The enigmatic loss of hyper-reactivity toward GSSG in structural protein cysteines during evolution: the case of transferrin superfamily.

Oxidative protein folding remains a subject of intense investigation, with ongoing debate surrounding the specific molecular actors driving this process. In this context, the involvement of oxidized glutathione (GSSG) is supported by the recent discovery of an enigmatic, previously undescribed hyper-reactivity toward this disulfide. This phenomenon is exhibited by structural cysteines within the reduced, molten-globule intermediates of several proteins, including bovine serum albumin, hen-egg lysozyme, human lysozyme, ribonuclease A, trypsinogen, chymotrypsinogen, and lactoferrin. Notably, four cysteines in lactoferrin recently demonstrated a thousand times higher reactivity toward GSSG - compared to that of unperturbed protein cysteines - suggesting that this glutathionylation event may trigger its oxidative folding. The present study focuses on the structural and kinetic properties of the molten globule status of serum transferrin and ovotransferrin, two iron binding proteins of the transferrin superfamily. These results are compared with previous findings for lactoferrin, a member of the same superfamily. Despite the evolutionary correlation, transferrin and ovotransferrin display cysteines with distinct kinetic properties and lack hyper-reactivity toward GSSG. Structural analysis and fluorometric evidence indicate that, over evolutionary time, these proteins lost the ability to properly bind GSSG, thereby abolishing hyper-reactivity. Conversely, pKa values of their cysteines are scattered across a wide range, resulting in a hierarchical reactivity toward GSSG that may be crucial for correct disulfide bond formation.