Xenobiotic Bi3+ Coordination by Cysteine-Rich Metallothionein-3 Reveals a Cooperatively Formed Thiolate-Sharing Bi2S5 Cluster

Bismuthbinding to apo-MT3 at physiological pH results inthe formation of a novel Bi2S5 species witha bridging cysteine, indicated by the appearance of a bright yellowcolor from absorption at 460 nm. This contrasts with low pH conditions,where the bridging cysteine is replaced with two terminal cysteines,resulting in a series of overlapping bands in the usual S-Bicharge transfer absorption band near 350 nm. The field of designing artificialmetalloproteins has yet to effectivelytackle the incorporation of multimetal clusters, which is a key componentof natural metalloproteins, such as metallothioneins (MTs) and calmodulin.MT is a physiological, essential, cysteine-rich metalloprotein thatbinds to a variety of metals but is only known to form metal-thiolateclusters with Cd2+, Zn2+, and Cu+. Bismuth is a xenobiotic metal and a component of metallodrugs usedto treat gastric ulcers and cancer, as well as an emerging metal usedin industrial practices. Electrospray ionization mass spectrometry,UV-visible spectroscopy, and extended X-ray absorption finestructure spectroscopy were used to probe the Bi3+ bindingsite structures in apo-MT3 (brain-located MT) at pH 7.4 and 2 andprovide the complete set of binding affinities. We discovered thehighly cooperative formation of a novel Bi3+ species, Bi(2)MT3, under physiological conditions, where each Bi3+ ion is coordinated by three cysteinyl thiolates, with one of thethiolates bridging between the two Bi3+ ions. This clusterstructure was associated with a strong visible region absorption band,which was disrupted by the addition of Zn2+ and reversiblydisrupted by acidification and increased temperature. This is thefirst reported presence of bridging cysteines for a xenobiotic metalin MT3 and the Bi2MT structure is the first Bi clusterfound in a metalloprotein.

Automated summary

At physiological pH, bismuth binds to apo-MT3 and forms a novel Bi2S5 species with a bridging cysteine, resulting in a bright yellow color from absorption at 460 nm. The binding affinity and structure of Bi3+ in apo-MT3 were investigated using various spectroscopic techniques, revealing the formation of a highly cooperative Bi(2)MT3 species that is coordinated by three cysteinyl thiolates, with one thiolate bridging between two Bi3+ ions. This discovery highlights the presence of bridging cysteines for bismuth in MT3 and the first reported Bi cluster in a metalloprotein.