NMR evidence for perturbation of the copper coordination sphere upon chemical modification of arginine 141 in bovine Cu,Zn superoxide dismutase.

The reaction of the Cu,Co derivative of bovine Cu,Zn superoxide dismutase with phenylglyoxal or butanedione, which are known to inactivate the enzyme by selectively binding to Arg 141, has been studied by 1H NMR. Several 1H NMR lines of the copper-liganding histidine residues were perturbed, reproducing an effect so far observed only in the case of binding of anions to this protein. The room temperature EPR spectrum of the modified Cu,Zn protein was altered very slightly, indicating that the geometry of the copper site was not grossly affected by the modification. NMR and EPR changes were reversed by dialysis in the case of the reversible butanedione adduct. These data show that the coordination of the copper in Cu,Zn superoxide dismutase can be destabilized by modifications occurring at a neighboring but not a metal-liganding residue. It is suggested that part of the NMR effects seen on copper ligands in the case of anion binding are produced by interaction of anions with Arg 141, rather than by direct ligand replacement.     

Identification of the residues responsible for the alkaline inhibition of Cu,Zn superoxide dismutase: a site-directed mutagenesis approach.

The catalytic rate of wild type, two single (Lys 120-->Leu, Lys 134-->Thr), and one double (Lys 120-->Leu-Lys 134-->Thr) mutants of Xenopus laevis B Cu,Zn superoxide dismutase has been studied by pulse radiolysis as a function of pH. The pH dependence curve of the wild-type enzyme can be deconvoluted by two deprotonation equilibria, at pH 9.3 (pK1) and at pH 11.3 (pK2). Catalytic rate measurements on single and double mutants indicate that pK1 is mainly due to the deprotonation of Lys 120 and Lys 134, with only a minor contribution from other surface basic residues, whereas pK2 is due to titration of the invariant Arg 141, likely coupled to deprotonation of the copper-bound water molecule. Accordingly, Brownian dynamics simulations carried out as a function of pH reproduce well the pH dependence of the catalytic rate, when the experimentally determined pKs are assigned to Lys 120, Lys 134, and Arg 141.     

Substitution of arginine for lysine 134 alters electrostatic parameters of the active site in shark Cu,Zn superoxide dismutase

The complete amino acid sequence was determined for the Cu,Zn superoxide dismutase from the shark Prionace glauca. The active site region shows the substitution of an Arg for Lys at position 134, which is important for electrostatic facilitation of the diffusion of O2- to the catalytically active copper. This change may be related to observed alterations of electrostatic parameters of the enzyme (pK of the pH dependence of the enzyme activity, rate of inactivation by H2O2), although it preserves a high efficiency of dismutation at neutral pH.     

1H and 31P NMR studies of the binding of low-affinity anions to Cu,Zn superoxide dismutase

Anions that do not coordinate to the catalytically active copper ion of Cu,Zn superoxide dismutase, but still affect the activity of the enzyme by weaker interactions with the protein moiety surrounding the active site (low affinity anions), uniformly perturbed the 1H NMR line of the NH group of the copper ligand His 46. This effect was detected on the enzyme having Co(II) substituted for the native Zn(II), in which the resonances of residues bound to the copper are detected because of the antiferromagnetic coupling between Cu(II) and Co(II). The interaction with the enzyme of phosphate, a good representative of low-affinity anions, was also studied by 31P NMR of the native enzyme and of enzyme samples covalently modified at all lysines or at the Arg 141, which is 5 A away from the copper. The results obtained indicate that Arg 141 is a likely candidate for binding of low-affinity anions in the vicinity of the copper and that the 1H NMR line of His 46 NH is diagnostic for such an interaction.     

