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Extensive use of the broad-spectrum tetracycline antibiotics (TCs) has resulted their wide spread in the environment and drive new microecological balances, including the infamous antibiotic resistance. TCs require metal ions for their antibiotic activity and resistance via interactions with ribosome and tetracycline repressor TetR, respectively, at specific metal-binding sites. Moreover, the Lewis-acidic metal center(s) in metallo-TCs can interact with Lewis-basic moieties of many bioactive secondary metabolites, which in turn may alter their associated chemical equilibria and biological activities. Thus, it is ultimately important to reveal detailed coordination chemistry of metallo-TC complexes. Herein, we report (a) conclusive specific Co 2+ , Ni 2+ , and Cu 2+ -binding of TC revealed by paramagnetic 1 H NMR, showing different conformations of the coordination and different metal-binding sites in solution and solid state, (b) significant metal-mediated activity of Cu-TC toward catechol oxidation with different mechanisms by air and H 2 O 2 (i.e., mono- and di-nuclear pathways, respectively), (c) interactions of metallo-TCs with bioactive salicylic acid and its precursor benzoic acid, and (d) noticeable change of TC antibiotic activity by metal and salicylic acid. The results imply that TCs may play broad and versatile roles in maintaining certain equilibria in microecological environments in addition to their well-established antibiotic activity. We hope the results may foster further exploration of previously unknown metal-mediated activities of metallo-TC complexes and other metalloantibiotics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Phosphate was proposed to be a bridging ligand in the structure 1xjo.pdb of Streptomyces dizinc aminopeptidase (sAP), which prompted further studies of phosphate binding to this enzyme. Phosphate inhibits sAP and its Co(2+)-substituted derivatives in a noncompetitive manner from pH 6.0 to 9.0, with strongest inhibition observed at lower pHs (K(i) = 0.6, 8.2, and 9.1 mM for ZnZn-, CoCo-, and CoZn-sAP, respectively, at pH 6.0), which indicates that phosphate does not compete with substrate binding to the dinuclear active site and that monobasic phosphate has a higher binding affinity. The inhibition K(i)-pH profiles for phosphate inhibition of both the native and the Co(2+)-substituted derivatives reveal a similar pK(a) around 7.0, reflecting that phosphate binding is not affected by the metal centers of different Lewis acidities. Modification of ZnZn- and CoCo-sAP with the arginine-specific reagent phenylglyoxal reveals a significant weakening in phosphate and substrate binding by showing approximately a 10-fold increase in the dissociation constant K(i) for phosphate binding and approximately 4-8-fold increase in K(m). The catalysis is also influenced by the modification as reflected by a significant decrease in k(cat) in both cases. Furthermore, phosphate and the transition-state inhibitor 1-aminobutyl phosphonate can protect arginine from the modification, strongly suggesting that Arg202 near the active site is involved in phosphate binding and in stabilizing the transition state. The effect on (31)P NMR relaxation of phosphate caused by the paramagnetic metal center in Co(2+)-substituted derivatives of sAP has been measured, which reveals that only one phosphate is bound to sAP with the Co(2+)-(31)P distance in the range of 4.1-4.3 A. The (1)H NMR relaxation of the bulk water signal in the CoCo-sAP sample remains unchanged in the presence of phosphate, further indicating that phosphate may not bind to the active-site metals to displace any metal-bound water/hydroxide. These results strongly support that the phosphate binding site is Arg202 and that this residue plays an important role in the action of sAP.

Serralysin is a bacterial Zn-endopeptidase which has been considered a virulence factor to cause tissue damage and anaphylactic response. It contains a coordinated Tyr that is unique to the astacin-like Zn enzymes. The coordinated Tyr has been proposed to play an important role in the action of this endopeptidase family. Several metal-substituted derivatives of serralysin (including Mn2+, Co2+, Ni2+, Cu2+, and Cd2+ derivatives) are found to exhibit significant activities. Particularly, the Co- and Cu-substituted derivatives exhibit much higher activities than the native serralysin toward the hydrolysis of the tripeptide mimic benzoyl-Arg- p-nitroanilide, i.e., 35 and 49 times higher in k(cat) and 33 and 26 times in k(cat)/ K(m), respectively. Such remarkably higher activities of metal-substituted derivatives, especially the Cu derivative, than that of the native Zn enzyme are rare in the literature, reflecting the uniqueness of this enzyme among all Zn enzymes. The significantly different k(cat) yet similar K(m) values among the several metal derivatives suggests that the metal center is involved in catalysis, but not necessarily in the binding of the substrate, whereas the dramatically different inhibition constants for Arg-hydroxamate binding to the metal-substituted derivatives indicates direct binding of this inhibitor to the metal center. The activity-pH profiles of serralysin and its Co2+ and Cu2+ derivatives and the optical-pH profile of Cu-serralysin have been obtained, in which the decrease in activity at higher pH values was found to be associated with a dramatic increase in the Tyr-to-Cu2+ charge transfer transitions. This observation suggests that the binding of Tyr216 to the metal is inhibitory. A metal-centered mechanism is proposed for serralysin catalysis based on the results presented here, in which the detachment of the coordinated Tyr and formation of a H-bond with the transition-state complex are considered essential for the stabilization of the transition state.

The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PC-PLC(Bc)) is a tri-Zn enzyme with two 'tight binding' and one 'loose binding' sites. The Zn2+ ions can be replaced with Co2+ and Cu2+ to afford metal-substituted derivatives. Two Cu2+-substituted derivatives are detected by means of 1H NMR spectroscopy, a 'transient' derivative and a 'stable' derivative. The detection of sharp hyperfine-shifted 1H NMR signals in the 'transient' derivative indicates the formation of a magnetically coupled di-Cu2+ center, which concludes that the Zn2+ ions in the dinuclear (Zn1 and Zn3) sites are more easily replaced by Cu2+ than that in the Zn2 site. This might possibly be the case for Co2+ binding. Complete replacement of the three Zn2+ ions can be achieved by extensive dialysis of the enzyme against excess Cu2+ to yield the final 'stable' derivative. This derivative has been determined to have five-coordinated His residues and an overall S'=1/2 spin state with NMR and EPR, consistent with the formation of a tri-Cu2+ center (i.e. a di-Cu2+/mono-Cu2+ center) in this enzyme. The binding of substrate to the inert tri-Cu2+ center to form an enzyme-substrate (ES) complex is clearly seen in the 1H NMR spectrum, which is not obtainable in the case of the native enzyme. The change in the spectral features indicates that the substrate binds directly to the trinuclear metal center. The studies reported here suggest that 1H NMR spectroscopy can be a valuable tool for the characterization of di- and multi-nuclear metalloproteins using the 'NMR friendly' magnetically coupled Cu2+ as a probe.