ametal ion bound in the active site [3]. ARD 0, which bindsFe2+ in the active site, catalyzes an oxidative carbon–car-bon bond cleavage yielding a-keto acid and formate (pathsB in Fig. 1), while ARD, which binds Ni2+ ion, catalyzes acleavage of two adjacent carbon–carbon bonds producingcarbon monoxide and two carboxylic acids (path A inFig. 1) [4,5]. The uncatalyzed reaction between acireduc-tone and dioxygen was reported to give the same productsas the reaction catalyzed by ARD 0, i.e., ketoacid and for-mate [1,6]. The intriguing metal-dependent product speci-ficity of the ARD enzymes together with the importanceof the methionine salvage pathway for cell division makethat the oxidation of ACR is a significant chemical reac-tion. From a medical perspective, an interesting observa-tion is that the level of ARD expression regulates celldeath of prostate cancer [7].

0166-1280/$ - see front matter � 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.theochem.2006.06.025

* Corresponding author. Tel.: +48 12 6395101; fax: +48 12 4251923.E-mail address: ncborows@cyf-kr.edu.pl (T. Borowski).

Relatively little is known about molecular mechanismsof ACR/O2 reactions. The results of isotope labeling exper-iments show that oxygen atoms derived from dioxygen arepresent in the acidic products, one atom per acid molecule.The carbon atom in CO originates exclusively from the C-2carbon of ACR [4,5]. Mechanistic studies employing a sub-strate analogue with a cyclopropyl substituent indicatedthat a radical intermediate could be present in the catalyticcycles of ARD and ARD 0 [6]. Based on these results, themechanisms depicted in Fig. 1 were proposed for ARDand ARD 0.

Thus, in the first step the enzyme-bound substrate is oxi-dized by triplet oxygen, which produces a radical pair thatsubsequently collapses and forms a peroxide species. Then,for ARD(Ni2+) the reaction involves a cyclic peroxide witha five-membered ring (path A), while ARD 0(Fe2+) eitherstabilizes a cyclic peroxide with a four-membered ring orfacilitates the Baeyer–Villiger rearrangement (paths B).

Formation of the peroxide with a five-membered ringhas an experimental precedent, since such a species is aproduct of a reaction between H2O2 and 2,4-pentanedione[8]. Moreover, the model triketone (2,3,4-pentanetrione)reacts rapidly with H2O2 producing exclusively CO andacetic acid, and this reaction most likely also involves anintermediate with a five-membered ring (see Fig. 2) [6].Thus, if in the uncatalyzed reaction between ACR and

Fig. 2. The model reaction between 2,3,4-pentanetrione and H2O2.

A

B

Fig. 3. The calculated energetics for the suggested mechanism of theuncatalyzed ACR/O2 reaction: (A) the radical initiation step, (B)propagation paths.

Fig. 1. The experiment-motivated mechanisms for the catalytic reactionsof: (A) ARD and (B) ARD 0.

90 T. Borowski et al. / Journal of Molecular Structure: THEOCHEM 772 (2006) 89–92

O2 the peroxo species were present, one would expect thatan intermediate with a five-membered ring is also formed.Such a species would decompose to CO and two carboxylicacids. However, the experimental results indicate that atpH 7.5 and 13 the only products are a-ketoacid and for-mate, and that the reaction at the higher pH is 66 timesfaster [6]. Results of the computational study, which arecommunicated here, provide a possible explanation forthe observed product specificity of the uncatalyzed reactionbetween acireductone and dioxygen.