Electrostatic control of the rate-determining step of the copper, zinc superoxide dismutase catalytic reaction

The dependence of the activity of bovine Cu,Zn superoxide dismutase on pH and ionic strength was extensively investigated in the ranges of pH 7.4-pH 12.3 and of ionic strength of 0.02-0.25 M. The results obtained indicate that two positively charged groups having pK values of approximately 10.1 and 10.8 are involved in the control of the activity. On the basis of previous work on the three-dimensional structure and on the chemically modified enzyme, these groups are likely to be lysine side chains, in particular Lys-120 and Lys-134. The oxidation state of the enzyme-bound copper ion at the steady state was found to be the same at either pH 7.4 or pH 11.5. The diffusion of superoxide ion into the active site, which is controlled by the positive charges around the active site itself, appears to be the rate-determining step of the dismutation reaction. NMR measurements of the relaxation rates of F- showed that this control also applies to the access of F- to the active site. Comparison of the nuclear relaxation rates of F- with the enzyme activity indicates that F- relaxation is controlled by the deprotonation of the group with pK approximately 10.8, which appears to be responsible for about 50% of the total activity measured at neutral pH.     

Investigating the cause of the alkaline transition of phytocyanins

The phytocyanins are a family of plant cupredoxins that have been subdivided into the stellacyanins, plantacyanins, and uclacyanins. All of these proteins possess the typical type 1 His(2)Cys equatorial ligand set at their mononuclear copper sites, but the stellacyanins have an axial Gln ligand in place of the weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. The stellacyanins exhibit altered visible, EPR, and paramagnetic (1)H NMR spectra at elevated pH values and also modified reduction potentials. This alkaline transition occurs with a pK(a) of approximately 10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study we demonstrate that the alkaline transition has a similar influence on the visible, EPR, and paramagnetic NMR spectra of cucumber basic protein (CBP), which is a plantacyanin. The mutation of the axial Gln95 ligand into a Met in umecyanin (UMC), the stellacyanin from horseradish roots, and the axial Met89 into a Gln in CBP have very limited, yet similar, influence on the pK(a) for the alkaline transition as judged from alterations in visible spectra. The complete removal of the axial ligand in the Met89Val variant of CBP results in a slightly larger decrease in the pK(a) for this effect, but similar spectral alterations are still observed at elevated pH. Thus, the axial Gln ligand is not the cause of the alkaline transition in Cu(II) stellacyanins, and alterations in the active site structures of the phytocyanins have a limited effect on this feature. The conserved Lys residue found adjacent to the axial ligand in the sequences of all phytocyanins, and implicated as the trigger for the alkaline transition, has been mutated to an Arg in UMC. The influence of increasing pH on the spectroscopic properties of Lys96Arg UMC is almost identical to those of the wild type protein, and thus, this residue is not responsible for the alkaline transition. However, a positively charged residue in this position seems to be important for the correct folding of UMC. Other possible triggers for the effects seen in the phytocyanins at elevated pH are discussed along with the relevance of the alkaline transition.     

Superoxide dismutase mimetic activity of cytokinin-copper(II) complexes

Dissociation constants of cytokinins, derivatives of purine which form complexes with cupric ion, were determined by spectrophotometry and the stability constants of their copper complexes by pH titration. The values found for kinetin were 3.76, 9.96, 7.8, and 15.3 for pK1, pK2, logk1, and log beta 2, respectively, and those for 6-benzylaminopurine were, in the same order, 3.90, 9.84, 8.3, and 15.9. The copper(II) complexes with kinetin and 6-benzylaminopurine had superoxide dismutase mimetic activity, and the reaction rate constants with superoxide, which were determined by polarography, were 2.3 X 10(-7) M-1 s-1 for kinetin and 1.5 X 10(-7) M-1 s-1 for 6-benzylaminopurine at pH 9.8 and 25 degrees C.     

The effects of pH and various salts upon the activity of a series of superoxide dismutases.

