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

Human serum albumin (HSA) has a single reduced cysteine residue, Cys34, whose acidity has been controversial. Three experimental approaches (pH-dependence of reactivity towards hydrogen peroxide, ultraviolet titration and infrared spectroscopy) are used to determine that the pK_a value in delipidated HSA is 8.1±0.2 at 37°C and 0.1M ionic strength. Molecular dynamics simulations of HSA in the sub-microsecond timescale show that while sulfur exposure to solvent is limited and fluctuating in the thiol form, it increases in the thiolate, stabilized by a persistent hydrogen-bond (HB) network involving Tyr84 and bridging waters to Asp38 and Gln33 backbone. Insight into the mechanism of Cys34 oxidation by H₂O₂ is provided by ONIOM(QM:MM) modeling including quantum water molecules. The reaction proceeds through a slightly asynchronous S_N2 transition state (TS) with calculated Δ^‡G and Δ^‡H barriers at 298K of respectively 59 and 54kJmol^-1 (the latter within chemical accuracy from the experimental value). A post-TS proton transfer leads to HSA-SO^- and water as products. The structured reaction site cages H₂O₂, which donates a strong HB to the thiolate. Loss of this HB before reaching the TS modulates Cys34 nucleophilicity and contributes to destabilize H₂O₂. The lack of reaction-site features required for differential stabilization of the TS (positive charges, H₂O₂ HB strengthening) explains the striking difference in kinetic efficiency for the same reaction in other proteins (e.g. peroxiredoxins). The structured HB network surrounding HSA-SH with sequestered waters carries an entropic penalty on the barrier height. These studies contribute to deepen the understanding of the reactivity of HSA-SH, the most abundant thiol in human plasma, and in a wider perspective, provide clues on the key aspects that modulate thiol reactivity against H₂O₂.