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.     

Interaction of anions with the active site of carboxypeptidase A

Studies of azide inhibition of peptide hydrolysis catalyzed by cobalt(II) carboxypeptidase A identify two anion binding sites. Azide binding to the first site (KI = 35 mM) inhibits peptide hydrolysis in a partial competitive mode while binding at the second site (KI = 1.5 M) results in competitive inhibition. The cobalt electronic absorption spectrum is insensitive to azide binding at the first site but shows marked changes upon azide binding to the second site. Thus, azide elicits a spectral change with new lambda max (epsilon M) values of 590 (330) and 540 nm (190) and a KD of 1.4 M, equal to the second kinetic KI value for the cobalt enzyme, indicating that anion binding at the weaker site involves an interaction with the active-site metal. Remarkably, in the presence of the C-terminal products of peptide or ester hydrolysis or carboxylate inhibitor analogues, anion (e.g., azide, cyanate, and thiocyanate) binding is strongly synergistic; thus, KD for azide decreases to 4 mM in the presence of L-phenylalanine. These ternary complexes have characteristic absorption, CD, MCD, and EPR spectra. The absorption spectra of azide/carboxylate inhibitor ternary complexes with Co(II)CPD display a near-UV band between 305 and 310 nm with epsilon M values around 900-1250 M-1 cm-1. The lambda max values are close to the those of the charge-transfer band of an aquo Co(II)-azide complex (310 nm), consistent with the presence of a metal azide bond in the enzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physicochemical properties of cloned nucleocapsid protein from HIV. Interactions with metal ions

The nucleocapsid (NC) protein (p15) of the human immunodeficiency virus (HIV) has been cloned and overproduced (under the control of a phage T7 promoter) in soluble form in an Escherichia coli host. The soluble NC protein is a fusion protein containing 15 amino acids from the T7 gene 10 and 7 amino acids from the HIV p24 protein at the N-terminus to make a protein of 171 amino acids. The plasmid containing the fusion gene is designated p15DF. A homogeneous product has been isolated from the induced cells and, when isolated under aerobic conditions, contains 0.3-0.5 mol of Zn/mol of protein and has only 2 titratable SH groups. Reduction and refolding in the presence of Zn(II) yields a protein containing 2.0 mol of Zn/mol of protein and 6 titratable SH groups. On the other hand, if the cells are sonicated in 2 mM CdCl2 and purified at pH 5.0, an unoxidized protein containing 2 mol of Cd/mol of protein is obtained. The Cd(II) ions can be exchanged with Zn(II), Co(II), or 113Cd(II). The Co(II)2 NC protein shows d-d electronic transitions at 695 nm [epsilon = 675 M-1 cm-1 per Co(II)] and 640 nm [epsilon = 825 M-1 cm-1 per Co(II)] compatible with regular tetrahedral geometry around both Co(II) ions. The Co(II)2 and Cd(II)2 NC proteins show intense charge-transfer bands in the near-UV, at 355 nm (epsilon = approximately 4000 M-1 cm-1) and 310 nm (epsilon = approximately 8000 M-1 cm-1) for the Co(II) protein and 255 nm (epsilon = approximately 10(4) M-1 cm-1) for the Cd(II)2 NC protein, compatible with -S- coordination. 113Cd NMR of the 113Cd(II)2 NC protein shows two 113Cd NMR signals at 659 and 640 ppm, respectively, each integrating to approximately 1 Cd(II) ion. The downfield chemical shifts suggest coordination of each 113Cd(II) ion to 3 sulfur donor atoms. The spectroscopic data fully support the prediction that the NC protein binds metal ions to each of the tandem repeats of the -Cys-X2-Cys-X4-His-X4-Cys- sequence contained in the N-terminal half of the molecule. 113Cd NMR shows, however, that the sites are not identical. Isolation of the NC protein under standard aerobic conditions results in oxidation of the sulfhydryl groups and loss of the coordinated Zn(II) ions, while preparation of the NC protein as the Cd(II) derivative at low pH protects the sulfhydryl groups from oxidation.     