The CuZn superoxide dismutases (SODs) from ox, sheep, pig and yeast were investigated by pulse radiolysis in order to evaluate the role of electrostatic interactions between O2.- and SOD proteins in the mechanism of action of the SOD enzymes. The protein net charge in this series varies, as evaluated by the protein pI values spanning over a large range of pH: 8.0 (sheep), 6.5 (pig), 5.2 (ox) and 4.6 (yeast). The amino acid sequences are largely conserved, with the three mammalian proteins being highly homologous and the yeast protein having some distinct variations in the region surrounding the active site. At pH 8.0 the activities of the SODs from various sources are similar, though the minor differences observed suggest that in the highly homologous mammalian series the most acidic protein is the most enzymically efficient one. The pH-dependences of the various activities in the pH range 7-12 are similar, and the related curves are best fitted by two pK values, which are approx. 9.2 and 11.0 for the mammalian enzymes and 9.1 and 11.4 for the yeast enzyme. The activities of the proteins at I 0.1 are decreased by approx. 20% when compared with the activity at I 0.02 at pH 8.5, whereas at pH above 10 the pH-dependence of the activity approaches that determined at I 0.02 and at pH 11.9 the activity is essentially independent of ionic strength. The dependence upon ionic strength also depends on the salt used, with perchlorate being more effective than phosphate or borate or Mops and still effective at pH above 10.5, where the effect of other salts becomes negligible. The dual and concerted dependence of the activities of different SODs on pH and salt concentration is explained with the encounter of O2.- with the active-site copper being governed by the protonation of two positively charged groups in the vicinity of the active site. The gradient between these localized charges and the rest of the protein may explain the different activities of the mammalian proteins at lower pH. On the basis of the sequence variation of the SODs examined it is not possible to definitely identify these groups. Likely candidates are conserved basic amino acid side chains in the vicinity (less than or equal to 1.2 nm) of the active site, i.e. Lys-134 and Arg-141, but co-ordination of OH- in the first copper co-ordination sphere may be an additional factor accounting for the higher pK.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biosynthesis and Cellular Distribution of the Two Superoxide Dismutases of Dactylium dendroides

The synthesis and subcellular localization of the two superoxide dismutases of Dactylium dendroides were studied in relation to changes in copper and manganese availability. Cultures grew normally at all medium copper concentrations used (10 nM to 1 mM). In the presence of high (10 μM) copper, manganese was poorly absorbed in comparison to the other metals in the medium. However, cells grown at 10 nM copper exhibited a 3.5-fold increase in manganese content, while the concentration of the other metals remained constant. Cultures grown at 10 nM copper or more had 80% Cu/Zn enzyme and 20% mangani enzyme; the former was entirely in the cytosol, and the latter was mitochondrial. Removal of copper from the medium resulted in decreased Cu/Zn superoxide dismutase synthesis with a concomitant increase in the mangani enzyme such that total cellular superoxide dismutase activity remained constant. The mangani enzyme in excess of the 20% was present in the non-mitochondrial fraction. The mitochondria, therefore, show no variability with respect to superoxide dismutase content, whereas the soluble fraction varies from 100 to 13% Cu/Zn superoxide dismutase. Copper-starved cells that were synthesizing predominantly mangani superoxide dismutase could be switched over to mostly Cu/Zn superoxide dismutase synthesis by supplementing the medium with copper during growth. Immunoprecipitation experiments suggest that the decrease in Cu/Zn activity at low copper concentration is a result of decreased synthesis of that protein rather than the production of an inactive apoprotein.     

pH dependence of the four individual transitions in the catalytic S-cycle during photosynthetic oxygen evolution

