Spectroscopic properties of the cobalt(II)-substituted alpha-fragment of rabbit liver metallothionein

The C-terminal segment of rabbit liver metallothionein 1 (alpha-fragment) containing four paramagnetic Co(II) ions was obtained by stoichiometric replacement of the originally bound diamagnetic Cd(II) ions. The latter form was prepared by limited proteolysis with subtilisin as described previously [Winge, D. R., & Miklossy, K. A. (1982) J. Biol. Chem. 257, 3471-3476]. Electronic absorption, magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) measurements were employed to monitor the stepwise incorporation of Co(II) ions into the metal-free fragment. Absorption and MCD spectra of the apofragment containing the first 3 Co(II) equiv show the typical features of tetrahedral tetrathiolate Co(II) coordination. However, in the d-d region only small changes in the visible and no apparent change in the near-infrared region are discernible when the fourth Co(II) is bound. This unusual spectral behavior was not seen in Co(II) substitution of native metallothionein [Vasák, M., & K?gi, J. H. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6709-6713] and may indicate a different cluster geometry. In the charge-transfer region, the binding of all 4 Co(II) equiv is accompanied by characteristic increments of the thiolate S----Co(II) bands. As in the formation of Co(II)7-metallothionein, the development of the charge-transfer and EPR spectral properties upon binding of the first 2 Co(II) equiv to the apofragment is indicative of isolated, noninteracting tetrahedral tetrathiolate Co(II) complexes. The binding of the additional Co(II) ion is accompanied by a red shift in the charge-transfer region and by the dramatic loss of paramagnetism in the EPR spectra, both diagnostic of the formation of metal-thiolate cluster structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic studies on cobalt(II)-substituted metallo-beta-lactamase ImiS from Aeromonas veronii bv. sobria

In an effort to probe the structure of a group Bb metallo-beta-lactamase, Co(II)-substituted ImiS was prepared and characterized by electronic absorption, NMR, and EPR spectroscopies. ImiS containing 1 equiv of Co(II) (Co(II)(1)-ImiS) was shown to be catalytically active. Electronic absorption studies of Co(II)(1)-ImiS revealed the presence of two distinct features: (1) an intense sulfur to Co(II) ligand to metal charge transfer band and (2) less intense, Co(II) ligand field transitions that suggest 4-coordinate Co(II) in Co(II)(1)-ImiS. (1)H NMR studies of Co(II)(1)-ImiS suggest that one histidine, one aspartic acid, and one cysteine coordinate the metal ion in Co(II)(1)-ImiS. The addition of a second Co(II) to Co(II)(1)-ImiS did not result in any additional solvent-exchangeable NMR resonances, strongly suggesting that the second Co(II) does not bind to a site with histidine ligands. EPR studies reveal that the metal ion in Co(II)(1)-ImiS is 4-coordinate and that the second Co(II) is 5/6 coordinate. Taken together, these data indicate that the catalytic site in ImiS is the consensus Zn(2) site, in which Co(II) (and by extrapolation Zn(II)) is 4-coordinate and bound by Cys221, His263, Asp120, and probably one solvent water molecule. These studies also show that the second, inhibitory metal ion does not bind to the consensus Zn(1) site and that the metal ion binds at a site significantly removed from the active site. These results give the first structural information on metallo-beta-lactamase ImiS and suggest that the second metal binding site in ImiS may be targeted for inhibitors.     

Use of 2-mercaptoethanol during chromatography of crab (Cancer pagurus) metallothionein on DEAE cellulose

The chromatographic properties of crab metallothionein appear particularly sensitive to oxidation, and this oxidation does not take place when 2-mercaptoethanol is present in the buffers. The chromatographic changes brought about by oxidation can be reversed by subsequent addition of 2-mercaptoethanol.     

Spectroscopic studies of zinc(II)- and cobalt(II)-associated Escherichia coli formamidopyrimidine-DNA glycosylase: extended X-ray absorption fine structure evidence for a metal-binding domain