Spectroscopic studies on the metal-ion-binding sites of Co2(+)-substituted D-xylose isomerase from Streptomyces rubiginosus

The coordination sphere of the two metal-binding sites/subunit of the homotetrameric D-xylose isomerase from Streptomyces rubiginosus has been probed by the investigation of the Co2(+)-substituted enzyme using electronic absorption, CD and magnetic circular dichroic spectroscopies in the visible region. The spectrum of the high-affinity site (B site) has an absorption coefficient, epsilon 545, of 18 M-1 cm-1, indicating a distorted octahedral complex geometry. The spectrum of the low-affinity site (A site) shows two absorption maxima at 505 nm and 586 nm with epsilon values of 170 M-1 cm-1 and 240 M-1 cm-1, respectively, which indicates a distorted tetrahedral or pentacoordinated complex structure as also observed for the enzyme from Streptomyces violaceoruber [Callens et al. (1988) Biochem. J. 250, 285-290] having the same feature but lower epsilon values. The first 4 mol Co2+ added/mol apoenzyme occupy both sites nearly equally. Subsequently the Co2+ located in the A site slowly moves into the B site. After equilibrium is reached, the next 4 mol Co2+/mol again occupy the A site with its typical spectrum, restoring full activity. Addition of 4 mol Cd2+ or Pb2+/mol Co4-loaded derivative displaces the Co2+ from the B site to form the Pb4/Co4 derivative containing Co2+ in the A site, reducing activity fourfold while the Pb4/Pb4 species is completely inactive. In contrast, Eu3+ displaces Co2+ preferentially from the A site. Thus, the high- and low-affinity sites may be different for different cations. After addition of the substrates D-xylose, D-glucose and D-fructose and the inhibitor xylitol the intense Co2+ A-site spectrum of both the active Co4/Co4 derivative and the less active Pb4/PCo4 derivative decreases, indicating that these compounds are bound to the A site, changing the distorted tetrahedral or pentacoordinated symmetry there to a distorted octahedral complex geometry.     

A continuous spectrophotometric method for the determination of diphenolase activity of tyrosinase using 3,4-dihydroxymandelic acid.

A continuous spectrophotometric method for the rapid determination of diphenolase activity of tyrosinase is described. It uses 3,4-dihydroxymandelic acid (DOMA) as the substrate of tyrosinase and measures the final product, 3,4-dihydroxybenzaldehyde (DOBA). The spectrum of this product shows a bathochromic displacement of its absorbance maximum when the pH increases. The optimization of the method is described by using tyrosinase from several biological sources, whose enzymatic activities show different optimal pH. Thus, the enzymatic activity of mushroom tyrosinase was assayed at pH 7.5 and monitored at 350 nm (epsilon 350 pH 7.5 (DOBA) = 15,200 M-1 cm-1), whereas the spectrophotometric experiments with grape tyrosinase were carried out at pH 3.0 and monitored at 310 nm (epsilon 310 pH 3.0 (DOBA) = 9200 M-1 cm-1). The method for mushroom tyrosinase was found to be 50-fold more sensitive than the commonly used dopachrome assay, whereas for grape tyrosinase the method was found to be threefold more sensitive than the commonly used o-quinone production assay. The great solubility and stability of the chromophoric product, DOBA, as well as its high molar absorptivities at any pH, enable the method to be employed to determine the diphenolase activity of tyrosinase from different biological sources.     

p10 single-stranded nucleic acid binding protein from murine leukemia virus binds metal ions via the peptide sequence Cys26-X2-Cys29-X4-His34-X4-Cys39