We have investigated the pH dependence for each individual redox transition in the S-cycle of the oxygen evolving complex (OEC) of photosystem II by electron paramagnetic resonance (EPR) spectroscopy. In the experiments, OEC is advanced to the appropriate S-state at normal pH. Then, the pH is rapidly changed, and a new flash is given. The ability to advance to the next S-state in the cycle at different pHs is determined by measurements of the decrease or increase of characteristic EPR signals from the OEC in different S-states. In some cases the measured EPR signals are very small (this holds especially for the S0 ML signal at pH >7.5 and pH <4.8). Therefore, we refrain from providing error limits for the determined pK's. Our results indicate that the S1 --> S2 transition is independent of pH between 4.1 and 8.4. All other S-transitions are blocked at low pH. In the acidic region, the pK's for the inhibition of the S2 --> S3, the S3 --> [S4] --> S0, and the S0 --> S1 transitions are about 4.0, 4.5, and 4.7, respectively. The similarity of these pK values indicates that the inhibition of the steady-state oxygen evolution in the acidic range, which occurs with pK approximately 4.8, is a consequence of similar pH blocks in three of the redox steps involved in the oxygen evolution. In the alkaline region, we report a clear pH block in the S3 --> [S4] --> S0 transition with a pK of about 8.0. Our study also indicates the existence of a pH block at very high pH (pK approximately 9.4) in the S2 --> S3 transition. The S0 --> S1 transition is not affected, at least up to pH 9.0. This suggests that the inhibition of the steady-state oxygen evolution, which occurs with a pK of 8.0, is dominated by the inhibition of the S3 --> [S4] --> S0 transition. Our results are obtained in the presence of 5% methanol (v/v). However, it is unlikely that the determined pK's are affected by the presence of methanol since our results also show that the pH dependence of the steady-state oxygen evolution is not affected by methanol. The results in the alkaline region are in good agreement with a model, which suggests that the redox potential of Y(Z*)/Y(Z) is directly affected by high pH. At high pH the Y(Z*)/Y(Z) potential becomes lower than that of S2/S1 and S3/S2. The acidic block, with a pK of 4-5 in three S-transitions, implies that the inhibition mechanism is similar, and we suggest that it reflects protonation of a carboxylic side chain in the proton relay that expels protons from the OEC.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactytium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, an extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (haloenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (< 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 μM, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 μM medium copper, holoenzyme secretion is maintained throughout cell growth.The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN^?-insensitive, manganese form of this enzyme. Cells grown at 10 μM copper shown 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactylium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, and extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (holoenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (less than 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 micrometer, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 micrometer medium copper, holoenzyme secretion is maintained throughout cell growth. The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN(-)-insensitive, manganese form of this enzyme. Cells grown at 10 micrometer copper show 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

Cu/Zn superoxide dismutase in excretory-secretory products of the human hookworm Necator americanus. An electron paramagnetic spectrometry study.

EPR spectrometry was used to investigate the effect of excretory/secretory product from Necator americanus on superoxide radical anions generated by xanthine/xanthine oxidase as a measure of excretory/secretory product superoxide dismutase activity. Using 1,1',5,5'-dimethylpyrollidine-N-oxide (DMPO) as a superoxide spin-trapping agent a 12-line EPR spectrum characteristic of the DMPO-OOH adduct was observed to decrease in the presence of excretory/secretory product. Superoxide dismutase activity was proportional to excretory/secretory protein concentration, was inhibited with cyanide treatment and was progressively destroyed with increasing time of heat denaturation of excretory/secretory product. Using a purpose-built chamber the superoxide dismutase activity of excretory/secretory product from live worms in culture was shown to accumulate with time to a maximum at 4 h. The electron paramagnetic resonance spectrum obtained for the frozen excretory/secretory product of N. americanus recorded at 77 K is typical of Cu(II) in a protein matrix. The results are consistent with the presence of an active Cu/Zn superoxide dismutase in excretory/secretory product from N. americanus and demonstrate a method for the unequivocal determination of the fate of superoxide anions in the presence of live worms.     

Two groups control light-induced schiff base deprotonation and the proton affinity of asp(85) in the Arg(82)His mutant of bacteriorhodopsin

Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 &mgr;s to 75 &mgr;s with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.     

The red light of the helium-neon laser reactivates superoxide dismutase

The effect of low-energy helium-neon laser (HNL) on enzymatic activity, absorbtion spectra and electron paramagnetic resonance (EPR) signals of superoxide dismutase (SOD) from bovine erythrocytes in acid medium were investigated. It was found that incubation during 2 hours at pH 5.9 led to eventually complete inactivation of the enzyme. The subsequent illumination of inactivated SOD by HNL brought about the enzyme reactivation. Both absorption and EPR-spectra were changed after incubation at pH 5.9. These changes may be attributed to protonation of histidine residue in the enzyme active site. After laser irradiation both absorption and EPR spectra were restored to those typical of native enzyme at pH 8.2. In a model system, copper-histidine complex, absorption maximum was shifted from 632-633 nm at pH 5.8 to 639-640 nm at pH 8.5-9.0. The similar long-wave length shift of the maximum was observed after illumination by HNL at pH 5.8. It may be postulated that the photoreactivation of SOD consists essentially in deprotonation of His-61 residue in the enzyme active site and subsequent recovery of imidazole bridge between copper and zinc which had been destroyed at low pH. Since many other enzymes possess similar copper-histidine structures in their active sites, one may expect diverse effects of red (laser) light on the enzyme activity.     

