Ulrich Weser

Mass fractionation processes of transition metal isotopes

Recent advances in mass spectrometry make it possible to utilise isotope variations of transition metals to address some important issues in solar system and biological sciences. Realisation of the potential offered by these new isotope systems however requires an adequate understanding of the factors controlling their isotope fractionation. Here we show the results of a broadly based study on copper and iron isotope fractionation during various inorganic and biological processes. These results demonstrate that: (1) naturally occurring inorganic processes can fractionate Fe isotope to a detectable level even at temperature V1000‡C, which challenges the previous view that Fe isotope variations in natural system are unique biosignatures; (2) multiple-step equilibrium processes at low temperatures may cause large mass fractionation of transition metal isotopes even when the fractionation per single step is small; (3) oxidation^reduction is an importation controlling factor of isotope fractionation of transition metal elements with multiple valences, which opens a wide range of applications of these new isotope systems, ranging from metal-silicate fractionation in the solar system to uptake pathways of these elements in biological systems; (4) organisms incorporate lighter isotopes of transition metals preferentially, and transition metal isotope fractionation occurs stepwise along their pathways within biological systems during their uptake. > 2002 Elsevier Science B.V. All rights reserved.

Superoxide dismutase activity of low molecular weight Cu2+-chelates studied by pulse radiolysis

The discovery of an accelerated superoxide dismutation [ 1 ] using erythrocuprein led to an overwhelming number of studies on this metalloprotein (for a review see ref. [2] ). The mechanism of this enzymic catalysed reaction was successfully studied by pulse radiolysis [3-51. It was intriguing to realize that erythrocuprein which has apparently evolved [6] to perform so simple a reaction proved, in some ways, far from simple. The situation turned out even more complicated with the knowledge that free Cu2 * catalyses superoxide dismutation in acidic media in a much faster way compared to the reactivity of erythrocuprein under physiological pH conditions [7] . Furthermore, it was demonstrated that low molecular weight copper chelates display marked superoxide dismutase activity at physiological pH values employing the cytochrome c reductase assay [X] . In this context it was of high importance to measure the rate constants for reaction between some of these cupric-peptide chelates and the superoxide ion generated by pulse radiolysis at pH 7 5. The reaction of chelated Cu2 * with superoxide was very fast throughout. The respective second order rate constants of either equivalent of Cu2+ ranged from 0.6 X IO9 M-r see-’ (Cu(ly~)~) up to 1.3 X IO9 M-’

Inhibition of lipid peroxidation in isolated inner membrane of rat liver mitochondria by superoxide dismutase

Lipid peroxidation in mitochondrial membranes results in loss of respiratory control, lack of contractibility and final lysis of mitochondria [l-3]. It may be induced by incubation of isolated mitochondria in the presence of oxygen with either chaotropic agents [4] or autoxidizable substances like ascorbate [2] or thiol compounds [3]. Besides, formation of lipid peroxides in livers of tocopherol deficient rats has been observed under in vim conditions [5 ] and may, therefore, have a pathophysiological significance. Two enzymes, GSH peroxidase (C-factor I) [l] and catalase (C-factor II) [ 11, were found to prevent peroxidation of unsaturated lipids in suspensions of mitochondria or “high amplitude swelling” which is related to lipid peroxidation [2, 3,6, 71. These observations suggest an involvement of H202 which may for instance be built according to the equation

Copper-thionein from fetal bovine liver.