Formamidopyrimidine-DNA glycosylase (Fpg) is a 30.2 kDa protein that plays an important role in the base excision repair of oxidatively damaged DNA in Escherichia coli. Sequence analysis and genetic evidence suggest that zinc is associated with a C4-type motif, C(244)-X(2)-C(247)-X(16)-C(264)-X(2)-C(267), located at the C-terminus of the protein. The zinc-associated motif has been shown to be essential for damaged DNA recognition. Extended X-ray absorption fine structure (EXAFS) spectra collected on the zinc-associated protein (ZnFpg) in the lyophilized state and in 10% frozen aqueous glycerol solution show directly that the metal is coordinated to the sulfur atom of four cysteine residues. The average Zn-S bond length is 2.33 +/- 0.01 and 2.34 +/- 0.01 A, respectively, in the lyophilized state and in 10% frozen aqueous glycerol solution. Fpg was also expressed in minimal medium supplemented with cobalt nitrate to yield a blue-colored protein that was primarily cobalt-associated (CoFpg). The profiles of the circular dichroism spectra for CoFpg and ZnFpg are identical, suggesting that the substitution of Co(2+) for Zn(2+) does not alter the structure of Fpg. A similar conclusion is reached upon the analysis of two-dimensional (15)N/(1)H HSQC spectra of uniformly (15)N-labeled samples of ZnFpg and CoFpg; the spectra are similar and display features characteristic of a structured protein. Biochemical assays with a 54 nt DNA oligomer containing 7, 8-dihydro-8-oxoguanine at a specific location show that CoFpg and ZnFpg are equally active at cleaving the DNA at the site of the oxidized guanine. EXAFS spectra of CoFpg indicate that the cobalt is coordinated to the sulfur atom of four cysteine residues with an average Co-S bond length of 2.28 +/- 0.01 and 2.29 +/- 0.01 A, respectively, in the lyophilized state and in 10% frozen aqueous glycerol solution. The structural similarity between CoFpg and ZnFpg suggests that it is biologically relevant to use the paramagnetic properties of Co(2+) as a structural probe.     

The partition of copper and cadmium between different charge-forms of metallothionein in the digestive tubules of the crab, Cancer pagurus

In the presence of 2-mercaptoethanol DEAE cellulose chromatography of desalted hepatopancreas cytosols of crabs rich in both copper and cadmium yielded three peaks, probably all due to metallothioneins, with very different copper to cadmium ratios. Addition of a copper glycine complex to a desalted crab cytosol containing cadmium metallothionein gave a chromatogram similar to that obtained by in vivo administration of copper and cadmium. The chromatographic distribution of the metals between the different charge-forms in the distal and proximal portions of the tubules was basically similar although the concentrations of the metals were different.     

Deoxyribonucleic acid of the crab Cancer pagurus. 3. Intracellular localization of poly d(A-T) of Cancer pagurus by hybridization in situ

The satellite DNA poly [d(AT) · d(TA)] of the crab Cancer pagurus has been localized in situ by DNA-DNA hybridization in the nuclei of various spermatogenetic, midgut gland, intestinal and tegument cells. The specificity of hybridization was checked by various tests before, during and after hybridization. The nuclear sites revealed by this method were compared with those shown by quinacrine mustard or Giemsa staining. The AT-rich satellite DNA appears to be highly dispersed and does not seem to have any preferential localization inside the crab interphasic nucleus. This situation was compared with that presented by mouse nuclei using similar methods.     

Visible and magnetic circular dichroism studies on cobalt(II)-substituted rhus vernicifera laccase

The visible and magnetic circular dichroism (MCD) spectra of Co(II) derivatives of Rhus vernicifera laccase are reported. Anaerobic incorporation of 1 g-atom of Co(II) into apolaccase gave bands at 528(ϵ = 248), 558 (254) and 589 nm (shoulder) attributable to dd transitions. The MCD spectrum in the corresponding region is similar to that of Co(II)-substituted hemocyanin, indicating that the Co(II) ion incorporated into apolaccase is tetrahedral. On increasing the amount of Co(II) ion acting on the apolaccase, both the intensities of the absorption and the MCD spectra increased, and 2 g-atoms of tetrahedral Co(II) ion were introduced into the apolaccase. Very similar absorption and MCD spectra were obtained when laccase whose type I copper site was occupied by Hg(II) and both type II and type III copper sites were vacant (TlHg apolaccase) was treated with Co(II); this clearly supports the hypothesis that Co(II) cannot be incorporated into a type I copper site but may possibly be incorporated into a type III copper site. A tetrahedral Co(II) ion was also introduced into a type II copper site of type II copper-depleted (T2D) laccase, although its MCD bands were shifted ca. 20 nm to the longer wavelength region from the MCD bands due to tetrahedral Co(II) ion incorporated into type III copper site(s). The present study demonstrate that a tetrahedral Co(II) ion is introduced into type II or type III copper site(s) of laccase.     

