Wojciech Bal

Specific structure-stability relations in metallopeptides

Abstract This review discusses the modes of coordination of oligopeptides by Cu(II) and Ni(II). Special attention is given to two general classes of peptides. The first part of the review deals with indirect effects introduced by special sequences of non-bonding side-chains. Unusual coordination modes resulting from the introduction of the break-point proline residues are also discussed. The second part of the review describes the binding properties of histidine peptides. The effects of the positioning of a His residue are discussed in the terms of cooperation and competition between potential metal anchoring sites. Special attention is given to His-3 peptides, modeling the biologically relevant albumin-like metal binding site. Finally, the coordination-related specific hydrolysis processes in histidine peptides are briefly discussed.

MULTI-METAL BINDING SITE OF SERUM ALBUMIN

Circular dichroism and electron spin resonance spectroscopy are used to investigate the second specific metal binding site on human, bovine and porcine albumins. Ni(II), Zn(II) and Cd(II) can displace Cu(II) from the second Cu(II) site but not from the first strong site of human and bovine albumins (the N-terminal site). The second Cu(II) binds more strongly than the other metal ions to the second site of all three proteins, except Zn(II) binding to porcine albumin which is ca. 10 x stronger than Cu(II). The second Cu(II) site appears to be a tetragonal ¿2N, 4O¿ site.

Induction of oxidative DNA damage by carcinogenic metals.

The metal ions carcinogenic to humans are As, Be, Cd, Cr and Ni, and the candidates also include Co, Cu, Fe and Pt. A range of molecular mechanisms was proposed for these metals, reflecting their diverse chemical properties. The oxidative concept in metal carcinogenesis proposes that some complexes of the above metals (Co, Cr, Cu, Fe, Ni) formed in vivo undergo redox cycling, yielding reactive oxygen species and/or high valence metal ions which oxidize DNA. Some of the products of oxidative DNA damage, including 8-oxoguanine and strand breaks, induce mutations, which may lead to neoplastic transformation. The establishment of metal-binding modes in the cell nucleus and of their reactivity is crucial for the understanding of molecular events in metal carcinogenesis. We have proposed the binding sites for Ni(II) and Cu(II) in core histones (H3, H2A) and sperm protamines (HP2) and, using molecular models, provided evidence for the generation of promutagenic oxidative DNA damage by the bound metals.

Binding of transition metal ions to albumin: sites, affinities and rates.

BACKGROUND Serum albumin is the most abundant protein in the blood and cerebrospinal fluid and plays a fundamental role in the distribution of essential transition metal ions in the human body. Human serum albumin (HSA) is an important physiological transporter of the essential metal ions Cu(2+), and Zn(2+) in the bloodstream. Its binding of metals like Ni(2+), Co(2+), or Cd(2+) can occur in vivo, but is only of toxicological relevance. Moreover, HSA is one of the main targets and hence most studied binding protein for metallodrugs based on complexes with Au, Pt and V. SCOPE OF REVIEW We discuss i) the four metal-binding sites so far described on HSA, their localization and metal preference, ii) the binding of the metal ions mentioned above, i.e. their stability constants and association/dissociation rates, their coordination chemistry and their selectivity versus the four binding sites iii) the methodology applied to study issues of items i and ii and iv) oligopeptide models of the N-terminal binding site. MAJOR CONCLUSIONS Albumin has four partially selective metal binding sites with well-defined metal preferences. It is an important regulator of the blood transport of physiological Cu(II) and Zn(II) and toxic Ni(II) and Cd(II). It is also an important target for metal-based drugs containing Pt(II), V(IV)O, and Au(I). GENERAL SIGNIFICANCE The thorough understanding of metal binding properties of serum albumin, including the competition of various metal ions for specific binding sites is important for biomedical issues, such as new disease markers and design of metal-based drugs. This article is part of a Special Issue entitled Serum Albumin.

Coordination of heavy metals by dithiothreitol, a commonly used thiol group protectant.

D,L-Dithiothreitol (DTT), known also as Cleland reagent, is a thiol group protectant, used commonly in peptide and protein chemistry. Therefore, it is often added at high concentrations in preparations of proteins relevant to heavy metal biochemistry. The coordination of five of these metal ions, Zn(II), Cd(II), Pb(II), Ni(II) and Cu(I) to DTT was studied by means of potentiometric titrations, and UV-Vis and NMR spectroscopies. It was found that DTT forms specific and very stable polymeric and monomeric complexes with all of these metal ions, using both of its sulfur donors. The quantitative description of these complexes in solution and the solid state provides the basis for predictions of interference from DTT in studies of metal ion binding of thiol-containing biomolecules.

Overexpression of phytochelatin synthase in tobacco: distinctive effects of AtPCS1 and CePCS genes on plant response to cadmium

