Sulfane sulfur, a chemical state of sulfur atoms with six valence electrons, has been a topic of interest in cellular redox homeostasis. However, its role in zinc regulation has been less explored. This study identifies growth inhibitory factor (GIF)/metallothionein-3 (MT-3) as a sulfane sulfur-binding protein, revealing an unexpected C-S-S-Zn structure. The oxidation of this zinc/persulfide cluster leads to zinc ion release, and the intramolecular tetrasulfide bridges in apo-GIF/MT-3 can be cleaved by thioredoxin, regenerating Zn7GIF/MT-3. Three-dimensional molecular modeling confirms the critical role of the persulfide group in the thermostability and Zn-binding affinity of GIF/MT-3.
Cysteine-rich proteins, like metallothionein (MT), act as 'redox switches' in biological systems, sensing oxidative stressors and free zinc ions. MT is involved in essential metal homeostasis, detoxification, and protection from oxidative stress. While the metal binding to MT is stable, oxidation of the thiolate cluster leads to metal release and the formation of MT-disulfide linkages. This study focuses on MT-3, originally identified as a growth inhibitory factor in the human brain, to understand the existence and content of sulfane sulfur in GIF/MT-3 and its role in zinc release and thermostability.
Using an Escherichia coli expression system, recombinant human Zn7GIF/MT-3 protein was prepared, and its molecular weight was measured with and without bound zinc using Fourier transform ion cyclotron resonance (FT-ICR)-MALDI-TOF/MS. The results indicated that zinc dissociates from the protein in acidic conditions. Raman spectroscopy was used to detect bonding changes, confirming the presence of sulfane sulfur atoms in both Zn7GIF/MT-3 and apo-GIF/MT-3.
To determine the sulfane sulfur atoms in Zn7GIF/MT-3, β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM) was used to derivatize the sulfane sulfur, allowing quantitative analysis using LC-MS/MS with a stable isotope-labeled standard. The results showed that each MT isoform possessed approximately 20 sulfane sulfurs, and all 20 sulfane sulfurs were bound to cysteine residues of Zn7GIF/MT-3.
The stability of sulfane sulfur in GIF/MT-3 with or without bound zinc was examined. Freshly prepared Zn7GIF/MT-3 and apo-GIF/MT-3 possessed similar amounts of sulfane sulfur, but in apo-GIF/MT-3, the content decreased markedly within 12 hours of incubation. The addition of zinc blocked any further decrease, suggesting that zinc ions stabilize sulfane sulfur atoms.
To explore the functional role of sulfane sulfur in GIF/MT-3, the interaction of sulfane sulfur species with KCN was used to yield thiocyanate and thiol products. KCN treatment decreased the sulfane sulfur atom content of Zn7GIF/MT-3, and subsequent addition of the reducing agent TCEP did not recover the level of sulfane sulfur in GIF/MT-3. This indicates that KCN indeed removed sulfane sulfur from GIF/MT-3.
Thioredoxin (Trx) is a master enzyme that reduces disulfide bonds in cellular proteins. Surprisingly, apo-GIF/MT-3 was a much more efficient substrate for Trx than insulin. Trx was unable to reduce Zn7GIF/MT-3, suggesting that the zinc/persulfide clusters in Zn7GIF/MT-3 may block the interaction with Trx.
Three-dimensional modeling of GIF/MT-3 with sulfane sulfur atoms was performed using the Molecular Operating Environment (MOE) software and Protein Data Bank (PDB) structures. The results showed that the addition of sulfane sulfur atoms to each cysteine residue increased the thermostability and metal-binding affinity of GIF/MT-3.
In conclusion, this study provides evidence that sulfane sulfur plays a central role in the hold-and-release regulation of zinc by GIF/MT-3. The findings open new directions of research in redox and metals biology, highlighting the importance of sulfane sulfur in hold-and-release regulation of zinc ions by zinc-binding proteins.