The RNA binding protein of 56 residues encoded by the extreme 3' region of the gag gene of Rauscher murine leukemia virus (MuLV) has been chemically synthesized by a solid-phase synthesis approach. Since the peptide contains a Cys26-X2-Cys29-X4-His34-X2-Cys39 sequence that is shared by all retroviral gag polyproteins which has been proposed to be a metal binding region, it was of considerable interest to examine the metal binding properties of the complete p10 protein. As postulated, p10 binds the metal ions Cd(II), Co(II), and Zn(II). The Co(II) protein shows a set of d-d absorption bands typical of a tetrahedral Co(II) complex at 695 (epsilon = 565 M-1 cm-1), 642 (epsilon = 655 M-1 cm-1), and 615 nm (epsilon = 510 M-1 cm-1) and two intense bands at 349 (epsilon = 2460 M-1 cm-1) and 314 nm (epsilon = 4240 M-1 cm-1) typical of Co(II)----(-)S- charge transfer. The ultraviolet absorption spectrum also indicates Cd(II) binding by the appearance of a Cd(II)----(-)S- charge-transfer band at 255 nm. The 113Cd NMR spectrum of 113Cd(II)-p10 reveals one signal at delta = 648 ppm. This chemical shift correlates well with that predicted for ligation of 113Cd(II) to three -S- from the three Cys residues of p10. The chemical shift of 113Cd(II)-p10 changes by only 4 ppm upon binding of d(pA)6, indicating that the chelate complex is little changed by oligonucleotide binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Spectral and kinetic studies of metal-substituted Aeromonas aminopeptidase: nonidentical, interacting metal-binding sites

Apoenzyme prepared by removal of the 2 mol of Zn2+/mol from Aeromonas aminopeptidase is inactive. Addition of Zn2+ reactivates it completely, and reconstitution with Co2+, Ni2+, or Cu2+ results in a 5.0-, 9.8-, and 10-fold more active enzyme than native aminopeptidase, respectively. Equilibrium dialysis and spectral titration experiments with Co2+ confirm the stoichiometry of 2 mol of metal/mol. The addition of only 1 mol of metal/mol completely restores activity characteristic of the particular metal. Interaction between the two sites, however, causes hyperactivation; thus, addition of 1 mol of Zn2+/mol subsequent to 1 mol of Co2+, Ni2+, or Cu2+ per mole increases activity 3.2-, 42-, or 59-fold, respectively. The cobalt absorption spectrum has a peak of 527 nm with a molar absorptivity of 53 M-1 cm-1 for 1 mol of cobalt/mol, which increases to 82 M-1 cm-1 for a second cobalt atom and is unchanged by further addition of Co2+. Circular dichroic (CD) and magnetic CD spectra indicate that the first Co2+ binding site is tetrahedral-like and that the second is octahedral-like. Stoichiometric quantities of 1-butylboronic acid, a transition-state analogue inhibitor of the enzyme [Baker, J. O., & Prescott, J. M. (1983) Biochemistry 22, 5322], profoundly affects absorption, CD, and MCD spectra, but n-valeramide, a substrate analogue inhibitor, has no effect. These findings suggest that the tetrahedral-like site is catalytic and the other octahedral-like site is regulatory or structural.     

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.     

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.     

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.     

Comparison of amino acid sequence and thermostability of tyrosinase from three wild type strains of Neurospora crassa

The thermostability of tyrosinase from three wild type strains of Neurospora crassa has been investigated. For this purpose a sequence comparison of two thermostable and one thermolabile tyrosinase isoenzyme was carried out. It revealed that at position 201 the thermostable enzyme forms share an aspartate residue in contrast to an asparagine residue in the thermolabile form. In addition, one of the thermostable isoenzymes displays five other substitutions. Since the relative stability of the thermostable forms as compared to the thermolabile one decreases with increasing ionic strength, the common aspartate residue is thought to bring about the additional stability of the thermostable isoenzymes by forming a salt bridge between aspartate 201 and a positively charged group of the protein. The strong pH-dependency of the thermostability with an apparent pKA of 6.6 indicates a histidinium side chain as the most likely ionic group to be involved in the salt bridge. This conjecture is also supported by measurements of the stability towards the chaotropic agent guanidinium chloride. The difference of the free energy change of denaturation delta GDH2O between the apoenzymes of a thermostable and a thermolabile isoenzyme was calculated as 2.5 kcal mol-1. Furthermore, it was shown that the copper ions of the native and the cobalt ions of Co(II)-substituted tyrosinase strongly enhance the stability of the protein as compared to its apoform.     

