The extraordinary catalytic ability of peroxiredoxins: a combined experimental and QM/MM study on the fast thiol oxidation step

Abstract

Peroxiredoxins (Prxs) catalyze the reduction of peroxides, a process of key relevance in a variety of cellular processes. The first step in the catalytic cycle of all Prxs is the oxidation of a cysteine residue to sulfenic acid, which occurs 103-107 times faster than in free cysteine. We present an experimental kinetics and hybrid QM/MM investigation to explore the reaction of Prxs with H2O2 using alkyl hydroperoxide reductase E from Mycobacterium tuberculosis as a Prx model. We report for the first time the reaction thermodynamical activation parameters of H2O2 reduction by a Prx, which show that the protein lowers significantly the activation enthalpy, with an unfavourable entropic effect, compared to the uncatalyzed reaction. The QM/MM simulations show that the remarkable catalytic effects responsible for the fast H2O2 reduction in Prxs are mainly due to an active-site arrangement, which establishes a complex hydrogen bond network activating both reactive species.

Selenocysteine oxidation in glutathione peroxidase catalysis: an MS-supported quantum mechanics study

“…It allows a charge separation that, irrespective of absence or presence of substrate, leads to abstraction of the proton from the catalytic selenol or thiol, respectively. Only in the presence of water, the DFT calculations yielded the selenolate or thiolate form of the GPx model, which is amply documented to prevail at physiological pH in natural GPxs [49,50] and analogous peroxiredoxins [54,55]. In the latter enzyme family, the active site thiolate is essentially generated by positive charges of conserved arginine residues and a Cys-coordinated Thr (Ser) [55,56].…”

“…Only in the presence of water, the DFT calculations yielded the selenolate or thiolate form of the GPx model, which is amply documented to prevail at physiological pH in natural GPxs [49,50] and analogous peroxiredoxins [54,55]. In the latter enzyme family, the active site thiolate is essentially generated by positive charges of conserved arginine residues and a Cys-coordinated Thr (Ser) [55,56]. Strong basic residues being missing in the active site of GPxs, it is here the water-mediated charge separation that provides the nucleophilicity to Sec (Cys) required to react with a hydroperoxide.…”

The thiol of human serum albumin: Acidity, microenvironment and mechanistic insights on its oxidation to sulfenic acid

“…Both thiol environments lack a positively charged entity proximal to stabilize the anionic TS by electrostatic interaction, a crucial factor largely recognized to enhance Cys nucleophilicity in different protein environments, in particular in more reactive ones present in other mammalian albumins [34] and peroxiredoxins [19,20,22,67]. Thus, enthalpy barriers are expected to be significantly higher for HSA−SH and free Cys in solution [66] than for peroxiredoxin active sites (10 3 -10 7 times faster than free cysteine) where wellconserved protonated Arg residues are proximal to the peroxidatic Cys.…”

“…Thus, enthalpy barriers are expected to be significantly higher for HSA−SH and free Cys in solution [66] than for peroxiredoxin active sites (10 3 -10 7 times faster than free cysteine) where wellconserved protonated Arg residues are proximal to the peroxidatic Cys. Details from calculated and experimental thermochemistry corresponding to hPrx5 [20] and MtAhpE [19] peroxiredoxins are also collected in Table 3. This data enables a more extended comparison, evidencing differences in the contributions that underlie a very similar outcome in terms of free-energy barriers, probably attributable to the absence/presence of water molecules at each active site and to differences in the nature and strength of the HB networks established at the corresponding Michaelis complex (RC) and TS pairs.…”

“…Following the recognition that the reactions of thiols with H2O2 can participate in catalysis and regulation [9,10], these processes have received considerable interest in the last decade, both from experimental and theoretical points of view. Within the latter, a wide variety of computational strategies have been applied to address the reactivity of thiols and the mechanisms of H2O2 reactions with different LMWTs [11][12][13][14], with reduced models mimicking protein environments [15][16][17], and within whole proteins under different conditions [18][19][20][21]. A consensus on the SN2-character of Cys thiolate oxidation by H2O2 emerges from the studies conducted on LMWTs [11,12,14] and proteins [18][19][20][21][22][23].…”