An x-ray absorption near edge structure spectroscopy study of metal coordination in Co(II)-substituted Carcinus maenas hemocyanin.

High-resolution x-ray absorption near edge structure spectroscopy was used to characterize the metal sites in three different cobalt-substituted derivatives of Carcinus maenas hemocyanin (Hc), including a mononuclear cobalt, a dinuclear cobalt and a copper-cobalt hybrid derivative. Co(II) model complexes with structures exemplifying octahedral, trigonal bipyramidal, pseudo-tetrahedral, and square planar geometries were also studied. The results provide structural information about the metal binding site(s) in the Co-Hcs that extend earlier results from EPR and optical spectroscopy (Bubacco et al. 1992. Biochemistry. 31: 9294-9303). Experimental spectra were compared to those calculated for atomic clusters of idealized geometry, generated using a multiple scattering approach. The energy of the dipole forbidden 1s-->3d transition and of the absorption edge in the spectra for all cobalt Hc derivatives confirmed the cobaltous oxidation state which rules out the presence of an oxygenated site. Comparisons between data and simulations showed that the mononuclear and dinuclear Co(II) derivatives, as well as the hybrid derivative, contain four-coordinate Co(II) in distorted tetrahedral sites. Although the spectra for Co(II) in dinuclear metal sites more closely resemble the simulated spectrum for a tetrahedral complex than do spectra for the mononuclear derivative, the Co(II) sites in all derivatives are very similar. The Cu K-edge high resolution x-ray absorption near edge structure spectrum of the hybrid Cu-Co-Hc resembles that of deoxy-Hc demonstrating the presence of three-coordinate Cu(I).     

Cobalt substitution of mouse R2 ribonucleotide reductase as a model for the reactive diferrous state: spectroscopic and structural evidence for a ferromagnetically coupled dinuclear cobalt cluster

The R2 dimer of mouse ribonucleotide reductase contains a dinuclear iron-oxygen cluster and tyrosyl radical/subunit. The dinuclear diferrous form reacts with dioxygen to generate the tyrosyl radical essential for the catalytic reaction that occurs at the R1 dimer. It is important to understand how the reactivity toward oxygen is related to the crystal structure of the dinuclear cluster. For the mouse R2 protein, no structure has been available with a fully occupied dinuclear metal ion site. A cobalt substitution of mouse R2 was performed to produce a good model for the very air-sensitive diferrous form of the enzyme. X-band EPR and light absorption studies (epsilon(550 nm) = 100 mm(-1) cm(-1)/Co(II)) revealed a strong cooperative binding of cobalt to the dinuclear site. In perpendicular mode EPR, the axial signal from mouse R2 incubated with Co(II) showed a typical S = 3/2 Co(II) signal, and its low intensity indicated that the majority of the Co(II) bound to R2 is magnetically coupled. In parallel mode EPR, a typical integer spin signal (M(s) = +/-3) with g approximately 12 is observed at 3.6 K and 10 K, showing that the two Co(II) ions (S = 3/2) in the dinuclear site are ferromagnetically coupled. We have solved the 2.4 A crystal structure of the Co(II)-substituted R2 with a fully occupied dinuclear cluster. The bridging Co(II) carboxylate ligand Glu-267 adopts an altered orientation compared with its counterpart Glu-238 in Escherichia coli R2. This might be important for proper O(2) activation of the more exposed native diferrous site in mouse R2 compared with E. coli R2.     

Co(II)-substituted Octopus vulgaris hemocyanin

Co(II)-substituted hemocyanin (Co(II)Hc) of the octopus, Octopus vulgaris, has been prepared by dialysis of apohemocyanin against Co(II·) ion and subsequent Chelex-treatment. The blue 50%-Co(II)Hc (half-apo Co(II)Hc), in which binuclear coppers are replaced in the hemocyanin by a single Co(II), exhibits two absorption maxima at 560 (?_Co=250) and 594 nm (?_Co=320 M^?1 cm^?1) and a shoulder near 610 nm, all of which are attributed to a dd transition of high spin Co(II) (S=3/2) with a tetrahedral geometry. The magnetic circular dichroism (MCD) spectrum in this region also suggests the existence of a tetrahedral Co(II) species in the protein. The visible absorption and MCD spectra of octopus 50%-Co(II)Hc are quite similar to those of squid 50%-Co(II)Hc described in the previous paper (S. Suzuki, J. Kino, M. Kimura, W. Mori and A. Nakahara, Inorg. Chim. Acta, 66, 41 (1982)). The formation of half-apo Co(II)Hc demonstrates that the binuclear copper sites in native octopus hemocyanin may differ from each other in coordination geometry, as in other molluscan hemocyanins, squid and snail hemocyanins. The coordination environment of the active-site Co(II) substituted for Cu in the octopus hemocyanin is the same as that of the corresponding active site of the squid hemocyanin.     

