Disulfide bridges in proteins

Disulfide (sulfur-sulfur) linkages between two cysteine residues are an integral component of the three-dimensional structure of many proteins. The interconversion between thiols and disulfide groups is a redox reaction: the thiol is the reduced state, and the disulfide is the oxidized state.

Notice that in the oxidized (disulfide) state, each sulfur atom has lost a bond to hydrogen and gained a bond to a sulfur - this is why the disulfide state is considered to be oxidized relative to the thiol state. The redox agent that mediates the formation and degradation of disulfide bridges in most proteins is glutathione, a versatile coenzyme that we have met before in a different context (section 14.2A). Recall that the important functional group in glutathione is the thiol, highlighted in blue in the figure below. In its reduced (free thiol) form, glutathione is abbreviated 'GSH'.

In its oxidized form, glutathione exists as a dimer of two molecules linked by a disulfide group, and is abbreviated 'GSSG'.

A new disulfide in a protein forms via a 'disulfide exchange' reaction with GSSH, a process that can be described as a combination of two S_N2-like attacks. The end result is that a new cysteine-cysteine disulfide forms at the expense of the disulfide in GSSG.

In its reduced (thiol) state, glutathione can reduce disulfides bridges in proteins through the reverse of the above reaction.

Disulfide bridges exist for the most part only in proteins that are located outside the cell. Inside the cell, cysteines are kept in their reduced (free thiol) state by a high intracellular concentration of GSH, which in turn is kept in a reduced state (ie. GSH rather than GSSG) by a flavin-dependent enzyme called glutathione reductase.

Disulfide bridges in proteins can also be directly reduced by another flavin-dependent enzyme called 'thioredoxin'. In both cases, NADPH is the ultimate electron donor, reducing FAD back to FADH₂ in each catalytic cycle.

In the biochemistry lab, proteins are often maintained in their reduced (free thiol) state by incubation in buffer containing an excess concentration of b-mercaptoethanol (BME) or dithiothreitol (DTT). These reducing agents function in a manner similar to that of GSH, except that DTT, because it has two thiol groups, forms an intramolecular disulfide in its oxidized form.