Selective substitution in vitro of an intrinsic zinc of Escherichia coli RNA polymerase with various divalent metals

A simple in vitro substitution method involving a sequential denaturation--reconstitution process was developed to substitute selectively one of the two intrinsic Zn ions in Escherichia coli RNA polymerase with Co, Mn, Ni, or Cu ion. The resultant metal hybrid Co-Zn, Mn-Zn, Ni-Zn, and Cu-Zn RNA polymerases possess 100, 100, 60, and 17% of the enzymatic activity of the reconstituted Zn-Zn enzyme, respectively. The substituted metal was found to be located in the beta subunit of the polymerase which contains the substrate binding site. The biochemical and physical properties of these metal-substituted polymerases were compared with those of the native Zn enzyme. Co-Zn and Ni-Zn core polymerases exhibit characteristic absorption spectra in the near-UV and visible region, while Mn-Zn and Cu-Zn enzymes do not. The Co-Zn enzyme shows two major peaks at 400 nm (epsilon = 3000) and 475 nm (epsilon = 2700), while the Ni-Zn enzyme exhibits a major peak at 462 nm (epsilon = 8000). The difference absorption spectrum of Ni-Zn core polymerase could be perturbed by the addition of substrate ATP but not by UTP in the absence of template and Mg(II) ion. These observations suggest that the substituted metal was located at the initiation site of the enzyme. The various metal hybrid enzymes do not differ appreciably in their abilities to incorporate noncomplementary nucleotide or deoxyribonucleotide into RNA product. It was found, however, that the difference in enzymatic activities of these metal hybrid enzymes resides at least partly in the initiation step of RNA synthesis.     

Preparation and spectroscopic characterization of a coupled binuclear center in cobalt(II)-substituted hemocyanin.

A binuclear cobalt derivative of arthropod hemocyanin (Hc) has been prepared by the reaction of apo-Hc with Co(II) in the presence of thiocyanate. The crude product of the reaction contains specifically and adventitiously bound metal, the latter being removable by EDTA treatment. The specifically bound Co(II) constitutes a binuclear metal center that exhibits optical and CD spectra typical in their absorption maxima and extinction coefficients of Co(II) complexes with near-tetrahedral geometry. The EPR spectrum of the binuclear Co(II) derivative contains a resonance at g approximately 13, which is characteristic of integer spin systems and indicates coupled metal ions; the excess Co(II) bound to crude products exhibits an EPR signal at g approximately 4. The time course of derivative formation was followed by EPR, optical and atomic absorption techniques, and by fluorimetry. The intensity of the optical absorption in the visible region due to Co(II) increases with increasing stoichiometry of specifically bound metal [up to 2 Co(II) per protein monomer], but the intensity of the Co(II) EPR signal increases only during the formation of a mononuclear derivative. As the reaction proceeds over approximately 100 h to the formation of the binuclear derivative, the EPR signal intensity decreases to 10% of the value expected for 2 mol of EPR-active Co(II)/mol of protein. The binuclear cobalt derivative cannot be reconstituted to native Hc with Cu(I), indicating the stable loading of Co(II) in the active site. EPR and optical spectroscopic evidence is presented showing that the binuclear derivative does not bind oxygen.     

Interactions between retinyl phosphate and bivalent cations.

In the presence of Mn(II) ions, the u.v. absorption spectrum of retinyl phosphate (Ret-P) solubilized in Triton X-100 micelles, phosphatidylcholine liposomes or rat liver microsomes exhibited a shift from the maximum of 330 nm to 287 nm. The effect of Mn(II) was reversed by adding EDTA or phosphate buffer. The same spectral change was found in the presence of poly-L-lysine in place of Mn(II) ions. The e.s.r. spectrum of Mn(II) in the presence or in the absence of Ret-P clearly showed that approx. 75% of the initial concentration of Mn(II) ions is bound to Ret-P when the molar ratio of Ret-P to Mn(II) ions is 4:1; no such binding occurred in the presence of retinol or retinoic acid. The appearance of two isosbestic points at 303 and 368 nm, in the presence of Mn(II) ions, suggests the existence of an equilibrium between an Mn(II)-bound monomer and an Mn(II)-bound dimer of Ret-P in Triton X-100 micelles. The same effect on the u.v.-absorption spectrum of Ret-P was also induced by Co(II), Cr(II), Zn(II) and Fe(II), but not by Mg2+ or Cu(II). The formation of the 'metachromatic complex' between Ret-P and Mn(II) or Co(II) inhibited the synthesis of retinyl phosphate mannose (Ret-P-Man) from exogenous and endogenous Ret-P and guanosine diphosphate [14C]mannose when bovine serum albumin was added after the metal ion. However, the order of addition did not influence Ret-P-Man synthesis in incubations containing MgCl2, which does not form the metachromatic complex with Ret-P. These results suggest that the bioavailability of proteins, polyamines and metal ions may control the extent to which Ret-P can be mannosylated in the intact membrane.     