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.     

Octahedral metal coordination in the active site of glyoxalase I as evidenced by the properties of Co(II)-glyoxalase I

Co(II)-glyoxalase I has been prepared by reactivation of apoenzyme from human erythrocytes with Co2+. The visible absorption spectrum showed maxima at 493 and 515 nm and shoulders at 465 and 615 nm. The absorption coefficients at 493 and 515 nm were 35 and 33 M-1 cm-1/cobalt ion, respectively; i.e. 70 and 66 M-1 cm-1 for the dimeric metalloprotein. The product of the enzymatic reaction, S-D-lactoylglutathione, although binding to Co(II)-glyoxalase I, had no demonstrable effect on the visible absorption spectrum, indicating binding outside the first coordination sphere of the metal. The EPR spectrum at 3.9 K was characterized by g1 approximately 6.6, g2 approximately 3.0, and g3 approximately 2.5, and eight hyperfine lines with A1 = 0.025 cm-1. Binding of the strong competitive inhibitor S-p-bromobenzylglutathione to Co(II)-glyoxalase I gave three g values: 6.3, 3.4, and 2.5, indicating a conformational change affecting the environment of the metal ion. Both optical and EPR spectra strongly suggest a high spin Co2+ with octahedral coordination in the active site of the enzyme. The similarities in kinetic properties between native Zn(II)-glyoxalase I and enzyme substituted with Mg2+, Mn2+, or Co2+ is consistent with the view that these enzyme forms have the same metal coordination in the protein.     

Cobalt tyrosinase: replacement of the binuclear copper of Neurospora tyrosinase by cobalt

The antiferromagnetically spin-coupled copper(II) pair in Neurospora tyrosinase was substituted by cobalt, yielding a stoichiometry of 2 mol of Co/mol of protein. The low magnitude of the high-spin Co(II) EPR signal indicates spin coupling of the two Co(II) ions similar to that observed in the native enzyme. The absorption spectrum with four transitions in the visible region of intermediate intensity (epsilon 607(670), epsilon 564(630), epsilon 526(465)), a shoulder at 635 nm, and the near-infrared bands at 1180 (epsilon 30) and 960 nm (epsilon 15) indicate tetrahedral coordination around the Co(II) center. The cobalt(II) tyrosinase is enzymatically inactive, and there is no evidence that it binds molecular oxygen. Upon addition of cyanide or the competitive tyrosinase inhibitors L-mimosine, benzoic acid, or benzhydroxamic acid te absorption spectrum changes in a characteristic manner. This optical perturbation shows that binding of these inhibitors (and presumably of the substrates) occurs at or near the metal site. One Co(II) ion can be removed preferentially by incubation with KCN at high pH, indicating the two ions not to be in an identical environment.     

Transition metal complexes of amino acids and derivatives containing disulphide bridges

The interaction of cobalt(II), nickel(II), copper(II) and zinc(II) with D-penicillamine disulphide, oxidized glutathione and L-cysteinylglycine disulphide were studied by pH-metric, spectrophotometric and EPR methods. D-Penicillamine disulphide forms binuclear complexes with all the metal ions studied. The formation of 1:1 complexes is characteristic of oxidized glutathione. L-Cysteinylglycine disulphide behaves like dipeptides, but the presence of two separate peptide moieties also results in the formation of various binuclear complexes. Metal ion-disulphide binding was not observed in any case.     

