Preparation and characterization of met apo hemocyanin: a single copper (II) active site.

A new derivative of $\text{Busycon canaliculatum}$ hemocyanin has been prepared for which one copper has been removed from the binuclear active site of the holoprotein and the remaining copper has been oxidized with a variety of small molecule oxidizing agents. This met apo derivative [( )…Cu(II)] binds a number of ligands; EPR spectra of several forms are reported and compared to those obtained for a singly oxidized (half met-L) derivative [Cu(I)…Cu(II)L]. The site of the oxidized copper for both forms is found to be quite similar in structure but shows large differences in ligand binding ability.     

Structural states of the binuclear copper cluster of Cancer magister methemocyanin.

The binuclear cupric copper cluster of $\text{Cancer}\text{magister}$ methemocyanin prepared from hemocyanin and hydrogen peroxide is diamagnetic (1). Upon treatment with azide, it is transformed into magnetic dipolar coupled (paramagnetic) Cu(II) pairs and then into magnetically isolated Cu(II) complexes. This progressive uncoupling of the binuclear cupric pairs in methemocyanin is interpreted in terms of a relaxation of superexchange through one or more bridging ligands.     

The redox properties of (1,6-bis(benzimidazol-2-yl)-2,5-dithiahexane) chloro-copper(II) chloride as studied by cyclic voltammetry

Cyclic voltammetry in acetonitrile has been used to study the redox properties of the compound [Cu-(BBDH)Cl] Cl under various conditions. The nature of the species in solution was studied with the aid of ligand-field spectra and ESR spectra. It appears that in CH₃CN solution two Cu(II) species are predominating, viz. [Cu(BBDH)(CH₃CN)_x]²⁺ and Cu(BBDH)-Cl⁺. Minor species are dimeric in nature, such as [(BBDH)CuCl₂Cu(BBDH)]²⁺, as easily seen from ESR. The relative amount of the two major species was varied using added LiCl, allowing us to determine the nature of both species. The redox potential of the species [Cu(BBDH)Cl]⁺ appeared to be 0.62 V (against a normal hydrogen electrode), which is very high for a Cu(II) compound and in the same area as found for the blue copper proteins. Re-oxidation of Cu(BBDH)⁺ in the presence of LiCl shows that C$\text{l}$ slowly recoordinates after reoxidation.     

Preparation and characterization of a stable half met derivative of type 2 depleted Rhus laccase: Exogenous ligand binding to the type 3 site

We report the preparation and characterization of a stable half met (Cu(II)Cu(I)) type 2 copper depleted derivative of $\text{Rhus}$ laccase. Anion binding studies to this mixed valent type 3 protein form indicate no tight binding of anions nor group 1 - group 2 ligand behavior. This suggests that, in contrast to the well-characterized hemocyanins and tyrosinase coupled binuclear sites, exogenous ligands do $\text{not}$ appear to bridge the type 3 binuclear copper ions in laccase.     

Affinity labeling of stellacyanin by Cr(II) aquo ions

Stellacyanin, the single blue copper protein from $\text{Rhus}$$\text{vernicifera}$, is reduced stoichiometrically by Cr(II)_aq ions yielding a 1:1 adduct between the Cr(III) produced and the reduced protein. This Cr(III)-labeled stellacyanin is substitution inert and no significant loss of the label occurs during extensive dialysis for more than a week. Oxidation by O₂ of the Cr(III)-labeled Cu(I) stellacyanin does not cause the loss of Cr(III) either. Furthermore, reduction of the Cr(III)-labeled stellacyanin Cu(II) by a second equivalent of Cr(II) may be attained without any further labeling. Thus, the one mole of Cr ions binds to stellacyanin during the first reduction step and is most probably coordinated at a specific locus on that protein.     

Chemical and spectroscopic conformation of an exogenous ligand bridge in half met hemocyanin.

