Disulfide bonds: cysteine and cystine template

A cysteine thiolate attacks an oxidised disulfide (in cells PDI or GSSG) and a second thiolate closes cystine. DTT reverses it: one thiolate cuts the S–S bond, the other closes DTT’s own six-membered ring.

Mechanism step by step

Two cysteine thiols are oxidised to the S–S bond of cystine; thiols such as DTT reduce it again by thiol–disulfide exchange

1. Thiol–disulfide exchange – Oxidation: a Cys thiolate attacks the oxidant’s S–S (R′ = glutathione or PDI)
2. Second exchange – Another Cys thiolate releases R′S⁻ and closes cystine
3. Reduction by DTT – A DTT thiolate cuts the S–S bond: a mixed disulfide
4. Ring closure – DTT’s second thiolate attacks: oxidised DTT (a 1,2-dithiane) and free cysteine

Key points

• Overall 2 R–SH ⇌ R–S–S–R + 2 H⁺ + 2 e⁻. The thiolate (Cys SH pKa ≈ 8.3) is the nucleophile, attacking S in an SN2-like way.
• Disulfides form in the oxidising ER (protein disulfide isomerase) and outside cells; the cytosol, rich in glutathione (GSH), keeps cysteines reduced.
• DTT (Cleland’s reagent) reduces completely at low concentration because its second step is intramolecular; β-mercaptoethanol needs a large excess.
• S–S bridges link insulin’s A and B chains and stabilise secreted proteins. They are reduced (and the thiols blocked) before SDS-PAGE or sequencing.

Drawn with 149 atoms, 13 arrows, 8 shapes – every one editable in MolDraw.

Sources

• Nelson & Cox, Lehninger Principles of Biochemistry, 8th ed., 2021, ch. 3 Textbook
• Berg, Tymoczko, Gatto & Stryer, Biochemistry, 9th ed., 2019 Textbook
• Cleland, Biochemistry 1964, 3, 480 Original paper