On the reaction of iron bleomycin with thiols and oxygen.

Iron(III)bleomycin undergoes a redox reaction with thiols. Evidence from epr and UV-visible absorbance spectra indicate that the metal complex forms an intermediate with sulfhydryl groups presumably by adding a sixth ligand. In the presence of oxygen iron bleomycin cycles between its two oxidation states to catalyze the generation of oxygen free radicals using thiols as a source of electrons.     

Characterization of the adduct formed from the reaction between homocysteine thiolactone and low-density lipoprotein: antioxidant implications

Homocysteine thiolactone is a cyclic thioester that is implicated in the development of atherosclerosis. This molecule will readily acylate primary amines, forming a homocystamide adduct, which contains a primary amine and a thiol. Here, we have characterized and evaluated the antioxidant potential of the homocystamide-low-density lipoprotein (LDL) adduct, a product of the reaction between homocysteine thiolactone and LDL. Treatment of LDL with homocysteine thiolactone resulted in a time-dependent increase in LDL-bound thiols that reached approximately 250 nmol thiol/mg LDL protein. The thiol groups of the homocystamide-LDL adduct were labeled with the thiol-reactive nitroxide, methanethiosulfonate spin label. Using paramagnetic relaxing agents and the electron spin resonance spin labeling technique, we determined that the homocystamide adducts were predominately exposed to the aqueous phase. The homocystamide-LDL adduct was resistant to myoglobin- and Cu2(+)-mediated oxidation (with respect to native LDL), as measured by the formation of conjugated dienes and thiobarbituric acid reactive substances, and the depletion of vitamin E. This antioxidant effect was due to increased thiol content, as the effect was abolished with N-ethylmaleamide pre-treatment. We conclude that the reaction between homocysteine thiolactone and LDL generates an LDL molecule that is more resistant to oxidative modification than native LDL. The potential relationship between the homocystamide-LDL adduct and the development of atherosclerosis is discussed.     

Interactions of iron bleomycin, phosphate or cyanide, and DNA: sequence-dependent conformations and reactions

The hypothesis was investigated that axial ligands bound to Fe(III)-bleomycin [Fe(III)Blm] are destabilized at specific 5'-guanine-pyrimidine-3' binding sites but are stable at nonselective dinucleotides. DNA oligomers and calf-thymus DNA were used in reactions with L-Fe(III)Blm, where phosphate and cyanide served as examples of large and small ligands (L). Both ligands underwent dissociation when L-Fe(III)Blm was bound to d(GGAAGCTTCC)2 (I) but not d(GGAAATTTCCC)2 (II) and at large ratios of calf-thymus DNA to drug. Fe(III)Blm is high spin in 20 mM phosphate buffer, signifying the presence of a phosphate adduct. In the titration of HPO4-Fe(III)Blm with calf-thymus DNA, a large excess of DNA was needed to reach the low-spin state, consistent with an equilibrium competition between phosphate and DNA for Fe(III)Blm. Equilibrium constants for binding Fe(III)Blm and CN-Fe(III)Blm to calf-thymus DNA (6.8x10(5) M(-1) and 5.9x10(4) M(-1), respectively, in HEPES buffer at 25 degrees C and pH 7.4) showed that the CN- ligand also reduced the affinity of DNA for the drug. The kinetics of dissociation of CN- from CN-Fe(III)Blm-DNA were slow and first order in bound drug. The reversible nature of these dissociation reactions was shown using 1H NMR spectroscopy of Fe(III)Blm-I in the absence and presence of large excesses of CN- or phosphate. The results are discussed in terms of a two-state hypothesis for the binding of L-Fe(III)Blm to specific and nonspecific dinucleotides. It is proposed that steric restrictions at specific sites inhibit binding of these ligands.     

Spectral and thermodynamic properties of methanobactin from γ-proteobacterial methane oxidizing bacteria: A case for copper competition on a molecular level

Methanobactin (mb) is a low molecular mass copper-binding molecule analogous to iron-binding siderophores. The molecule is produced by many methanotrophic or methane oxidizing bacteria (MOB), but has only been characterized to date in one MOB, Methylosinus trichosporium OB3b. To explore the potential molecular diversity in this novel class of metal binding compound, the spectral (UV-visible, fluorescent, and electron paramagnetic resonance) and thermodynamic properties of mb from two γ-proteobacterial MOB, Methylococcus capsulatus Bath and Methylomicrobium album BG8, were determined and compared to the mb from the α-proteobacterial MOB, M. trichosporium OB3b. The mb from both γ-proteobacterial MOB differed from the mb from M. trichosporium OB3b in molecular mass and spectral properties. Compared to mb from M. trichosporium OB3b, the extracellular concentrations were low, as were copper-binding constants of mb from both γ-proteobacterial MOB. In addition, the mb from M. trichosporium OB3b removed Cu(I) from the mb of both γ-proteobacterial MOB. Taken together the results suggest mb may be a factor in regulating methanotrophic community structure in copper-limited environments.     