Co(II) derivatives of Cu,Zn-superoxide dismutase with the cobalt bound in the place of copper. A new spectroscopic tool for the study of the active site

Co(II) derivatives of Cu,Zn-superoxide dismutase having cobalt substituted for the copper (Co,Zn-superoxide dismutase and Co,Co-superoxide dismutase) were studied by optical and EPR spectroscopy. EPR and electronic absorption spectra of Co,Zn-superoxide dismutase are sensitive to solvent perturbation, and in particular to the presence of phosphate. This behaviour suggests that cobalt in Co,Zn-superoxide dismutase is open to solvent access, at variance with the Co(II) of the Cu,Co-superoxide dismutase, which is substituted for the Zn. Phosphate binding as monitored by optical titration is dependent on pH with an apparent pKa = 8.2. The absorption spectrum of Co,Zn-superoxide dismutase in water has three weak bands in the visible region (epsilon = 75 M-1 X cm-1 at 456 nm; epsilon = 90 M-1 X cm-1 at 520 nm; epsilon = 70 M-1 X cm-1 at 600 nm) and three bands in the near infrared region, at 790 nm (epsilon = 18 M-1 X cm-1), 916 nm (epsilon = 27 M-1 X cm-1) and 1045 nm (epsilon = 25 M-1 X cm-1). This spectrum is indicative of five-coordinate geometry. In the presence of phosphate, three bands are still present in the visible region but they have higher intensity (epsilon = 225 M-1 X cm-1 at 544 nm; epsilon = 315 M-1 X cm-1 at 575 nm; epsilon = 330 M-1 X cm-1 at 603 nm), whilst the lowest wavelength band in the near infrared region is at much lower energy, 1060 nm (epsilon = 44 M-1 X cm-1). The latter property suggests a tetrahedral coordination around the Co(II) centre. Addition of 1 equivalent of CN- gives rise to a stable Co(II) low-spin intermediate, which is characterized by an EPR spectrum with a highly rhombic line shape. Formation of this CN- complex was found to require more cyanide equivalents in the case of the phosphate adduct, suggesting that binding of phosphate may inhibit binding of other anions. Titration of the Co,Co-derivative with CN- provided evidence for magnetic interaction between the two metal centres. These results substantiate the contention that Co(II) can replace the copper of Cu,Zn-superoxide dismutase in a way that reproduces the properties of the native copper-binding site.     

Cobalt substitution studies on bovine erythrocyte superoxide dismutase: evidence for a novel cobalt-superoxide dismutase derivative

Three cobalt derivatives of bovine erythrocyte superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) have been prepared under different pH conditions using a cobalt-thiocyanate complex which has already proved to yield specific substitutions on other copper proteins. The cobalt-protein derivatives have been characterized by optical, circular dichroism and fluorescence spectroscopies. One derivative, referred to as Co2Co2-protein, contains Co(II) ions specifically bound at both Zn(II) and Cu(II) sites. On the basis of their spectroscopic properties, the other two derivatives can be referred as E2Co2- and Co2E2-superoxide dismutase, with cobalt substituting, respectively, at the zinc and the copper sites leaving the contiguous site empty (E). The Co2E2-protein complex represents a novel derivative, since it has never been described in literature. The optical spectrum in the visible region of Co2-Co2-protein well corresponds to the sum of the spectra of the other two derivatives. The circular dichroism spectrum of Co2Co2-derivative, however, is not the sum of individual E2Co2- and Co2E2-proteins, suggesting that the presence of Co(II) in one site strongly affects the geometry of the neighbouring site. Some discrepancies between our spectroscopic data and those reported in literature are discussed. The results obtained from fluorescence experiments indicate that Co(II) ions exert a different quenching effect on the tyrosine emission, depending on whether they are located in the Zn(II) or in the Cu(II) site. The fluorescence quenching can be attributed to a 'heavy atom' and 'paramagnetic ion' effect by Co(II) ions.     

Spectroscopic characterization of the metal-binding sites in the periplasmic metal-sensor domain of CnrX from Cupriavidus metallidurans CH34

CnrX, the dimeric metal sensor of the three-protein transmembrane signal transduction complex CnrYXH of Cupriavidus metallidurans CH34, contains one metal-binding site per monomer. Both Ni and Co elicit a biological response and bind the protein in a 3N2O1S coordination sphere with a nearly identical octahedral geometry as shown by the X-ray structure of CnrXs, the soluble domain of CnrX. However, in solution CnrXs is titrated by 4 Co-equiv and exhibits an unexpected intense band at 384 nm that was detected neither by single-crystal spectroscopy nor under anaerobiosis. The data from a combination of spectroscopic techniques (spectrophotometry, electron paramagnetic resonance, X-ray absorption spectroscopy) showed that two sites correspond to those identified by crystallography. The two extra binding sites accommodate Co(II) in an octahedral geometry in the absence of oxygen and are occupied in air by a mixture of low-spin Co(II) as well as EPR-silent Co(III). These extra sites, located at the N-terminus of the protein, are believed to participate to the formation of peroxo-bridged dimers. Accordingly, we hypothesize that the intense band at 384 nm relies on the formation of a binuclear μ-peroxo Co(III) complex. These metal binding sites are not physiologically relevant since they are not detected in full-length NccX, the closest homologue of CnrX. X-ray absorption spectroscopy demonstrates that NccX stabilizes Co(II) in two-binding sites similar to those characterized by crystallography in its soluble counterpart. Nevertheless, the original spectroscopic properties of the extra Co-binding sites are of interest because they are susceptible to be detected in other Co-bound proteins.     

