Conservative and nonconservative mutations of the transmembrane CPC motif in ZntA: effect on metal selectivity and activity.

ZntA from Escherichia coli belongs to the P1B-ATPase transporter family and mediates resistance to toxic levels of selected divalent metal ions. P1B-type ATPases can be divided into subgroups based on substrate cation selectivity. ZntA has the highest selectivity for Pb2+, followed by Zn2+ and Cd2+; it also shows low levels of activity with Cu2+, Ni2+, and Co2+. It has two high-affinity metal-binding sites, one each in the N-terminus and the transmembrane domains. Ligands to the transmembrane metal site in ZntA include the cysteine residues of the conserved 392CPC394 motif in the sixth transmembrane helix. Pro393 is invariant in all P-type ATPases. For ZntA homologues with different metal ion selectivity, the cysteines are replaced by serine, histidine, and threonine. To test the effect on activity and metal ion selectivity, single alanine, histidine, and serine substitutions at Cys392 or Cys394 in ZntA were characterized, as well as double substitutions of both cysteines by histidine or serine. P393A was also characterized. C392A, C394A, and P393A lost the ability to bind a metal ion with high affinity in the transmembrane domain. Histidine and serine substitutions at Cys392 and Cys394 resulted in loss of binding of Pb2+ at the transmembrane site, indicating that both cysteines of the CPC motif are required for binding Pb2+ with high affinity in ZntA homologues. However, C392H, C392S, C394H, C394S, C392S/C394S, and C392H/C394H could bind other divalent metal ions at the transmembrane site and retained low but measurable activity. Interestingly, these mutants lost the predominant selectivity for Zn2+ and Cd2+ shown by wtZntA. Therefore, conserved residues contribute to metal selectivity by supplying ligands that bind metal ions not only with high affinity, as for Pb2+, but also with the most favorable binding geometry that results in efficient catalysis.

Comparative study of copper(II)-curcumin complexes as superoxide dismutase mimics and free radical scavengers.

Two stoichiometrically different copper(II) complexes of curcumin (stoichiometry, 1:1 and 1:2 for copper:curcumin), were examined for their superoxide dismutase (SOD) activity, free radical-scavenging ability and antioxidant potential. Both the complexes are soluble in lipids and DMSO. The formation constants of the complexes were determined by voltammetry. EPR spectra of the complexes in DMSO at 77K showed that the 1:2 Cu(II)-curcumin complex is square planar and the 1:1 Cu(II)-curcumin complex is distorted orthorhombic. Cu(II)-curcumin complex (1:1) with larger distortion from square planar structure shows higher SOD activity. These complexes inhibit gamma-radiation induced lipid peroxidation in liposomes and react with DPPH acting as free radical scavengers. One-electron oxidation of the two complexes by radiolytically generated azide radicals in Tx-100 micellar solutions produced phenoxyl radicals, indicating that the phenolic moiety of curcumin in the complexes participates in free radical reactions. Depending on the structure, these two complexes possess different SOD activities, free radical neutralizing abilities and antioxidant potentials. In addition, quantum chemical calculations with density functional theory have been performed to support the experimental observations.

Conserved aspartic acid 714 in transmembrane segment 8 of the ZntA subgroup of P1B-type ATPases is a metal-binding residue.

ZntA from Escherichia coli is a member of the P1B-type ATPase family that confers resistance specifically to Pb2+, Zn2+, and Cd2 salts by active efflux across the cytoplasmic membrane. P1B-type ATPases are important for homeostasis of metal ions such as Cu+, Ag+, Pb2+, Zn2+, Cd2+ Cu2+, and Co2+, with different subgroups showing specificity for different metal ions. Sequence alignments of P1B-type ATPases show that ZntA and close homologues have a strictly conserved Asp714 in the eighth transmembrane domain that is not conserved in other subgroups of P1B-type ATPases. However, in the sarcoplasmic reticulum Ca2+-ATPase, a structurally characterized P-type ATPase, the residue corresponding to Asp714 is a metal-binding residue. Four site-specific mutants at Asp714, D714E, D714H, D714A, and D714P, were characterized. A comparison of their metal-binding affinity with that of wtZntA revealed that Asp714 is a ligand for the metal ion in the transmembrane site. Thus, Asp714 is one of the residues that determine metal ion specificity in ZntA homologues. All four substitutions at Asp714 in ZntA resulted in complete loss of in vivo resistance activity and complete or large reductions in ATPase activity, though D714E and D714H retained the ability to bind metal ions with high affinity at the transmembrane site. Thus, the ability to bind metal ions with high affinity did not correlate with high activity. The metal-binding affinity of the N-terminal site remained unchanged in all four mutants. The affinities of the two metal-binding sites in wtZntA determined in this study are similar to values reported previously for the individual sites in isolated ZntA fragments.

Metal-binding affinity of the transmembrane site in ZntA: implications for metal selectivity.