Phytochelatins, heavy-metal-binding polypeptides, are synthesized by phytochelatin synthase (PCS) (EC 2.3.2.15). Previous studies on plants overexpressing PCS genes yielded contrasting phenotypes, ranging from enhanced cadmium tolerance and accumulation to cadmium hypersensitivity. This paper compares the effects of overexpression of AtPCS1 and CePCS in tobacco (Nicotiana tabacum var. Xanthi), and demonstrates how the introduction of single homologous genes affects to a different extent cellular metabolic pathways leading to the opposite of the desired effect. In contrast to WT and CePCS transformants, plants overexpressing AtPCS1 were Cd-hypersensitive although there was no substantial difference in cadmium accumulation between studied lines. Plants exposed to cadmium (5 and 25 μM CdCl2) differed, however, in the concentration of non-protein thiols (NPT). In addition, PCS activity in AtPCS1 transformants was around 5-fold higher than in CePCS and WT plants. AtPCS1 expressing plants displayed a dramatic accumulation of γ-glutamylcysteine and concomitant strong depletion of glutathione. By contrast, in CePCS transformants, a smaller reduction of the level of glutathione was noticed, and a less pronounced change in γ-glutamylcysteine concentration. There was only a moderate and temporary increase in phytochelatin levels due to AtPCS1 and CePCS expression. Marked changes in NPT composition due to AtPCS1 expression led to moderately decreased Cd-detoxification capacity reflected by lower SH:Cd ratios, and to higher oxidative stress (assessed by DAB staining), which possibly explains the increase in Cd-sensitivity. The results indicate that contrasting responses to cadmium of plants overexpressing PCS genes might result from species-dependent differences in the activity of phytochelatin synthase produced by the transgenes.

Molecular models in nickel carcinogenesis.

Nickel compounds are known human carcinogens, but the exact molecular mechanisms of nickel carcinogenesis are not known. Due to their abundance, histones are likely targets for Ni(II) ions among nuclear macromolecules. This paper reviews our recent studies of peptide and protein models of Ni(II) binding to histones. The results allowed us to propose several mechanisms of Ni(II)-inflicted damage, including nucleobase oxidation and sequence-specific histone hydrolysis. Quantitative estimations of Ni(II) speciation, based on these studies, support the likelihood of Ni(II) binding to histones in vivo, and the protective role of high levels of glutathione. These calculations indicate the importance of histidine in the intracellular Ni(II) speciation.

Cu(II) Affinity for the Alzheimer’s Peptide: Tyrosine Fluorescence Studies Revisited

Copper(II) binding to the amyloid-β peptide has been proposed to be a key event in the cascade leading to Alzheimers disease. As a direct consequence, the strength of the Cu(II) to Aβ interaction, that is, the Cu(II) affinity of Aβ, is a very important parameter to determine. Because Aβ peptide contain one Tyr fluorophore in its sequence and because Cu(II) does quench Tyr fluorescence, fluorescence measurements appear to be a straightforward way to obtain this parameter. However, this proved to be wrong, mainly because of data misinterpretation in some previous studies that leads to a conflicting situation. In the present paper, we have investigated in details a large set of fluorescence data that were analyzed with a new method taking into account the presence of two Cu(II) sites and the inner-filter effect. This leads to reinterpretation of the published data and to the determination of a unified affinity value in the 10(10) M(-1) range.

Monomethylarsonous Acid Destroys a Tetrathiolate Zinc Finger Much More Efficiently than Inorganic Arsenite: Mechanistic Considerations and Consequences for DNA Repair Inhibition

Arsenic compounds are human carcinogens. The ingested inorganic arsenic is metabolized to methylated derivatives, which are considered to be more toxic than the inorganic species. Interactions of trivalent arsenicals with thiol groups of proteins are believed to be important for arsenic carcinogenesis, but inorganic arsenite appears to bind to thiol groups more strongly than the methylated As (III) species. Inhibition of the nucleotide excision repair pathway of DNA repair (NER) is likely to be of primary importance in arsenic carcinogenesis. Previously, we demonstrated that methylated As (III) compounds are more efficient than arsenite in releasing zinc from ZnXPAzf, the zinc finger of XPA, a crucial member of the NER complex [Schwerdtle, T., Walter, I., and Hartwig, A. (2003) Arsenite and its biomethylated metabolites interfere with the formation and repair of stable BPDE-induced DNA adducts in human cells and impair XPAzf and Fpg. DNA Repair (Amsterdam) 2, 1449-1463]. In this work, we used ESI-MS to compare aerobic reactivities of arsenite and monomethylarsonous acid (MMA (III)) toward ZnXPAzf on the molecular level. We demonstrated that equimolar MMA (III) released Zn (II) from ZnXPAzf easily, forming mono- and diarsenical derivatives of XPAzf. This reaction was accompanied by oxidation of unprotected thiol groups of the monomethylarsinated peptide to intramolecular disulfides. The estimated affinity of MMA (III) to XPAzf is 30-fold higher than that established previously for arsenite binding to the thiol groups. No binding of arsenite to the thiol groups of XPAzf was observed under our experimental conditions, and a 10-fold excess of arsenite was required to partially oxidize ZnXPAzf. These results indicate a particular susceptibility of tetrathiolate zinc fingers to MMA (III), thereby providing a novel molecular pathway in arsenic carcinogenesis.

Spectroscopic and thermodynamic determination of three distinct binding sites for Co(II) ions in human serum albumin.

Human serum albumin (HSA) is the most abundant protein of blood serum, involved in the transport of metal ions, including Co(II). Using circular dichroism spectroscopic titrations we characterized three distinct Co(II) binding sites in HSA. Applying Cu(II), Ni(II) and Cd(II) ions as competitors we determined that these sites are identical with three binding sites known for other metal ions. We ordered these sites according to their binding affinities as cadmium site B (CdB)>multi-metal binding site (MBS)>N-terminal binding site (NTS). Using isothermal titration calorimetry (ITC) we confirmed the presence of these three binding sites and determined their conditional binding constants at pH 7.4 as 9+/-5, 1.1+/-0.5, and 0.9+/-0.3x10(4)M(-1), respectively. The impact of these results on the albumin cobalt binding (ACB) clinical assay for myocardial ischemia is discussed.