Structural and electronic mimics of the active site of cobalt(II)-substituted zinc metalloenzymes

Complexes of cobalt(II) and zinc(II) which involve monodentate coordination of two alkyl carboxylate and two imidazole ligands in a slightly distorted tetrahedral fashion have visible and magnetic circular dichroism spectra remarkably similar to the cobalt(II)-substituted proteolytic enzymes thermolysin and carboxypeptidase A. Single crystal x-ray structure determinations on [Co(C₂H₅COO)₂Im₂], Im = imidazole, and its zinc counterpart reveal only minor structural differences between the cobalt and zinc species. Electron paramagnetic resonance spectra of cobalt(II) doped into zinc(II) complexes with known structures demonstrate the extreme sensitivity of the g-values to minor structural differences.     

Spectral studies of cobalt (II)- and Nickel (II)-metallothionein

The zinc and cadmium of native rabbit metallothionein-1 were replaced stoichiometrically with either cobalt (II) or nickel (II). The electronic, magnetic circular dichroic (MCD), and electron spin resonance spectra of Co (II)-metallothionein reflect distorted tetrahedral coordination of the cobalt atoms. Both the d-d and charge-transfer spectral regions closely resemble those of simple cobalt-tetrathiolate complexes, implying that their coordination chemistry is analogous. Ni (II) complex ions and Ni (II)-metallothionein similarly exhibit analogous MCD bands in the d-d region. The circular dichroic bands associated with ligand-metal charge-transfer transitions in the non-d-d region of Co (II)- and Ni (II)-metallothionein afford additional evidence for the similarity in tetrahedral microsymmetry of the two metal derivatives. The known ratio of 20 thiolate ligands to 7 metal ions, in conjunction with the spectral evidence for tetrathiolate coordination in metallothionein, represents good evidence that these metal thiolates are organized in clusters.     

Spectroscopic studies on metal distribution in Co(II)/Zn(II) mixed-metal clusters in rabbit liver metallothionein 2.

Metal selectivity of metal-thiolate clusters in rabbit liver metallothionein (MT) 2 has been studied by examining the metal distribution of two similarly sized divalent metal ions, cobalt and zinc, which have different thiolate affinity. The forms of mixed-metal cluster species in (Co/Zn)7-MT generated with different ratios of both metal ions offered to the metal-free protein were investigated using EPR, ultraviolet/visible absorption and MCD spectroscopy. The results demonstrated that the distribution of these metals between the two metal-thiolate clusters is not random. Thus, the EPR absorption intensities of the bound Co(II) ions in the Zn-cluster matrix increased linearly up to a ratio of Co(II)/Zn(II) equivalents of 3:4, with the final EPR intensity of three non-interacting Co(II)-binding sites. This EPR behaviour is consistent with a binding scheme in which one Co(II) ion occupies a metal-binding site within the three-metal cluster and the remaining two Co(II) ions occupy two distinctly separate sites in the four-metal cluster. With four or more Co(II) ions in the cluster matrix, magnetic coupling between adjacent, sulphur-bridged Co(II) ions was observed. In previous studies on mixed-metal clusters in MT formed with Co(II)/Cd(II), Zn(II)/Cd(II) and Cd(II)/Fe(II), changes in the respective cluster volumes were shown to be a significant factor dictating the widely differing metal distributions in these systems. Based on the results of the current study, it is suggested that both the sizes of the two metal ions and their relative affinities towards the cysteine-thiolate ligands are important in the formation of mixed-metal clusters in MT.     

Iron(II)-substituted metallothionein: evidence for the existence of iron-thiolate clusters

Metallothioneins (MT's) are unique low molecular weight (Mr 6000-7000) metal- and cysteine-rich proteins characterized by two tetrahedral tetrathiolate clusters containing three and four metal ions. Naturally occurring proteins usually contain the diamagnetic metal ions Zn(II) and/or Cd(II). We have now succeeded in substituting these ions by paramagnetic Fe(II). Rabbit liver MT-1 in which all seven metal binding sites were occupied by Fe(II) ions displays absorption features typical of tetrahedral tetrathiolate Fe(II) coordination. This is documented by the presence of a ligand field 5E----5T2 transition in the near-infrared region centered at about 1850 nm (epsilon Fe approximately 100 M-1 cm-1) and a broad charge-transfer absorption in the UV region with a shoulder at 314 nm. A metal-thiolate cluster structure is inferred from the 7 to 20 ratio of metal ions to cysteine residues and from spectral studies in which successive increments of Fe(II) were incorporated into the metal-free protein. Thus, to about 4 equiv, the charge-transfer absorption and magnetic circular dichroism (MCD) features of the complexes formed resemble closely those of reduced rubredoxin from Desulfovibro gigas in which tetrahedral tetrathiolate Fe(II) coordination is documented. However, upon further addition of Fe(II) ions, the charge-transfer absorption bands undergo a progressive red-shift until the full metal occupancy of seven Fe(II) ions per molecule is reached. The bathochromic shift which is also manifested in the MCD spectra can be ascribed to the transformation of some of the terminal thiolate ligands to bridging when the full complement of Fe(II) is bound.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosorption of copper(II) and cobalt(II) from aqueous solutions by crab shell particles