A series of DFT calculations were performed with amodel compound (R = CH3; see Fig. 1), which is a non-physiological substrate of both ARD enzymes [6]. Thetwo hydroxyl groups of ACR have pKas of 4.0 and 12.2,and these values are very close to the pKas of ascorbic acid,which are 4.2 and 11.6. Thus, one might suspect that thetwo reductants may have similar properties. In the biolog-ically relevant range of pH acireductone exists as a mono-anion species [6], and for this reason the ACR/O2 reactionwas modeled assuming a single ionization of the organicsubstrate. All geometry optimizations and frequency calcu-lations were done at the B3LYP/6-31+G* level of theory.The solvation effects were calculated at the UHF/6-31+G* level with the CPCM [9] method employing theUAKS atomic radii. This level of approximation hasrecently been shown to give reliable solvation energetics[10]. All calculations were done with Gaussian03 [11].The energies reported in Fig. 3 are Gibbs free energies cal-culated as a sum of electronic energy (B3LYP/6-31+G*),solvation free energy (UHF/6-31+G*), and thermal correc-tion dG calculated from harmonic frequencies (B3LYP/6-31+G*). For reactions involving proton release, theenergies were calculated from the computed values forthe protonated species, i.e., HO2, acetic and pyruvic acids.The free energy changes caused by ionization of these

species were calculated from the experimental pKa valuesand were added to the reaction free energies computedfor neutral compounds; it was assumed that the reactionstake place at pH 7.0 and 298 K (pKa = 4.68, 4.76 and2.39 for HO2, acetic and pyruvic acid, respectively). Eventhough the pKa of the ACR radical is currently unknown,it seems reasonable to assume that it is close to the pKa ofthe ascorbate radical, which is �0.86. Accordingly, in allcalculations species 2 is not protonated.

Several plausible mechanisms for the reaction in waterbetween ACR (1) and O2 were tested. The suggestedmechanism, which involves the lowest activation barrier,leads to ketoacid and formate, i.e., the experimentallyobserved products. This mechanism starts with oxidationof ACR by O2 (Fig. 3A), which yields the radical anion 2

and superoxide. The calculated DG of this reaction isexactly zero, while the activation free energy for H-atomtransfer between ACR and O2 is 8.9 kcal/mol. This initi-ation reaction might also proceed by an electron transferfrom ACR to O2 synchronous with a proton release tobulk water, but such a process cannot be studied withthe computational methods and models used here. Never-theless, the energies of TS1 and 2 show that the radicalspecies 2 can be formed easily. Notably, this initial stepis identical with the first step of the mechanisms proposedfor the enzymatic reactions (Fig. 1). In the subsequentstep, the radical anion 2 reacts with triplet dioxygen,

T. Borowski et al. / Journal of Molecular Structure: THEOCHEM 772 (2006) 89–92 91

and this process takes place on the doublet potential ener-gy surface (Fig. 3B). There are three groups of possibleproducts which can be formed in this process, and dueto the asymmetry in TS structures, i.e., the lengths ofthe two oxygen–carbon bonds formed in this step aremarkedly different, there are two transition states leadingto a given product, i.e., six TSs in total. Most important-ly, TS2 which leads to the ketoacid radical 3 and formatehas the lowest energy (12.6 kcal/mol), which is also mark-edly lower than the barriers for the competing reactions(the second lowest barrier: TS4, 19.8 kcal/mol). Oncethe ketoacid radical 3 is formed, it reacts with 1 yieldingthe ketoacid anion 5 and the acireductone radical 2; i.e., itis a radical propagation reaction. For this step TS couldnot be optimized because already for a model system withmarkedly separated reactants the electron transfer is com-pleted. Thus, it is very likely that the rate of the propaga-tion reaction is limited by diffusion. As already noticed,the alternative reactions of the ACR radical 2 and tripletO2, leading through TS3 and TS4, involve considerablyhigher activation barriers (Fig. 3B). The reaction involv-ing TS4 leads to the acid 6, formate and CO, i.e., theproducts of the enzymatic reaction catalyzed byARD(Ni2+), while TS3 leads to HCOCOO�, i.e., a prod-uct not observed for any of the ACR/O2 reactions. Usingthe transition state theory for bimolecular reactions in gasphase and the calculated barrier height (12.6 kcal/mol),the rate constant for the ACR/O2 reaction can be estimat-ed. Assuming a reasonable value for the Arrhenius pre-exponential factor A (4 · 1011 M�1 s�1) the predicted rateconstant is 230 M�1 s�1, which should be compared withthe experimental value of 0.12 M�1 s�1. This experimentalrate constant corresponds to a barrier of 17.0 kcal/mol,which means that the calculations underestimate thebarrier by 4.4 kcal/mol.