ZntA, a P1B-type ATPase, confers resistance specifically to Pb2+, Zn2+, and Cd2 in Escherichia coli. Inductively coupled plasma mass spectrometry measurements show that ZntA binds two metal ions with high affinity, one in the N-terminal domain and another in the transmembrane domain. Both sites can bind monovalent and divalent metal ions. Two proteins, deltaN-ZntA, in which the N-terminal domain is deleted, and C59A/C62A-ZntA, in which the N-terminal metal-binding site is disabled by site-specific mutagenesis, can only bind one metal ion. Because C59A/C62A-ZntA can bind a metal ion at the transmembrane site, the N-terminal domain does not block direct access of metal ions to it from the cytosol. A third mutant protein, C392A/C394A-ZntA, in which cysteines from the conserved CPC motif in transmembrane helix 6 are altered, binds metal ions only at the N-terminal site, indicating that both these cysteines form part of the transmembrane site. The metal affinity of the transmembrane site was determined in deltaN-ZntA and C59A/C62A-ZntA by competition titration using a metal ion indicator and by tryptophan fluorescence quenching. The binding affinity for the physiological substrates, Zn2+, Pb2+, and Cd2+, as well as for the extremely poor substrates, Cu2+, Ni2+, and Co2+, range from 10(6)-10(10) M(-1), and does not correlate with the metal selectivity shown by ZntA. Selectivity in ZntA possibly results from differences in metal-binding geometry that produce different structural responses. The affinity of the transmembrane site for metal ions is of similar magnitude to that of the N-terminal site [Liu J. et al. (2005) Biochemistry 44, 5159-5167]; thus, metal transfer between them would be facile.

Kinetic analysis of metal binding to the amino-terminal domain of ZntA by monitoring metal-thiolate charge-transfer complexes.

ZntA, a P(1B)-ATPase transporter from Escherichia coli, mediates resistance specifically to Pb(2+), Zn(2+), and Cd(2+) by active efflux. ZntA has a hydrophilic N-terminal domain that binds one metal ion. This domain, approximately 120 residues long, contains the GXXCXXC motif that has been shown to be the binding site for metal ions such as Cu(+) and Zn(2+) in P(1B)-type ATPases, and an additional cysteine-rich motif, CCCDGAC. We report here that binding of Pb(2+) and Cd(2+) to this domain produces changes in the absorbance spectrum in the 250-400 nm range indicative of metal-thiolate charge-transfer complexes. The spectral changes indicate that only two cysteines are ligands to Cd(2+), but three or more cysteines are involved in binding Pb(2+); this confirms earlier results that the GXXCXXC sequence is not sufficient to bind Pb(2+), which likely involves residues from the CCCDGAC motif. The absorbance changes were used to measure metal binding kinetics of the N-terminal domain using stopped-flow techniques. Binding was described by simple second-order kinetics with a rate constant, k(on), of approximately 10(6)-10(7) M(-)(1) s(-)(1), at 4 degrees C. The activation energy of binding is similar for both Pb(2+) and Cd(2+); however, the entropy change is greater for Pb(2+). The surprisingly large rate constant for metal binding to the N-terminal domain of ZntA, compared to its low turnover rate, indicates that this step is not rate limiting in the overall transport mechanism. These results, in conjunction with earlier studies, suggest that metal binding to the transmembrane site in ZntA or metal release from the transporter is the slow step in the reaction cycle.

Antioxidant and antiproliferative activity of curcumin semicarbazone.

A new semicarbazone derivative of curcumin (CRSC) was synthesized and examined for its antioxidant, antiproliferative, and antiradical activity and compared with those of curcumin (CR). The antioxidant activity was tested by their ability to inhibit radiation induced lipid peroxidation in rat liver microsomes. The antiproliferative activity was tested by studying the in vitro activity of CRSC against estrogen dependant breast cancer cell line MCF-7. Kinetics of reaction of (2,2'-diphenyl-1-picrylhydrazide) DPPH, a stable hydrogen abstracting free radical was studied to measure the antiradical activity using stopped-flow spectrophotometer. Finally one-electron oxidized radicals of CRSC were generated and characterized by pulse radiolysis. The results suggest that the probable site of attack for CRSC is both the phenolic OH and the imine carbonyl position. CRSC shows efficient antioxidant and antiproliferative activity although its antiradical activity is less than that of CR.

Transition metal complexes of phenanthrenequinone thiosemicarbazone as potential anticancer agents: synthesis, structure, spectroscopy, electrochemistry and in vitro anticancer activity against human breast cancer cell-line, T47D.

The thiosemicarbazone derivative of 9,10-phenanthrenequinone, 1, and its metal complexes were synthesized. The X-ray crystal structure for 1 confirms the presence of the E tautomeric arrangement in this compound. Its copper complex shows 1:1 stoichiometry while nickel and cobalt compounds show 1:2 stoichiometry. The X-ray crystal structure of the nickel complex indicates two tridentate ligands coordinating in the thiolato form yielding an octahedral geometry for the 'mer' isomer. The copper complex exhibits maximum antiproliferative activity against human breast cancer cell-line, T47D probably due to inhibition of steroid binding to the cognitive receptor or by preventing dimerization of the estrogen receptor.