Inactivation of thiol-dependent enzymes by hypothiocyanous acid: role of sulfenyl thiocyanate and sulfenic acid intermediates

Abstract

Myeloperoxidase (MPO) forms reactive oxidants including hypochlorous and hypothiocyanous acids (HOCl and HOSCN) under inflammatory conditions. HOCl causes extensive tissue damage and plays a role in the progression of many inflammatory-based diseases. Although HOSCN is a major MPO oxidant, particularly in smokers, who have elevated plasma thiocyanate, the role of this oxidant in disease is poorly characterized. HOSCN induces cellular damage by targeting thiols. However, the specific targets and mechanisms involved in this process are not well defined. We show that exposure of macrophages to HOSCN results in the inactivation of intracellular enzymes, including creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In each case, the active-site thiol residue is particularly sensitive to oxidation, with evidence for reversible inactivation and the formation of sulfenyl thiocyanate and sulfenic acid intermediates, on treatment with HOSCN (less than fivefold molar excess). Experiments with DAz-2, a cell-permeable chemical trap for sulfenic acids, demonstrate that these intermediates are formed on many cellular proteins, including GAPDH and CK, in macrophages exposed to HOSCN. This is the first direct evidence for the formation of protein sulfenic acids in HOSCN-treated cells and highlights the potential of this oxidant to perturb redox signaling processes.

Role of Myeloperoxidase Oxidants in the Modulation of Cellular Lysosomal Enzyme Function: A Contributing Factor to Macrophage Dysfunction in Atherosclerosis?

“…This conclusion is supported by the observation that HOSCN-induced cathepsin inactivation could be reversed by DTT addition, which is consistent with the formation of Cys-derived oxidation products, such as sulfenic acids, as observed previously in macrophages exposed to this oxidant [40]. No evidence was obtained for oxidant-induced inactivation of cathepsin D, which has similar considerable sequence homology to cathepsins B and L, but has an Asp residue, rather than Cys in its active site, which is not reactive with HOSCN [39].…”

Biological chemistry and functionality of protein sulfenic acids and related thiol modifications

“…The factors driving the reactivity of thiols towards oxidants have been eloquently reviewed and are not discussed here [1]. In biological systems the pool of oxidants responsible for -SOH formation include hydroxyl radical (HO • ) [2], hydrogen peroxide (H 2 O 2 ) [3], peroxynitrite (ONOO − ) [3], hypochlorous acid (HOCl) [4], and hypothiocyanous acid (HOSCN) [5]. Oxygen atom transfer from nitrite to –SH during the reduction of Fe(III)-NO 2 complex to Fe(II)-NO can also generate sulfenic acid [6,7].…”

Pseudomonas aeruginosa defense systems against microbicidal oxidants

“…Nevertheless, HOCl and HOBr are both associated with numerous diseases, including initiation and progression of various inflammatory diseases, such as atherosclerosis (Klebanoff, ; Davies et al ., ; Davies, ). HOSCN appears to react with great specificity with thiols (e.g., GSH; protein cysteines) and selenogroups (Skaff et al ., ), resulting in a number of reversible oxidation products, including sulfenyl thiocyanates, sulfenic acids or disulfides (Barrett et al ., ). High concentrations of HOSCN, however, result in non‐reversible thiol products including sulfinic/sulfonic acids (Barrett and Hawkins, ).…”