Half $\text{met-N}{}_3{}^{\mathrm{?}}$ hemocyanin is shown to undergo a unique change at the Cu(II)?Cu(I) active site with temperature, exhibiting class II mixed valent properties at low temperature (The appearance of an intense near IR intervalence-transfer transition and a delocalized EPR spectrum). This requires a Cu(II)NNNCu(I) bridging geometry. The effects of CO coordination to half $\text{met-N}{}_3{}^{\mathrm{?}}$, combined with the presence of a low energy $\text{N}{}_3{}^{\mathrm{?}}\text{→ Cu(II)}$ charge transfer transition, demonstrate that azide is also bridging at room temperature. Finally, half $\text{met-N}{}_3{}^{\mathrm{?}}$ is found to be capable of coordination of a second $\text{N}{}_3{}^{\mathrm{?}}$ at the copper(II) site.     

Do copper ions influence the reduction of ferricytochrome C by O−2?

Recently, it was suggested that the measured rate of reduction of ferricyto chrome $\text{C}$ by O^?₂ below pH 8, was too high in the presence of high concentrations of formate (Koppenol, W.H., Van Buuren, K.J.H., Butler J. and Braams, R. (1976) Biochim. Biophys. Acta 449, 157–168).The high values were attributed to the presence of impurities of copper, which compete for O^?₂. This assumption is consistent with either a decrease in the reduction yield of ferricytochrome $\text{C}$ in the presence of copper, or with a very fast reaction of Cu(I) with ferricytochrome $\text{C}$.It was previously shown by us and by others that the reduction yield of ferricytochrome $\text{C}$ by O^?₂ is 100%. We measured the rate of reduction of ferricytochrome $\text{C}$ by Cu(I), and found that this reaction is slow: $\text{k = (1.5±0.5) · 10}{}^3\text{M}{}^{\mathrm{?1}}\text{) ·}\text{s}{}^{\mathrm{?1}}$.Therefore, our results rule out the possibility that below pH 8 copper impurities affect the measured rate constant of the reduction of ferricytochrome $\text{C}$ by O^?₂.     

Conformation of 1- and 3-deazaadenosines in solution as studied by 1H nuclear magnetic resonance spectroscopy.

¹H nuclear magnetic resonance spectra of 1 - (II) and 3-deazaadenosines (III) together with adenosine (I) in dimethylsulfoxide have been examined. Features of coupling constants indicate that the furanose rings of I, II, and III have similar conformational preferences and that conformations about the 4′-C–5′-C bond are preferentially $\text{gauche-gauche}$. Nuclear Overhauser effect and spin-lattice relaxation-time measurements demonstrate that II predominantly adopts the $\text{syn}$-conformation similar to that of I, whereas that of III has a greater $\text{anti}$ (freely rotating) component. The results suggest that the $\text{syn}$-conformation in II as well as I is stabilized presumably through a hydrogen bond between the 3-N and 5′-hydroxyl group.     

The conformation and orientation of copper(II)-bleomycin intercalated with DNA

EPR data are used to describe the conformation and identity of the atoms coordinated to Cu(II) in Cu(II)-bleomycin bound to oriented DNA fibers. The fibers were slowly drawn from viscous solutions of Cu(II)-bleomycin-DNA containing one Cu(II)-bleomycin to 200 basepairs. EPR measurements were made at room temperature and 90 K for different orientations of the external magnetic field with respect to the helical axes of the fibers. The g-values ($\text{g}{}_{\mathrm{}}\text{=2.21}$, g_⊥ =2.04) and the hyperfine constant ($\text{A}{}_{\mathrm{}}\text{=175}\text{G}$) are consistent with values expected for Cu(II) chelated to a square planar array of ligands. In the oriented fibers, the square planar arrays do not all have the same orientations with respect to the fiber axes. At room temperature the chelated ions have rotational freedom in which the normal to the planar array has almost complete freedom of rotation about axes perpendicular to the DNA fiber axes. The normal maintains an angle of 75° with respect to the axis, in the plane of the basepair, about which it rotates. Nine superhyperfine peaks on the high field side of the EPR spectrum were partially resolved. The number and splitting (12 G) of these superhyperfine peaks indicate that four nitrogen atoms are chelated to Cu(II) in a square planar array. These data on Cu(II)-bleomycin bound to DNA give information on the orientation of the metal-containing portion of bleomycin which lies outside the double helix.     