Divalent metal binding properties of the methionyl aminopeptidase from Escherichia coli

The metal-binding properties of the methionyl aminopeptidase from Escherichia coli (MetAP) were investigated. Measurements of catalytic activity as a function of added Co(II) and Fe(II) revealed that maximal enzymatic activity is observed after the addition of only 1 equiv of divalent metal ion. Based on these studies, metal binding constants for the first metal binding event were found to be 0.3 +/- 0.2 microM and 0.2 +/- 0.2 microM for Co(II)- and Fe(II)-substituted MetAP, respectively. Binding of excess metal ions (>50 equiv) resulted in the loss of approximately 50% of the catalytic activity. Electronic absorption spectral titration of a 1 mM sample of MetAP with Co(II) provided a binding constant of 2.5 +/- 0.5 mM for the second metal binding site. Furthermore, the electronic absorption spectra of Co(II)-loaded MetAP indicated that both metal ions reside in a pentacoordinate geometry. Consistent with the absorption data, electron paramagnetic resonance (EPR) spectra of [CoCo(MetAP)] also indicated that the Co(II) geometries are not highly constrained, suggesting that each Co(II) ion in MetAP resides in a pentacoordinate geometry. EPR studies on [CoCo(MetAP)] also revealed that at pH 7.5 there is no significant spin-coupling between the two Co(II) ions, though a small proportion ( approximately 5%) of the sample exhibited detectable spin-spin interactions at pH values > 9.6. EPR studies on [Fe(III)_(MetAP)] and [Fe(III)Fe(III)(MetAP)] also suggested no spin-coupling between the two metal ions. (1)H nuclear magnetic resonance (NMR) spectra of [Co(II)_(MetAP)] in both H(2)O and D(2)O buffer indicated that the first metal binding site contains the only active-site histidine residue, His171. Mechanistic implications of the observed binding properties of divalent metal ions to the MetAP from E. coli are discussed.     

Reaction of N,N-diethyldithiocarbamate and other bidentate ligands with Zn, Co and Cu bovine carbonic anhydrases. Inhibition of the enzyme activity and evidence for stable ternary enzyme-metal-ligand complexes

The reactions with N,N-diethyldithiocarbamate (DDC) of zinc, cobalt and copper carbonic anhydrase from bovine erythrocytes were investigated. The native zinc enzyme was inhibited by DDC, but no removal of zinc could be detected even at a very high [ligand]/[protein] ratio. At identical pH values a larger inhibitory effect was found for the cobalt enzyme. The metal was removed by DDC from the protein at pH less than 7.0. No cobalt removal occurred at pH 10, where a stable ternary complex with the enzyme-bound Co(II) was detected. Its optical and EPR spectra are indicative of five-coordinate Co(II). The reaction of the Cu(II) enzyme with stoichiometric chelating agent was marked by the appearance of an electronic transition at 390 nm (epsilon = 4300 M-1 X cm-1). Metal removal from the copper enzyme readily occurred as the ligand was in excess over the metal, with parallel appearance of a band at 440 nm, which was attributed to the free Cu(II)-DDC complex. Also, in the case of the copper enzyme an alkaline pH was found to stabilize the ternary adduct with the diagnostic 390 nm band. EPR spectra showed that the ternary adduct is a mixture of two species, both characterized by the presence in the EPR spectrum of a superhyperfine structure from two protein nitrogens and by a low g parallel value, indicative of coordination to sulfur ligands. It is suggested that the two species contain the metal as penta- and hexacoordinated, respectively. Measurements of the longitudinal relaxation time, T1, of the water protons suggested that water coordination is retained in the latter case. Hexacoordination with retention of water is also proposed for the Cu(II) derivatives with the bidentate oxalate and bicarbonate anions, unlike the corresponding Co(II) derivatives, which are pentacoordinated. Different coordination of Co(II) and Cu(II) adducts may be relevant to the difference of activity of the two substituted enzymes.     