Copper coordination in the full-length,recombinant prion protein

The prion protein (PrP) binds divalent copper at physiologically relevant conditions and is believed to participate in copper regulation or act as a copper-dependent enzyme. Ongoing studies aim at determining the molecular features of the copper binding sites. The emerging consensus is that most copper binds in the octarepeat domain, which is composed of four or more copies of the fundamental sequence PHGGGWGQ. Previous work from our laboratory using PrP-derived peptides, in conjunction with EPR and X-ray crystallography, demonstrated that the HGGGW segment constitutes the fundamental binding unit in the octarepeat domain [Burns et al. (2002) Biochemistry 41, 3991-4001; Aronoff-Spencer et al. (2000) Biochemistry 39, 13760-13771]. Copper coordination arises from the His imidazole and sequential deprotonated glycine amides. In this present work, recombinant, full-length Syrian hamster PrP is investigated using EPR methodologies. Four copper ions are taken up in the octarepeat domain, which supports previous findings. However, quantification studies reveal a fifth binding site in the flexible region between the octarepeats and the PrP globular C-terminal domain. A series of PrP peptide constructs show that this site involves His96 in the PrP(92-96) segment GGGTH. Further examination by X-band EPR, S-band EPR, and electron spin-echo envelope spectroscopy, demonstrates coordination by the His96 imidazole and the glycine preceding the threonine. The copper affinity for this type of binding site is highly pH dependent, and EPR studies here show that recombinant PrP loses its affinity for copper below pH 6.0. These studies seem to provide a complete profile of the copper binding sites in PrP and support the hypothesis that PrP function is related to its ability to bind copper in a pH-dependent fashion.     

Structures of HO(2)-Co(III)bleomycin A(2) bound to d(GAGCTC)(2) and d(GGAAGCTTCC)(2): structure-reactivity relationships of Co and Fe bleomycins

HO(2)-Co(III)bleomycin is a model for HO(2)-Fe(III)bleomycin, which initiates single and double strand cleavage of DNA. In order to enlarge the understanding of its structure and reactivity, three-dimensional structures of HO(2)-Co(III)bleomycin bound to two DNA oligomers, d(GAGCTC)(2) (I) and d(GGAAGCTTCC)(2) (II), that have 5'-GC-3' binding sites, have been determined by nuclear magnetic resonance (NMR) methods. Besides previously recognized determinants of binding selectivity, a probable hydrogen bond was detected between the pyrimidinyl acetamido NH(2) and the carbonyl of cytosine base paired to G at the recognition site. Another hydrogen bond between the NH of the dimethylsulfonium R group and N7 of guanine opposite cytosine at the GC site may contribute to specification of the pyrimidine. Substitution of G with inosine shifted HO(2)-Co(III)Blm A(2)[bond]I and Fe(III)Blm[bond]I into fast exchange on the NMR time scale, supporting the role of the 2-amino group in site specification for each molecule. The conformationally stable metal-domain linker established a close-packed adduct with the minor groove in which the hydroperoxide ligand occupies a sterically constrained pocket that is isolated from the solvent. The hydroperoxide group is directed toward one of the two cytosine H4' hydrogens but is sterically blocked from access to the other by the drug. These findings enlarge the structural understanding of selective binding of Co(III)/Fe(III)Blm species at G-pyrimidine sites. They also rationalize the instability of a number of ligands bound to Co(III)/Fe(III)Blm at specific binding sequences and the relative unreactivity of Fe(III)Blm[bond]I with ascorbate as well as its lack of interaction with spin labels.     

Kinetics of reaction of DNA-bound Fe(III)bleomycin with ascorbate: interplay of specific and non-specific binding