Evidence for the involvement of (Cu-ATP)2− in the inhibition of human erythrocyte (Ca2+ + Mg2+-ATPase by copper

The effects of copper on the activity of erythrocyte $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ have been tested on membranes stripped of endogenous calmodulin or recombined with purified calmodulin. The interactions of copper with Ca²⁺, calmodulin and (Mg-ATP)^2? were determined by kinetic studies. The most striking result is the potent competitive inhibition exerted by (Cu-ATP)^2? against (Mg-ATP)^2?$\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 2.8 μ}\text{M}$), while free copper gives no characteristic inhibition. Our results also demonstrate that copper does not compete with calcium either on the enzyme or on calmodulin. The fixation of calmodulin on the enzyme is not altered in the presence of copper as shown by the fact that the dissociation constant remains unaffected. It may be speculated that (Cu-ATP)^2? is the active form of copper, which could plausibly be at the origin of some of the pathological features of erythrocytes observed in conditions associated with excess copper.     

Effects of desmethylimipramine and diphenylpropene derivatives on synthesis of rat brain lipids in vivo

The effects of antidepressant compounds on the synthesis of brain lipids from [1-¹⁴C] acetate $\text{in vivo}$ in 15 day old rats have been investigated. Compounds used included the drug desmethylimipramine (DMI), the tetrabenazine antagonist 3-methylamino-1:1-diphenylprop-1-ene (II) and the primary (I) and tertiary (III) amine analogues of (II). Compound (II), the most potent tetrabenazine antagonist in the diphenylpropene series, significantly increased lipogenesis, whereas the remaining compounds did not. The results from fractionation of the lipid extract from rats treated with (II) indicated that the incorporation of radioactivity from [1-¹⁴C] acetate increased proportionally in all neutral lipids and phospholipids. Tetrabenazine also increased brain lipogenesis $\text{in vivo}$ and altered the distribution within lipid classes of radioactivity from [1-¹⁴C] acetate. Using [¹⁴C] labelled compound, the concentration of (II) in the brain under the present experimental conditions has been determined.     

The amyloidogenic region of the human prion protein contains a high affinity (Met)2(His)2 Cu(I) binding site

The prion protein (PrP^c) is a cuproprotein implicated in a number of human neurodegenerative diseases. Although many physiological functions have been ascribed to PrP, its potential to act as a neuronal antioxidant, based in part on its copper binding ability, is controversial and unresolved. A number of studies have shown that copper bound to PrP^c is not redox silent, and recent data shows that the Cu(II) sites at histidines 96 and 111 display reversible electrochemistry. Reversible electrochemistry implies redox cycling whilst the metal remains bound and with the absence of permanent oxidation or reduction of the protein. Despite this indirect evidence of Cu(I) binding to PrP, the nature of the Cu(I) binding site/s is unclear, although previous extended X-ray absorption fine structure (EXAFS) data has implicated methionines in the Cu(I) binding site. Using spectroscopic techniques we find that the PrP region encompassing histidines 96 and 111 can bind a Cu(I) ion in a site comprising His 96, His 111, Met 109 and Met 112. The four-coordinate (His)₂(Met)₂ Cu(I) site has a K_d = 10⁻¹⁵–10⁻¹² M indicative of high affinity. Mutation of histidine residues reduces the Cu(I) affinity. Although alluding to the fact the PrP could act in a direct superoxide dismutase-like fashion, the Cu(I)–PrP(91–124) site and affinity is comparable to that observed for bacterial periplasmic Cu(I) transporters.     

The crystal and molecular structure of cytosine-glycyl-glycine-copper(II) complex,a biologically important ternary coordination complex

The title compound was prepared and studied to gain some insight into the structural basis for the protein-nucleic acid-metal ion interaction. The crystal structure has been determined from three-dimensional diffractometer X-ray data using Cu Kα radiation. The crystals are monoclinic, space group $\text{P2}{}_1\text{c}$, with cell dimension; a=10.642(1)Å, b=8.081(1)Å, c=17.792(1)Å, β=124.29(1)^o, z=4. Amino and amide nitrogen, carboxyl O(8) of glycylglycine, N(3) of cytosine and O(2) of adjacent cytosine molecule coordinate to the central copper ion to form a square pyramid. An additional weak interaction in complex molecule between copper and O(2) of cytosine is also observed. The complex molecules are held together by hydrogen- and coordination-bonds in crystalline state.     