Biosorption of each of the heavy metals, copper(II) and cobalt(II) by crab shell was investigated in this study. The biosorption capacities of crab shell for copper and cobalt were studied at different particle sizes (0.456-1.117 mm), biosorbent dosages (1-10 g/l), initial metal concentrations (500-2000 mg/l) and solution pH values (3.5-6) in batch mode. At optimum particle size (0.767 mm), biosorbent dosage (5 g/l) and initial solution pH (pH 6); crab shell recorded maximum copper and cobalt uptakes of 243.9 and 322.6 mg/g, respectively, according to Langmuir model. The kinetic data obtained at different initial metal concentrations indicated that biosorption rate was fast and most of the process was completed within 2h, followed by slow attainment of equilibrium. Pseudo-second order model fitted the data well with very high correlation coefficients (>0.998). The presence of light and heavy metal ions influenced the copper and cobalt uptake potential of crab shell. Among several eluting agents, EDTA (pH 3.5, in HCl) performed well and also caused low biosorbent damage. The biosorbent was successfully regenerated and reused for five cycles.     

Spectroscopic characterization of Co(II)-, Ni(II)-, and Cd(II)-substituted wild-type and non-native retroviral-type zinc finger peptides

The nucleocapsid protein (NCP) from Mason-Pfizer monkey virus (MPMV) contains two evolutionary invariant Cys-X₂-Cys-X₄-His-X₄-Cys retroviral-type zinc finger structures, where the Cys and His residues provide ligands to a tetrahedrally coordinated Zn(II) ion. The N-terminal zinc finger (F1) of NCP from MPMV contains an immediately contiguous Cys in the -1 position relative to the start of this conserved motif: Cys-Cys-X₂-Cys-X₄-His-X₄-Cys. Metal complexes of 18-amino acid peptides which model the native zinc finger sequence, SER-Cys-X₂-Cys-X₄-His-X₄-Cys (F1_SC), and non-native Cys-SER-X₂-Cys-X₄-His-X₄-Cys (F1_CS) and SER-SER-X₂-Cys-X₄-His-X₄-Cys (F1_SS) sequences have been spectroscopically characterized and compared to the native two-zinc-finger protein fragment, MPMV NCP 21-80. All Co(II)-substituted peptide complexes adopt tetrahedral ligand geometries and have S^-MCo(II) ligand-to-metal charge-transfer (LMCT) transition intensities consistent with three Co(II)-S bonds for F1_SC and F1_CS. The non-native F1_CS peptide binds Co(II) with K_Co=1.5᎒⁶ M^-1, comparable to that of the native complex, and 걄-fold tighter than F1_SS. Like the Co(II) derivative, the absorption spectrum of Ni(II)-substituted NCP 21-80 is most consistent with tetrahedral Ni(II) complexes with multiple thiolate donors. In contrast, Ni(II) complexes of F1_SC and F1_CS exhibit a single absorption band in the 400-550 nm region ()겨-300 M^-1 cm^-1), distinct in the two complexes, assignable to a degenerate d-d transition envelope characteristic of non-native square-planar coordination geometry, and an intense LMCT transition in the UV ()₂₅₅ᄾ,000 M^-1 cm^-1). Cd(II) complexes have intense absorption in the UV (5_max=233 nm), with absolute intensities consistent with 񬩈 M^-1 cm^-1 per Cd(II)-S bond. ¹¹³Cd NMR spectroscopy of ¹¹³Cd MPMV NCP gives '=649 ppm, consistent with S₃N coordination. Co(II) and Cd(II) complexes of non-native F1_CS peptides are more sensitive to oxidation by O₂, relative to F1_SC, suggestive of a higher lability in the non-native chelate. The implications of these findings for the evolutionary conservation of this motif are discussed.     

