Xenobiotic Bi Coordination by Cysteine-Rich Metallothionein-3 Reveals a Cooperatively Formed Thiolate-Sharing BiS 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, Zn, 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 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 species, BiMT3, under physiological conditions, where each Bi ion is coordinated by three cysteinyl thiolates, with one of the thiolates bridging between the two Bi ions. This cluster structure was associated with a strong visible region absorption band, which was disrupted by the addition of Zn 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 BiMT structure is the first Bi cluster found in a metalloprotein.