The aerobic redox reaction of Fe(III)bleomycin (Blm) and ascorbate was examined in the absence of DNA and in the presence of 7.5 and 25 calf thymus DNA base pairs per-drug molecule, in order to investigate the effect of DNA binding on the properties of FeBlm activation and DNA strand cleavage. Under these successive conditions, the rate of initial reduction of Fe(III)Blm became progressively slower and biphasic. Using 7.5 base pairs per-molecule of FeBlm, 2-3 times as much drug reacted in the faster step as with the larger DNA to drug ratio. In each case, the more rapid process was identified with the reaction of high spin Fe(III)Blm-DNA. With the smaller ratio, dioxygen consumption, formation of HO(2)-Fe(III)Blm-DNA, and production of DNA strand breaks as measured by the formation of base propenal were largely rate limited by the initial reaction of ascorbate with Fe(III)Blm-DNA. After a burst of reaction with the larger ratio of base pairs to Fe(III)Blm, a small fraction of the total Fe(III)Blm, representing high spin Fe(III)Blm, entered a steady state as HO(2)-Fe(III)Blm-DNA. Thereafter, reaction of dioxygen and base propenal formation occurred slowly with similar first-order rate kinetics. In order to explain these results, it is hypothesized that the metal domain-linker of Fe(III)Blm adopts two conformations with respect to DNA. One, at specific binding sites, is relatively unreactive with ascorbate. The other, present at non-specific sites as HPO(4)-Fe(III)Blm, is readily reactive with ascorbate to generate HO(2)-Fe(III)Blm-DNA. At the larger base pair to drug ratio, movement of Fe(III)Blm between specific and non-specific sites to generate HO(2)-Fe(III)Blm is a necessary part of the mechanism of strand scission.     

DNA-fiber EPR study of the orientation of Cu(II) complexes of 1,10-phenanthroline and its derivatives bound to DNA: mono(phenanthroline)-copper(II) and its ternary complexes with amino acids

The orientation of mono(1,10-phenanthroline)copper(II), [Cu(phen)]2+, and the ternary complexes with amino acids, [Cu(phen)X(aa)]n+, where X(aa) stands for an alpha-amino acid, has been investigated by electron paramagnetic resonance (EPR) spectra of the complexes on DNA fibers. It has been revealed that these complexes bind to DNA with several different binding modes. The observation of a species whose g axis is almost parallel to the DNA double helical axis has suggested that the phenanthroline moiety intercalates to DNA. An absence of the intercalated species for the corresponding 2,2'-bipyridine complex has shown that the three-fused aromatic rings in phenanthroline are critical for the intercalative binding of the complexes. The intercalative binding was promoted by 5,6-dimethyl groups on the phenanthroline ring, whereas it was disturbed by 2,9-dimethyl groups, indicating that the planarity of the coordination sphere is important for the intercalative binding. In all cases, the amount of the non-intercalated species was larger than that of the intercalated one. The amino acids in the ternary complexes of glycine, leucine, serine, threonine, cysteine, methionine, and asparagine were partly substituted with some coordinating groups in DNA, whereas the ternary complexes of lysine, arginine, and glutamine remained intact on DNA.     

Direct probing of copper active site and free radical formed during bicarbonate-dependent peroxidase activity of bovine and human copper, zinc-superoxide dismutases. Low-temperature electron paramagnetic resonance and electron nuclear double resonance studies

Using X-band electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopy at liquid helium temperatures, the Cu(II) coordination geometry at the active site of bovine and human copper,zinc-superoxide dismutases (bSOD1 and hSOD1) treated with H(2)O(2) and bicarbonate (HCO(3)(-)) was examined. The time course EPR of wild type human SOD1 (WT hSOD1), W32F hSOD1 mutant (tryptophan 32 substituted with phenylalanine), and bSOD1 treated with H(2)O(2) and HCO(3)(-) shows an initial reduction of active site Cu(II) to Cu(I) followed by its oxidation back to Cu(II) in the presence of H(2)O(2). However, HCO(3)(-) induced a Trp-32-derived radical from WT hSOD1 but not from bSOD1. The mutation of Trp-32 by phenylalanine totally eliminated the Trp-32 radical signal generated from W32F hSOD1 treated with HCO(3)(-) and H(2)O(2). Further characterization of the free radical was performed by UV irradiation of WT hSOD1 and bSOD1 that generated tryptophanyl and tyrosyl radicals. Both proton ((1)H) and nitrogen ((14)N) ENDOR studies of bSOD1 and hSOD1 in the presence of H(2)O(2) revealed a change in the geometry of His-46 (or His-44) and His-48 (or His-46) coordinated to Cu(II) at the active site of WT hSOD1 and bSOD1, respectively. However, in the presence of HCO(3)(-) and H(2)O(2), both (1)H and (14)N ENDOR spectra were almost identical to those derived from native bSOD1. We conclude that HCO(3)(-)-derived oxidant does not alter significantly the Cu(II) active site geometry and histidine coordination to Cu(II) in SOD1 as does H(2)O(2) alone; however, the oxidant derived from HCO(3)(-) (i.e. carbonate anion radical) reacts with surface-associated Trp-32 in hSOD1 to form the corresponding radical.