Reactions of cisplatin (cis-[PtCl₂(NH₃)₂]) with albumin are thought to play an important role in the metabolism of this anticancer drug. They are investigated here via (i) labeling of cisplatin with ¹⁵N and use of two-dimensional¹H,¹⁵N NMR spectroscopy, (ii) comparison of natural human serum albumin with recombinant human albumin (higher homogeneity and SH content), (iii) chemical modification of Cys, Met, and His residues, (iv) reactions of bound platinum with thiourea, and (v) gel filtration chromatography. In contrast to previous reports, it is shown that the major sulfur-containing binding site involves Met and not Cys-34, and also a N ligand, in the form of an S,N macrochelate. Additional monofunctional adducts involving other Met residues and Cys-34 are also observed. During the later stages of reactions of cisplatin with albumin, release of NH₃ occurs due to the strong trans influence of Met sulfur, which weakens the Pt-NH₃ bonds, and protein cross-linking is observed. The consequences of these findings for the biological activity of cisplatin-albumin complexes are discussed.

Zinc is essential for many cellular processes, including DNA synthesis, transcription, and translation, but excess can be toxic. A zinc-induced gene,smtA, is required for normal zinc-tolerance in the cyanobacteriumSynechococcusPCC 7942. Here we report that the protein SmtA contains a cleft lined with Cys-sulfur and His-imidazole ligands that binds four zinc ions in a Zn₄Cys₉His₂cluster. The thiolate sulfurs of five Cys ligands provide bridges between the two ZnCys₄and two ZnCys₃His sites, giving two fused six-membered rings with distorted boat conformations. The inorganic core strongly resembles the Zn₄Cys₁₁cluster of mammalian metallothionein, despite different amino acid sequences, a different linear order of the ligands, and presence of histidine ligands. Also, SmtA contains elements of secondary structure not found in metallothioneins. One of the two Cys₄-coordinated zinc ions in SmtA readily exchanges with exogenous metal (¹¹¹Cd), whereas the other is inert. The thiolate sulfur ligands bound to zinc in this site are buried within the protein. Regions of β-strand and α-helix surround the inert site to form a zinc finger resembling the zinc fingers in GATA and LIM-domain proteins. Eukaryotic zinc fingers interact specifically with other proteins or DNA and an analogous interaction can therefore be anticipated for prokaryotic zinc fingers. SmtA now provides structural proof for the existence of zinc fingers in prokaryotes, and sequences related to the zinc finger motif can be identified in several bacterial genomes.

Summary: Zinc is essential but toxic in excess. Bacterial metallothionein, SmtA from Synechococcus PCC 7942, sequesters and detoxifies four zinc ions per molecule and contains a zinc finger structurally similar to eukaryotic GATA. The dearth of other reported bacterial metallothioneins has been surprising. Here we describe related bacterial metallothioneins (BmtA) from Anabaena PCC 7120, Pseudomonas aeruginosa and Pseudomonas putida that bind multiple zinc ions with high stability towards protons. Thiol modification demonstrates that cysteine coordinates zinc in all of these proteins. Additionally, ¹¹¹Cd‐NMR, and ¹¹¹Cd‐edited ¹H‐NMR, identified histidine ligands in Anabaena PCC 7120 BmtA, analogous to SmtA. A related Escherichia coli protein bound only a single zinc ion, via four cysteine residues, with low stability towards protons; ¹¹¹Cd‐NMR and ¹¹¹Cd‐edited ¹H‐NMR confirmed exclusive cysteine‐coordination, and these cysteine residues reacted rapidly with 5,5′‐dithiobis‐(2‐nitrobenzoic acid). ¹H‐NMR of proteins from P. aeruginosa, Anabaena PCC 7120 and E. coli generated fingerprints diagnostic for the GATA‐like zinc finger fold of SmtA. These studies reveal first the existence of multiple bacterial metallothioneins, and second proteins with SmtA‐like lone zinc fingers, devoid of a cluster, and designated GatA. We have identified 12 smtA‐like genes in sequence databases including four of the gatA type.