Xenobiotic Bi 3+ Coordination by Cysteine-Rich Metallothionein-3 Reveals a Cooperatively Formed Thiolate-Sharing Bi 2 S 5 Cluster.

The field of designing artificial metalloproteins has yet to effectively tackle the incorporation of multimetal clusters, which is a key component of natural metalloproteins, such as metallothioneins (MTs) and calmodulin. MT is a physiological, essential, cysteine-rich metalloprotein that binds to a variety of metals but is only known to form metal-thiolate clusters with Cd 2+ , Zn 2+ , and Cu + . Bismuth is a xenobiotic metal and a component of metallodrugs used to treat gastric ulcers and cancer, as well as an emerging metal used in industrial practices. Electrospray ionization mass spectrometry, UV-visible spectroscopy, and extended X-ray absorption fine structure spectroscopy were used to probe the Bi 3+ binding site structures in apo-MT3 (brain-located MT) at pH 7.4 and 2 and provide the complete set of binding affinities. We discovered the highly cooperative formation of a novel Bi 3+ species, Bi 2 MT3, under physiological conditions, where each Bi 3+ ion is coordinated by three cysteinyl thiolates, with one of the thiolates bridging between the two Bi 3+ ions. This cluster structure was associated with a strong visible region absorption band, which was disrupted by the addition of Zn 2+ and reversibly disrupted by acidification and increased temperature. This is the first reported presence of bridging cysteines for a xenobiotic metal in MT3 and the Bi 2 MT structure is the first Bi cluster found in a metalloprotein.