It was of interest to examine whether or not a low molecular weight copper-rich metal-thionein was present in biological species which received no metal pretreatment at all. From bovine fetal liver an 8 Cu 2 Zn-thionein having a molecular weight of 11 500 was successfully isolated. 16% of the total copper present in the whole liver were recovered in this protein. During the isolation process anaerobic conditions had to be maintained to avoid uncontrolled oxidation leading to polymeric species and the loss of most of the copper. The similarity of both the present copper-thionein and the polymeric neonatal type mitochondrocuprein was shown. A comparison of different copper-thioneins containing variable amounts of copper was possible when xiCu from 280 nm to longer wavelength was determined. With respect to the ultraviolet properties there were no detectable differences between copper-thioneins prepared either in vivo or in vitro and the fetal copper-thionein. Furthermore, the positions of the Cotton effects as deduced from circular dichroism measurements were rather similar although the magnitude of the observed Cotton extrema was less pronounced and sometimes the signs were reversed. X-ray photoelectron spectrometric studies revealed a Cu(2p3/2) binding energy value of 932.9 eV. Unlike the S(2p1/2,3/2) value near 162 eV using Cu-thioneins from chicken liver or yeast the higher S(20p1/2,3/2) binding energy of 163.0 eV employing fetal Cu-thionein was attributed to partial oxidation of the protein moiety and/or a particular chemical environment. The second S(2p1/2,3/2) peak was assigned to the copper catalyzed oxidation of sulphur via OH to yield RSO-3. In the X-ray photoelectron spectrum of the apoprotein one homogeneous S(2p1/2,3/2) band at 163.7 eV was seen attributable to RSSR.

CONVERSION OF METALLOTHIONEIN INTO CU-THIONEIN, THE POSSIBLE LOW MOLECULAR WEIGHT FORM OF NEONATAL HEPATIC MITOCHONDROCUPREIN

Our knowledge on the preparation and physicochemica1 properties of metallothionein has improved in recent years [l-l 51. On the other hand, a highly polymeric sulphur and copper rich protein called neonatal hepatic mitochondrocuprein was described by Porter [ 16-221. Although this protein bears the name cuprein, it proved completely different from those intracellular cupreins containing 2 g-atoms of each of copper and zinc and which display superoxide dismutase and singlet oxygen decontaminating activities (for a review see [23] ). The neonatal type cuprein normally being present under physiological conditions in newborn species was also found in mitochondria of the livers of adults suffering from Wilson’s disease. Apart from these polymeric sulphur rich copper proteins some low molecular weight species of similar compositions were found [22,24,25] . It was assumed that the copper is in the cuprous state [24,25] and bound with the cysteine sulphur. Due to the similar amino acid composition of both the metallothionein and neonatal hepatic mitochondrocuprein Porter suggested some relationship to metallothionein [22]. In this context we were highly interested in the possibility of a direct conversion of metallothionein into Cu-thionein. The partially loaded Cu-thionein was expected to polymerize via disulphide bridges in the presence of oxygen to form the ‘neonatal type

Imidazole-bridged copper complexes as Cu2Zn2—superoxide dismutase models

Abstract Crystalline binuclear imidazole-bridged copper- and zinc-copper chromophores ([Me(pip)(im)Me(pip)](NO 3 ) 3 · x H 2 O) were synthesized and compared with the active site of Cu 2 Zn 2 —superoxide dismutase. The electron absorption profiles were of striking similarity with those of the copper chromophore of the native enzyme. This similarity was also seen in the electron paramagnetic properties (g ⊥ = 2.031 ± 0.009, g 1 = 2.230 ± 0.004 and |A 1 | = (14.8 ± 1.0) × 10 −3 cm −1 ). X-ray photoelectron spectrometry of the oxidized or the reduced superoxide dismutase models revealed the existence of 3d 9 Cu(II) (∼ 934.5 eV) and 3d 10 (Cu(I)) (∼ 931.8 eV). By way of contrast the 2p 3 2 binding energy of the copper in the Cu 2 Zn 2 —enzyme remained constant at 932 eV. In an indirect assay system for superoxide dismutation, the xanthine/xanthine oxidase-mediated reduction of nitroblue tetrazolium indicated that either enzyme model reacted at a rate of 10 8 M −1 s −1 . The rate constant of the binuclear copper—zinc complex was more than three times higher than the dicopper complex in pulse radiolysis. Rate constants determined by this method ( k 2 = 3.8 ± 2.1) × 10 8 M −1 s −1 ) were in reasonable agreement with those determined in the indirect test system.