Actin in the acrosome of the spermatozoa of the crab,Cancer pagurus L. (decapoda,crustacea)

The cytoskeletal protein actin was identified in the mature spermatozoon of the European edible crab, Cancer pagurus Linnaeus, by indirect immunofluoresce with monoclonal and polyclonal anti-actin antibodies, fluorescent phalloidin, and DNAase I. The actin was localized in two distinct concentric rings within the acrosome vesicle of the spermatozoon and appeared to correlate with the internal zonation of the vehicle. Modifications of the fluorescent pattern for actin were observed in sperm cells which were undergoing changes associated with the acrosome reaction. In these cases, fluorescent staining was observed in the nucleocytoplasm immediately subjacent to the perforatorial column and sometimes in the perforatorial column within the acrosome vesicle. Equally intense fluorescence was observed in an apical perforatorial projection. SDS-PAGE of C. pagurus sperm confirmed the presence of actin in the cells. A single band of actin (approximately 43 kDa) comigrated with rabbit muscle actin when immunoblotted onto nitrocellulose with mouse monoclonal anti-actin. The actin-associated cytoskeletal proteins α-actinin, tropomyosin, and spectrin were also identified within the spermatozoon of C. pagurus using specific polyclonal antibodies, but their presence was not confirmed by SDS-PAGE and immunoblotting. © 1994 Wiley-Liss, Inc.     

Preparation and spectroscopic studies of cobalt(II)-substituted cucumber basic blue protein "plantacyanin"

The cobalt(II) derivative of cucumber basic blue copper protein "plantacyanin" has been prepared. The visible absorption, circular dichroic and magnetic circular dichroic spectra of Co(II)-plantacyanin are similar to those of Co(II)-plastocyanin, indicating that the stereochemistry of Co(II) is tetrahedral and at least one cysteinyl ligand around Co(II) ion is responsible for the strong charge transfer bands at 331 and ca. 390 nm.     

Spectroscopic studies on the copper(II) complexes of carnosine

Carnosine complexes with copper(II) ions were studied with magnetic resonance techniques over a wide range of ligand to metal ratios at various pH values. Water proton relaxation rates increased with decreasing carnosine to copper ratios until a molar ratio of 48 was reached. Over the ratio range of 48–2 carnosine molecules per copper ion, the relaxation rate decreased so that in the 2:1 carnosine-copper(II) complex, the water-copper(II) distance was estimated to be 1.92 Å. Proton NMR studies revealed the broadening of imidazole proton lines at high mole ratios followed by other histidyl protons as the ratio decreased. The β-alanyl methylene protons were the last to be broadened by the addition of copper(II) ions. Carbon to copper(II) distances were determined for the carnosine to copper mole ratios of 500:1 and 5000:1. EPR spectra obtained at 93°K revealed the probable existence of four carnosine imidazoles as the sole coordinated ligands to copper(II) at high dipeptide-to-metal ratios (>10). At mole ratios below four, nuclear hyperfine lines characteristic of both monomeric and dimeric carnosine-copper(II) forms were observed. These results reveal that imidazole from carnosine is the sole ligand contributed to copper(II) for coordination over the pH range 5 to 7 at high carnosine to copper(II) ratios     

Spectroscopic studies on the copper(II)—Inosine system

Interactions of inosine derivatives with copper(II) were studied in the pH range 1.4–13 in 50% H₂O-50% DMSO solution. The distinct pH dependence of the optical spectra observed in copper(II)-inosine complexes are correlated to their respective EPR changes as a function of pH. It was concluded that a simple 1:1 complex of copper(II)-inosine is formed in the pH range 1.4–5.0 and bis complexes are present in the pH 5.0–6.2 region solutions of inosine and Cu(II). From pH 6.2 to 7.8 a diamagnetic, hydroxybridged complex dominates. At pH 7.8–9.2 an insoluble, oxybridged species is formed in addition to the soluble paramagnetic Cu(NI)₄ complex. Starting from pH 9.1 the N-polymeric complex is formed which is stable up to pH 12.5, and above pH 12.5 the only species is the Cu(ribose)₂ complex.     

An electron microscope study on the developing oocytes of the crab Cancer pagurus L. with special reference to yolk formation (Crustacea)

Summary The developing oocytes of the crab Cancer pagurus L. were studied with the light and electron microscope.Protein yolk formation was found to take place in two different ways. Yolk precursors of type 1 accumulate within the cisternae of an extensively developed granular endoplasmic reticulum. Also further growth and transformation into the definite yolk body occur within the reticular membranes. There is no structural indication that any other cell organelle contributes to the synthesis of this type of yolk building.Protein yolk formation of type 2 involves accumulation and transformation of material within a limiting membrane of the smooth type. The enclosed material is presumably derived from micropinocytosis, enclosed cellular elements and vesicles originating from the Golgi complex.It thus appears that the cell organelles play an important role in the process of drotein yolk formation in the growing oocytes of Cancer pagurus.