The catalytic effect of tyrosinase upon oxidation of 2-hydroxyestradiol in presence of catechol

The hydroxylating activity of mushroom tyrosinase has been utilized for over a decade in the preparation of 2-hydroxyestradiol from estradiol, yet this same enzyme is known to function as an oxidant of o-dihydric compounds to the corresponding o-quinones. It was questioned why catechol estrogens do not react further, particularly since the tyrosinase activity (hydroxylating) is exceeded many fold by the diphenol oxidase activity of the enzyme. This report describes that the catechol estrogen will react in presence of enzyme but only if catechol is also present. Diphenol oxidase activity was measured either by the polarographic oxygen-utilization technique or by changes in the absorption spectrum at 206 and 256 nm. The enzyme activity was standardized with catechol (Km = 5.2 X 10(-4) M). The steroid did not react with the enzyme if catechol was absent. With catechol, the steroid reacted rapidly and completely (Km = 4.2 X 10(-4) M). The consumption of oxygen with catechol and 2-hydroxyestradiol was additive and stoichiometric, 1 g-atom oxygen/mol of either substrate. Kinetic analysis shows that catechol functions as an activator of the tyrosinase.     

Isolation and characterization of an Fe,-S8 ferredoxin (ferredoxin II) from Clostridium thermoaceticum.

A second ferredoxin protein was isolated from the thermophilic anaerobic bacterium Clostridium thermoaceticum and termed ferredoxin II. This ferredoxin was found to contain 7.9 +/- 0.3 iron atoms and 7.4 +/- 0.4 acid-labile sulfur atoms per mol of protein. Extrusion studies of the iron-sulfur centers showed the presence of two [Fe4-S4] centers per mol of protein and accounted for all of the iron present. The absorption spectrum was characterized by maxima at 390 nm (epsilon 390 = 30,400 M-1cm-1) and 280 nm (epsilon 280 = 41.400 M-1 cm-1) and by a shoulder at 300 nm. The ration of the absorbance of the pure protein at 390 nm to the absorbance at 280 nm was 0.74. Electron paramagnetic resonance data showed a weak signal in the oxidized state, and the reduced ferredoxin exhibited a spectrum typical of [Fe4-S4] clusters. Double integration of the reduced spectra showed that two electrons were necessary for the complete reduction of ferredoxin II. Amino histidine, and 1 arginine, and a molecular weight of 6,748 for the native protein. The ferredoxin is stable under anaerobic conditions for 60 min at 70 degrees C. The average oxidation-reduction potential for the two [Fe4-S4] centers was measured as -365 mV.     

Absorption and circular dichroism spectra of different forms of mushroom tyrosinase.

The circular dichroism spectrum of resting mushroom tyrosinase between 800 and 400 nm showed two bands at 755, and 653 nm. The CD spectrum of resting tyrosinase between 400 and 250 nm showed oxygen-sensitive changes at 350 nm upon treatment of tyrosinase with hydroxylamine or hydrogen peroxide. These were similar to changes observed on regeneration of aged hemocyanin by similar procedures. A structural relationship between the active sites of hydroxylamine- or hydrogen peroxide-treated tyrosinase and hemocyanin is suggested by these observations, confirming inferences based upon other studies (Jolly, Jr., R.L., Evans, L.H., Makino, N. and Mason, H.S. (1974) J. Biol. Chem. 249, 335-345 and Schoot Uiterkamp, A.J.M. and Mason, H.S. (1973) Proc. Natl. Acad, Sci. U.S. 70, 993-996).