Competitive inhibition of Cu, Zn superoxide dismutase by monovalent anions.

Polarographic measurements showed that N₃^? and halides $\text{in}$ hibit the activity of bovine Cu, Zn superoxide dismutase in a competitive fashion, as previously demonstrated for CN^? and OH^?. All anions increase the spin-lattice nuclear magnetic relaxation time (T₁) of aqueous solutions of the enzyme as well, but the stability constants measured from T₁ data are lower than those calculated from activity data. The results suggest that substrate and anionic inhibitors bind during the catalytic action at the water coordination position of the enzyme copper, and that these inhibitors may have a greater affinity for the cuprous form of the enzyme which is generated in the catalytic cycle.     

Iron(II)-bleomycin. Biochemical and spectral properties in the presence of radical scavengers

Consistent with a recent literature report (Repine, J. E. $\text{et}$$\text{al.}$ (1981) $\text{Proc.}$$\text{Nat.}$$\text{Acad.}$$\text{Sci.}$$\text{USA}$$\text{7}\text{?}$$\text{8}\text{?}$, 1001–1003), the release of [³H]-thymine from PM-2 DNA by Fe(II)-H₂O₂-generated ·OH was suppressed by dimethyl sulfoxide. In contrast, DMSO did not affect [³H]-thymine release mediated by Fe(II)-bleomycin. Under aerobic conditions in the presence of $\text{t}$-butyl phenylnitrone, Fe(II)-BLM produces an epr signal that has been presumed to arise by transfer of ·OH or $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ from the “active complex” of bleomycin to the spin trap. Remarkably, high concentrations (80 mM) of PBN had no effect on the ability of Fe(II)-BLM to solubilize [³H]-thymine, although the ability of authentic ·OH to degrade DNA was completely suppressed under these condition. The suproxide dismutase catalyst tetrakis(4-N-methylpyridyl)porphineiron(III) also failed to suppress BLM-mediated DNA degradation. Moreover, the epr signal observed with 1.6 mM Fe(II)-BLM in the presence of 80 mM PBN was found to be much less intense than that produced by 1.6 mM Fe(II) and 290 mM H₂O₂, but equivalent in intensity to that obtained with 45 mM Fe(II) and exoess H₂O₂. We conclude that the fragmentation of DNA produced by Fe(II)-BLM can be due neither to free ·OH nor to $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$. We suggest that DNA degradation is initiated by an “active complex” consisting of BLM, metal and oxygen that functions by abstracting H· from susceptible sites on DNA.     

Spectroscopic studies on the active site of Sepioteuthis lessoniana hemocyanin.

The absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectra in the visible region have been measured for $\text{Sepioteuthis lessoniana}$ hemocyanin at 77, 198, and 293K. From the temperature dependence of the CD spectra of oxyhemocyanin, the bands observed at 450, 565, and 700 nm were resolved into those centered at 430, 490, 565, 600, and 700 nm. Since these five peaks are most probably due to the d-d transitions, the two copper ions at the oxygenated active center are inferred to be Cu(II) ions each in a non-equivalent coordination geometry of very low symmetry. The MCD spectral data confirm the view and reasonably explain the diamagnetism of oxyhemocyanin.     

Sequence homology of bacterial and mitochondrial cytochrome c oxidases: Partial sequence data of cytochrome c oxidase from Paracoccus denitrificans

The aerobic electron transport chain of $\text{Paracoccus denitrificans}$ is very similar to that of mitochondria. It has therefore been suggested that this bacterium might be evolutionarily related to mitochondria. The two subunits (Mr 45.000 and 28.000) of the $\text{Paracoccus}$ cytochrome c oxidase were isolated and partially sequenced. The sequences were found to be surprisingly homologous to sequences of the subunits I and II of mitochondrial cytochrome c oxidases. The data provide a molecular basis for the symbiotic origin of mitochondria and strongly support the notion that in eucaryotic oxidases subunits I and/or II carry the redox centers, heme and copper.