Superoxide dismutase activity of Cu2+ —amino acid chelates

The disproportionation of 0, * into H,O* and O2 was found enzymically catalysed by erythrocuprein [l-4]. Due to this reactivity the long known copper protein [.5] was renamed superoxide dismutase [I]. Detailed enzymic studies [6,7] led to the proposal of another important function of erythrocuprein. namely the scavenging of singlet oxygen in many oxidation reactions. At the moment no decision can be made which of these enzymic functions will be given privilege in metabolism. Before a final designation of this Cu-protein is suggested further studies regarding the specificity of the above mentioned biochemical reactions ought to be carried out. We thought it worthwhile to examine the question as to which degree copper chelates using both the free amino acids or some low molecular weight peptides would be able to display similar enzymic activities. All amino acids being present in bovine erythrocuprein [8] were employed. The Cu*+-complexes of lysine, histidine, tyrosine or some diand tripeptides thereof displayed considerable biochemical activities. The superoxide dismutase activity of both the native erythrocuprein and different Cu*+-amino acid complexes were compared on a molar basis of chelated Cu*+. In the cytochrome c reductase assay [ 1,4] enzymic activities of up to 5% Cu(Lys)* compared to native erythrocuprein were determined. Slightly higher activities were observed using the chemiluminescence test [6, 71. In this alternative test Cu(Lys), displayed 7.5%1 enzymic activity. As in the cytochrome c reductase assay only the Cu*+-chelates of

Substrate-induced redox change of selenium in glutathione peroxidase studied by X-ray photoelectron spectroscopy

Glutathione peroxidase showed an X-ray photoelectron spectroscopy signal of the Se 3d (3/2, 5/2) electrons at 54.4 eV. After the addition of the acceptor substrate H2O2, a marked shift of this signal to a value of 58.0 eV was observed. Upon subsequent treatment with the donor substrate glutathione, this chemical shift was reversed and the original signal was obtained. These data demonstrate that the enzyme-bound selenium moiety participates in the catalytic process. From the chemical shift obtained it is concluded that the enzyme shuttles between a selenol or selenol derivative in its reduced form and a seleninyl or selenonyl compound in its oxidized form.

Pulse radiolytically generated superoxide and Cu(II)-salicylates

Abstract The rate constants of the reactions between pulse radiolytically produced superoxide anions and the Cu(II) chelates of salicylate, acetylsalicylate, p-aminosalicylate and diisopropylsalicylate were determined at pH 7.5 and found to range from 0.8 to 2.4 × 109 M−1 sec−1. It was intriguing to note that they had a superoxide dismutase activity identical with that of native cuprein-copper (k245 = 1.3 × 109 M−1 sec−1 per g-atom of Cu). These measurements confirm our earlier observations using indirect assays that all copper salicylates act as perfect model superoxide dismutases and favour the proposal that the activity of anti-inflammatory agents might be assigned to their in vivo formed Cu complexes.

Superoxide dismutase activity of copper-penicillamine: possible involvement of Cu(I) stabilized sulphur radical.

Abstract The ability of [Cu14(D-penicillamine)12 Cl]5− to act as a superoxide dismutating agent was examined. The red-violet complex proved capable of inhibiting various reactions mediated by superoxide and perhaps other excited oxygen species, such as the reduction of both cytochrome c and nitroblue tetrazolium by the xanthine-xanthine oxidase-system as well as the formazan formation by KO2. These results favour the hypothesis, that one of the modes of action of anti-inflammatory drugs is the scavenging of the oxygen species responsible for the inflammatory response by their copper chelates formed in vivo. X-ray photoelectron spectrometric studies revealed that the actual oxidation state of copper before and after the reaction with O2− was +1 while the binding energy of the 2p core electrons of sulphur remained around 163 eV. The possibility that a stabilized sulphur radical, present as Cu(I)·SR, is the species undergoing the redox cycle rather than the copper is suggested.