Two alternative mechanism, different from the one dis-cussed above, are presented in Fig. 4. In the first mecha-nism (Fig. 4A), triplet O2 attacks the acireductone anion 1and forms a biradical species, which through an intersys-tem crossing transforms to the peroxide intermediate. Thecalculated barrier connected with TS5 is 15.6 kcal/mol,i.e., it is 3 kcal/mol higher than the barrier for the sug-

A

B

Fig. 4. Two alternative mechanisms for ACR/O2 reaction: (A) directreaction between ACR and triplet O2, (B) recombination of the radicals.

gested mechanism shown in Fig. 3. In agreement withexperimental findings [6,8], the calculations indicate thatthe peroxide intermediate easily forms the species with afive-membered ring, which decomposes to CO and twocarboxylic acids. Other reactions of the singlet peroxideintermediate, that lead to a-ketoacid and formate, involveconsiderably higher activation barriers. In other words,formation of the singlet peroxide intermediate favorsrelease of CO.

In the second alternative mechanism, the singlet peroxospecies is formed in a recombination reaction betweensuperoxide and 2 (Fig. 4B). Since both of these speciesare negatively charged, it is expected that at least one ofthem has to be protonated to avoid unfavorable electro-static interaction. From the known pKa of HO2 (4.68), itcan be calculated that protonation of superoxide is ender-gonic by 3.9 kcal/mol at the experimental [6] pH 7.5(3.2 kcal/mol at pH 7.0). Once HO2 is formed, it can easilyreact with 2 to produce the singlet peroxide species. Start-ing with the radical pair in the electronic configuration ofan open-shell singlet (Fig. 4B), many attempts were madeto locate the proper TS for this recombination process,but all of them failed. Thus, the enthalpy barrier for thereaction shown in Fig. 4B could not be calculated due tothe very early character of the TS. However, even if theenthalpy barrier for this reaction was zero, the free energybarrier calculated with respect to the true reactants, i.e.,O2� and 2, would be 13.9 kcal/mol, since protonation of

superoxide is endergonic by 3.9 kcal/mol, and the entropycontribution to the barrier of such a reaction is usuallyaround 10.0 kcal/mol (dG calculated for TS5 = 10.0 kcal/mol). Thus, the estimated barrier for the process presentedin Fig. 4B is 1.3 kcal/mol higher than the barrier for therate-limiting step in the suggested mechanism (TS2,Fig. 3B). The question whether the recombination reactioncould compete with the radical mechanism in more acidicsolutions remains open.

In conclusion, the results of the DFT calculations indi-cate that the uncatalyzed reaction of ACR and O2 followsthe radical mechanism involving species: 1, TS1, 2, TS2, 3

and 5 (Fig. 3). Importantly, the ketoacid 5 is produced onlyin the reaction of the radical anion 2 with triplet O2. Othermechanisms lead to the singlet peroxide species and favorformation of CO and two acids. It is believed that theseresults provide new insight into the chemistry of acireduc-tone and will prove useful in the studies on the reactions ofthe ARD/ARD 0 enzymes.

Acknowledgement

T.B. acknowledges the support from the Polish StateCommittee for Scientific Research (Grant 2 P04A 042 26).

Appendix A. Supplementary data

Supporting Information Available. Cartesian coordi-nates, absolute energies, solvation energies, free energy

92 T. Borowski et al. / Journal of Molecular Structure: THEOCHEM 772 (2006) 89–92

corrections and imaginary frequencies for transition states.Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.theochem.2006.06.025.

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