Metal cofactors of lysine-2,3-aminomutase.

Lysine-2,3-aminomutase from Clostridium SB4 contains iron and sulfide in equimolar amounts, as well as cobalt, zinc, and copper. The iron and sulfide apparently constitute an Fe-S cluster that is required as a cofactor of the enzyme. Although no B12 derivative can be detected, enzyme-bound cobalt is a cofactor; however, the zinc and copper bound to the enzyme do not appear to play a role in its catalytic activity. These conclusions are supported by the following facts reported in this paper. Purification of the enzyme under anaerobic conditions increases the iron and sulfide content. Lysine-2,3-aminomutase purified from cells grown in media supplemented with added CoCl2 contains higher levels of cobalt and correspondingly lower levels of zinc and copper relative to enzyme from cells grown in media not supplemented with cobalt. The specific activity of the purified enzyme increases with increasing iron and sulfide content, and it also increases with increasing cobalt and with decreasing zinc and copper content. The zinc and copper appear to occupy cobalt sites under conditions of insufficient cobalt in the growth medium, and they do not support the activity of the enzyme. The best preparations of lysine-2,3-aminomutase obtained to date exhibit a specific activity of approximately 23 units/mg of protein and contain about 12 g atoms of iron and of sulfide per mol of hexameric enzyme. These preparations also contain 3.5 g atoms of cobalt per mol, but even the best preparations contain small amounts of zinc and copper. The sum of cobalt, zinc, and copper in all preparations analyzed to date corresponds to 5.22 +/- 0.75 g atoms per mol of enzyme. An EPR spectrum of the enzyme as isolated reveals a signal corresponding to high spin Co(II) at temperatures below 20 K. The signal appears as a partially resolved 59Co octet centered at an apparent g value of 7. The 59Co hyperfine splitting (approximately 35 G) is prominent at 4.2 K. These findings show that lysine-2,3-aminomutase requires Fe-S clusters and cobalt as cofactors, in addition to the known requirement for pyridoxal 5'-phosphate and S-adenosylmethionine.     

Cobalt-substituted derivatives ofCarcinus hemocyanin

Summary Four derivatives ofCarcinus maenas hemocyanin containing Co(II) in the active site have been prepared under different experimental conditions. Two of them contain one Co(II) ion/active site and most probably represent isomeric forms containing Co(II) either in the fast-reacting or in the slow-reacting position within the active site. A third derivative contains two Co(II) ions active site, which reproduces the metal/protein stoichiometry of native hemocyanin. The fourth derivative is a metal hybrid form containing one Cu(I) ion and one Co(II) ion/active site. The derivatives have been characterized by absorption, circular dichroic and fluorescence spectroscopies. The results indicate that in all derivatives the metal is bound with a low coordination number, in agreement with the presence of three histidine residues/copper ion in the native protein. The two alternative metal-binding positions have different structures as shown by the different spectroscopic properties of the bound Co(II) ions. A marked hyperchromic effect on the optical absorption of Co(II) is observed as a result of the presence of a metal ion in the neighbouring metal-binding position in the active site.     

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.     

A five-coordinate metal center in Co(II)-substituted VanX

In an effort to structurally probe the metal binding site in VanX, electronic absorption, EPR, and extended x-ray absorption fine structure (EXAFS) spectroscopic studies were conducted on Co(II)-substituted VanX. Electronic spectroscopy revealed the presence of Co(II) ligand field transitions that had molar absorptivities of approximately 100 m(-1) cm(-1), which suggests that Co(II) is five-coordinate in Co(II)-substituted VanX. Low temperature EPR spectra of Co(II)-substituted VanX were simulated using spin Hamiltonian parameters of M(S) = |+/-1/2), E/D = 0.14, g(real(x,y)) = 2.37, and g(real(z)) = 2.03. These parameters lead to the prediction that Co(II) in the enzyme is five-coordinate and that there may be at least one solvent-derived ligand. Single scattering fits of EXAFS data indicate that the metal ions in both native Zn(II)-containing and Co(II)-substituted VanX have the same coordination number and that the metal ions are coordinated by 5 nitrogen/oxygen ligands at approximately 2.0 angstroms. These data demonstrate that Co(II) (and Zn(II) from EXAFS studies) is five-coordinate in VanX in contrast to previous crystallographic studies (Bussiere, D. E., Pratt, S. D., Katz, L., Severin, J. M., Holzman, T., and Park, C. H. (1998) Mol. Cell 2, 75-84). These spectroscopic studies also demonstrate that the metal ion in Co(II)-substituted VanX when complexed with a phosphinate analog of substrate D-Ala-D-Ala is also five-coordinate.     