Identification of a copper-sensitive ascorbate peroxidase in the unicellular green alga Selenastrum capricornutum

Extracts from the unicellular green alga Selenastrum capricornutum exhibit high superoxide dismutase activity, but only traces of catalase activity. The excess hydrogen peroxide (HO) generated by the superoxide dismutase in S. capricornutum may be degraded by a unique peroxidase. This peroxidase has a high specificity for ascorbate as its electron donor. The enzyme has an optimum pH at 8, is insensitive to cyanide and is inhibited by oxine. Addition of low concentrations of copper to algal cultures stimulates the peroxidase activity threefold. This enzymatic system could be used as a sensitive bioindicator for copper in fresh water.     

Catalytic and structural role of a metal-free histidine residue in bovine Cu-Zn superoxide dismutase

Cu-Zn superoxide dismutase (SOD) contains a conserved, metal-free His residue at an opening of the backbone beta-barrel in addition to six Cu- and/or Zn-bound His residues in the active site. We examined the protonation and hydrogen bonding state of the metal-free His residue (His41) in bovine SOD by UV Raman spectroscopy. Analysis of the His Raman intensity at 1406 cm(-1) in a D2O solution has shown that His41 has a pKa of 9.4, consistent with the NMR and X-ray structures at acidic to neutral pH, in which two imidazole nitrogen atoms of cationic His41 are hydrogen bonded to the main chain C=O groups of Thr37 and His118. Upon deprotonation of His41 at pH 9.4, the Thr37-His41-His118 hydrogen bond bridge breaks on the His118 side and SOD loses 70% of its activity. Concomitantly, hydrogen-deuterium exchange is accelerated for amide groups of beta-strands, indicating an increased conformational fluctuation of the beta-barrel. Thr37 and His41 are in direct contact with Leu36, whose hydrophobic side chain closes off the opening of the beta-barrel, while His118 is indirectly connected to Arg141 that assists the docking of superoxide to Cu. These Raman findings strongly suggest that the His41-mediated hydrogen bond bridge plays a crucial role in keeping the protein structure suitable for highly efficient catalytic reactions. The catalytic and structural role of His41 is consistent with the observation that the mutation of His43 in human SOD (equivalent to His41 in bovine SOD) to Arg largely reduces the dismutase activity and the protein structural stability.     

Two copper-containing proteins from white and gray matter of brain.

The procedure for the isolation of two water soluble copper-containing proteins from the white and gray matter of bovine brain is described. One of the proteins, cerebrocuprein I, is superoxide dismutase; and three molecular forms of this enzyme are to be found in brain. The other protein present in gray and white matter is devoid of superoxide dismutase and amine oxidase activities. The amino acid composition, molecular weight, isoelectric point and copper content of this protein were determined. The effect of some agents, pH and thermal treatment of the optical and EPR spectra of the protein were also studied. The copper of the protein may be removed and the holoprotein reconstituted again from apoprotein and copper. The results obtained led to the conclusion that in brain a new copper protein is discovered, which is named neurocuprein.     

Metal sites of copper-zinc superoxide dismutase.

Silver-copper and silver-cobalt proteins have been prepared in which Ag+ resides in the native copper site of superoxide dismutase and either Cu2+ of Co2+ reside in the zinc site. The electron paramagnetic resonance (EPR) spectrum of the copper and the visible absorption spectrum of the cobalt greatly resemble those of either Cu4 of Cu2,Cu2,Co2 proteins, respectively, in which the copper of the native copper sites has been reduced. It was found that, unlike cyanide, azide anion would not perturb the EPR spectrum of Ag2,Cu2 protein. Since azide produces the same perturbation upon the EPR spectrum of native and Cu2 proteins, it must bind to the copper and not the zinc of superoxide dismutase. A model of the metal sites of the enzyme has been fitted to a 3-A electron-density map using an interactive molecular graphics display. The model shows that histidine-61, which appears to bind both copper and zinc, does not lie in the plane of the copper and its three other histidine ligands, but occupies a position intermediate between planar and axial. This feature probably accounts for the rhombicity of the EPR spectrum and the activity of the enzyme.