Corrinoid catalysis of thiol oxidation

Abstract Under appropriate conditions, thiols can react with corrinoids to form relatively stable complexes [1–7], or reduced corrinoids [1, 2, 8–11]. If alkyl halides are present during the reduction of corrinoids by thiols, alkyl corrinoids are produced [4, 9, 12, 13]. In the presence of oxidizing substrates, corrinoids will efficiently catalyze the oxidation of thiols to their corresponding disulfides [14–18]. In view of the important role that enzyme sulfhydryl groups play in corrinoid–coenzyme-dependent catalysis [19, 20], we have further characterized aerobic thiol oxidation catalyzed by a selected group of biologically important corrinoids and have determined the stoichiometry of the reaction. Hydrogen peroxide and superoxide have been identified as reaction products during the aerobic catalysis of 2-mercaptoethanol [ME, 21] by the corrinoids listed in Table I. The reactions were conducted in a polarographic cell equipped with a Clark O 2 electrode [22]. The corrinoids used to initiate the reactions were prepared as described previously [23, 24] and their purity was determined by hplc [25]. H 2 O 2 was detected by adding catalase to the reaction system. This resulted in an abrupt increase in O 2 concentration which is consistent with the catalytic activity of the enzyme. As anticipated, the pseudo-first order rate constant (k 1 ) of O 2 consumption in the presence of catalase decreased 50 per cent. Using a similar approach, superoxide dismutase was used in an attempt to detect the presence of O − 2 as a reaction product. This was unsuccessful except for one corrinoid, namely Aq-Cbl (Table I). Thus, during the catalysis of ME oxidation by Aq-Cbl, O − 2 is the primary reaction product. In the presence of dismutase, k 1 for the Aq-Cbl-catalyzed reaction decreased approximately 50 per cent which is consistent with the catalytic activity of this enzyme. H 2 O 2 was also detected during Aq-Cbl-catalyzed oxidation of ME and may have been produced by the spontaneous dismutation of O − 2 . Disulfide bond formation was monitored spectrophotometrically [26] during the oxidation of DTE. Pseudo-first-order rate constants for O 2 disappearance and for DTE ox appearance were in good agreement (Table II). These results, which differ from previously published studies [16, 17, 27], suggest that the reactions and their stoichiometries for aerobic oxidation of mono- and dithiols by corrinoids are: 2 RSH + O 2 → RSSR + H 2 O 2 (1) t001 . Pseudo-First-Order Rate Constants, pH Optima, Products and Photosensitivity for Catalysis of ME Oxidation by Corrinoids. a Group Catalyst k 1 , sec −1 pH Optimum Reduced Product k 1 , sec −1 (after photolysis) b I AdoCbl 0.003 Broad H 2 O 2 0.102 (20 sec) MeCbl 0.003 7.0 c H 2 O 2 0.184 (20 sec) II CNCbl 0.080 8.0 c H 2 O 2 0.080 (60 sec) AqCbl 0.180 8.4 HO 2 0.180 (20 sec) AdoCbi 0.23 H 2 O 2 177.0 (60 sec) MeCbi 0.56 11–13 H 2 O 2 122.0 (90 sec) III CNCbi 191 8.5–9.5 H 2 O 2 190.0 (30 sec) (Aq) 2 Cbi 211 8.8 H 2 O 2 210.0 (60 sec) a ME = 2.5 × 10 −2 ; 25 ± 0.2 °C. b At the midway point of O 2 consumption, the polarographic cell was irradiated with light from a tungsten-filament high intensity lamp for the time indicated. c Measurement was made at the indicated pH only. t002 . Pseudo-First-Order Rate Constants for the Disappearance of O 2 and the Formation of Oxidized DTE. 1 Catalyst O 2 Disappearance k 1 , sec −1 2 DTE ox Formation k 1 , sec −1 3 Ratio O 2 DTE ox AqCbl 0.57 0.63 0.91 AdoCbi 0.49 0.46 1.07 (Aq) 2 Cbi 567 597 0.95 1 Initial substrate DTE = 1.25 × 10 −2 M ; pH = 8.0; 25 ± 0.2 °C. 2 The rate of O 2 disappearance was determined polarographically. 3 The rate of oxidized DTE formation was determined spectrophotometrically. The results of dismutase study and the data in Table II suggest that, in the case of AqCbl, the reactions are: Autocatalytic behavior was not observed for reactions catalyzed by the alkylcobalamins (AdoCbl and MeCbl) and the alkylcobinamides (AdoCbi and MeCbi) suggesting that the carboncobalt bond remains intact during the catalytic cycle. Similar observations have been made by Pellizer {et al.} [28] for the catalysis of cysteine oxidation by the model compound [MeCo(tn)H 2 O] + . The corrinoids in Table I have been grouped according to their catalytic activity. The alkylcobalamins (AdoCbl and MeCbl; Group I) are poor catalysts. The second group of corrinoids includes CNCbl, AqCbl, AdoCbi and MeCbi. The most active catalysts (CNCbi and (Aq) 2 Cbi; Group III) are cobinamides which lack covalently attached ligands. Thus, the catalytic activity of corrinoids with respect to thiol oxidation appears to be determined by the number (1 vs. 2) and accessibility (coordinate vs. covalent) of axial-ligand positions.