Spectroscopic studies of cobalt-substituted ferredoxin fromClostridium pasteurianum

Summary Ferredoxin fromClostridium pasteurianum substituted with two Co atoms did not give any cobalt EPR signal at 8 K as isolated, but upon reduction with sodium dithionite, a broad signal appeared withg values that indicate highspin (S=3/2) Co(II). These signals were distinct from Co(II)-dithiothreitol signals, and disappeared upon reoxidation with air. Under anaerobic incubation of apoferredoxin with Co(II), a green derivative showed a visible spectrum typical of tetrahedral Co(Il)-thiolate coordination, which shifted dramatically upon exposure to air. The¹H-NMR spectrum of the aerobically isolated protein is reported at 300 MHz; magnetic susceptibility measurements were indicative of a diamagnetic species. These spectroscopic studies indicate that Co(II)-substituted ferredoxin is oxidized to low-spin Co(III)-ferredoxin in the presence of sulfide and oxygen. The diamagnetic Co(III) state could reversibly be reduced to highspin Co(II) by sodium dithionite.     

The interaction of ascorbate oxidase with L-dopa,L-tyrosine and 3,4-dihydroxycinnamic acid. Evidence for irreversible damage of the enzyme during catechol oxidase activity

The aerobic interaction between ascorbate oxidase and L-tyrosine, L-3,4-dihydroxyphenylalanine or 3,4-dihydroxycinnamic acid in 1:10 molar ratio was followed by optical absorption, CD and EPR spectroscopy in 0.1 M phosphate buffer at pH 5.0. While the spectra of the system ascorbate oxidase—L-tyrosine remain practically unaffected after several hours, indicating that no oxidation of the amino acid occurs in the conditions employed, rather drastic changes can be observed in the spectra of the ascorbate oxidase-catechol systems. In particular, while the optical absorption below 500 nm increases markedly due to the formation of the substrate oxidation products, an irreversible decrease in intensity of the absorption, CD and EPR spectral features associated with the blue copper(II) chromophores indicates that a partial loss of Type 1 copper by ascorbate oxidase has occurred during this secondary catechol oxidase activity. A copper species characterized by weak positive CD activity at 370 nm and EPR signal at intermediate field between those of the Type 2 and Type 1 coppers can be detected in the early stages of the reaction. The irreversible damage undergone by the protein during catechol oxidase activity may have biological significance and accounts for the low yield of purified enzyme obtained when the crude enzyme extract is left in prolonged contact with low molecular weight cell components, rich in σ-diphenolic compounds.     

Metal-directed affinity labeling of zinc(II), cobalt(II) and cadmium(II) horse liver alcohol dehydrogenases

The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition of bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn ? Cd ? Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co ? Zn ? Cd.     

Exchange inert Co (III)-carboxypeptidase A: a catalytically inactive metallocarboxypeptidase.

Exposure of cobalt (II) carboxypeptidase A_α, [(CPD)Co(II)], to small molar excesses of the oxidizing agent m-chloroperbenzoate rapidly destroys (< 30 sec) both its peptidase and esterase activities in parallel. Concomitantly, the characteristic Co(II) electron paramagentic resonance (EPR) signal is abolished. [(CPD)Co(III)], isolated from the reaction mixture, has the same molecular weight and amino acid composition as [(CPD)Co(II)], contains 0.95 g-atom of Co and 0.01 g-atom of Zn per mole of protein, does not exhibit an EPR spectrum and is catalytically completely inactive towards both peptide and ester substrates. Identical treatment of the native zinc enzyme affects neither its catalytic activity nor its metal content. The reaction of m-chloroperbenzoate with [(CPD)Co(II)] follows saturation kinetics and is prevented by the inhibitor β-phenylpropionate. Furthermore, under the conditions found to oxidize [(CPD)Co(II)] effectively, there is no reaction with Co(II) $\text{E. coli}$ alkaline phosphatase. Thus, m-chloroperbenzoate has the characteristics of an active-site directed oxidizing reagent for [(CPD)Co(II)].