The proton magnetic resonance spectra of a cobalt(II) azurin

The proton magnetic resonance spectrum of a cobalt(II) derivative of $\text{Pseudomonas}\text{aeruginosa}$ azurin is reported. The temperature dependence of 26 resonances is described together with a study of the pH¹ titration behaviour over a range 4.7 to 9.3. A few resonances are observed shifted by more than 30 ppm from their diamagnetic positions. Of the remainder most extrapolate to the aliphatic region at T = ∞. Two lines are assigned to the C2 and C4 protons of a freely titrating histidine residue far from, and only slightly affected by, the Co(II) centre. A further two lines are assigned to the C2 hydrogen of protonated and deprotonated forms of a histidine residue in slow exchange with bulk aquaeous protons and closer to, but not bound to, the cobalt. The structure of the protein in the vicinity of the paramagnetic centre is found to be essentially insensitive to pH¹ over the range 4.7 to 9.3.     

Effect of nickel(II) substitution on the resonance Raman and NMR spectra of Alcaligenes xylosoxidans azurin II: implications for axial-ligand bonding interactions in cupredoxin active sites

Assignment of the resonance Raman (RR) spectrum of Ni(II)-substituted azurin II from Alcaligenes xylosoxidans (NCIMB 11015) using Ni isotope substitution reveals an anomalously low Ni-S(Cys) stretching frequency of 349?cm^–1, suggesting the presence of significant axial-ligand bonding interactions. The X-ray crystal structure of Ni(II)-substituted azurin from Pseudomonas aeruginosa shows that there are two potential axial ligands to the Ni ion: a peptide carbonyl O at a distance of 2.46?Å, together with a long-range interaction from a methionine sulfur (S′) at a distance of 3.30?Å. Comparison of the RR properties of Ni(II)-substituted azurin II with stellacyanin (which contains an axial carbonyl ligand, but no methionine) suggests that the interaction from the carbonyl oxygen ligand alone is not sufficient to account for the weak Ni azurin metal-thiolate bond. Instead, it appears that a Ni-methionine bonding interaction is also required to explain the low Ni-S(Cys) stretching frequency in Ni(II)-substituted azurin II. This hypothesis is supported by NMR studies which show a large paramagnetic shift for the protons of the methionine side-chain. Thus, it appears that Ni-substituted azurin II is best described as five-coordinate, and that significant Ni(II)-methionine bonding interactions can occur at a distance of 3.3?Å.     

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.     

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.     

A 13C nuclear magnetic resonance study of the interaction of ligands with arginine residues in dihydrofolate reductase.

¹³C nmr spectra of $\text{Streptococcus faecium}$ dihydrofolate reductase containing [¹³C-guanidino] arginine and ligand complexes with the labeled enzyme are reported. The spectrum of the native enzyme shows 5 well-resolved resonances (the enzyme contains 8 Arg) with a chemical shift range of 1.2 ppm. Addition of ligands to the enzyme produces distinct changes in the enzyme spectrum, and demonstrates that ¹³C nmr of labeled protein can be used in studies of protein-ligand interactions. An example of the use of ¹³C-depleted material is also presented.     

A resonance raman study of native stellacyanin and its Ni(II) derivative. On the origin of the 450-nm electronic absorption

The resonance Raman spectra of native stellacyanin and its Ni(II) derivative has been investigated. Raman intensity as a function of the exciting frequency shows minima at about 440–460 nm. Moreover, the resonance Raman spectra of the Ni(II) derivative indicate similar symmetry and nature of ligands, namely, one cysteine and at least one histidine. Optical electronegativity of the ligand involved in the intense visible absorption band of native stellacyanin and the corresponding transition of the Ni(II) derivative confirm this assumption. The origin of the 450 nm band is also discussed.     

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.     

Purification and preliminary crystallographic studies on azurin and cytochrome c' from Alcaligenes denitrificans and Alcaligenes sp. NCIB 11015.

Purification and crystallisation procedures are reported for azurin and cytochrome c′ from Alcaligenes denitrificans and Alcaligenes sp. NCIB 11015. The azurin crystals from A. denitrificans are suitable for high-resolution X-ray structure analysis. They are orthorhombic, space group C222₁ (with marked tetragonal pseudo-symmetry), cell dimensions $\text{a = 75.0}\text{A}\text{?}\text{, b = 74.1}\text{A}\text{?}\text{, c = 99.5}\text{A}\text{?}$, with two molecules per asymmetric unit. The cytochrome c′ crystals from both species are hexagonal, space group P6₁22 (or P6₅22), cell dimensions $\text{a = b = 54.7}\text{A}\text{?}\text{, c ~ 185}\text{A}\text{?}$, γ = 120 °, with one subunit (molecular weight 14,000) in the asymmetric unit.     

Preparation and immunochemical properties of methoxypolyethylene glycol-coupled and N-carboxymethylated derivatives of ragweed pollen allergen, antigen E.

A methoxypolyethylene glycol (PEG)-coupled and several N-carboxymethylated (N-CM) derivatives of antigen E, the major allergenic protein of ragweed pollen, were prepared. The PEG derivative contained seven residues of PEG groups (residue weight about 2100) per molecule of protein and the groups were linked to the lysyl residues of antigen via the 2,6-positions of 4-hydroxy-triazine nucleus. The maximally N-CM derivative contained, respectively, 10, 6, and 2 residues of mono-CM, di-CM, and unmodified lysyl residues per molecule of protein. The CM groups were introduced reductively on reaction with glyoxylic and sodium cyanoborohydride and the extent of mono- and dicarboxymethylation was controlled more by the concentration of cyanoborohydride than by that of glyoxylic acid. The molar allergenic activities of the PEG and the N-CM derivatives in man were, respectively, 0.02 and 0.5 of that of the native antigen. Rabbits immunized with the PEG derivative gave antibody titers about $\text{1}\text{8}$th of those obtained with animals immunized with the native antigen. However, the rabbits preimmunized with the PEG derivative gave a vigorous secondary response on challenge with the native antigen and their titers approached those of rabbits preimmunized with the native antigen. The immunogenicity of the reduced and S-carboxymethylated derivative of antigen E which has the denatured conformation was studied as a control. Rabbits immunized with the S-CM derivative gave antibody titers $\text{1}\text{34}$th of those obtained with animals immunized with the native antigen; on secondary challenge with the native antigen, these rabbits gave antibody titers about $\text{1}\text{6}$th of those of animals preimmunized with the native antigen.     

The presence of redox-sensitive nickel in the periplasmic hydrogenase from Desulfovibrio gigas

A new and improved method for the purification of the periplasmic hydrogenase from $\text{Desulfovibrio}\text{gigas}$is described. This preparation of hydrogenase was found to contain 0.64 g atom of nickel per mole of protein. In the oxidized state, the hydrogenase exhibited an isotropic signal at g = 2.02 and a characteristic Ni(III) signal with g-values at 2.31, 2.20 and ～2.0. The EPR spectrum of the reduced enzyme consisted of multiple species. One set of g-values are determined as 2.17, 2.08 and 2.04. The other minor species exhibited a resonance at g = 2.28. On partial reoxidation of the hydrogenase, the initial Ni(III) signals reappeared along with additional signals attributed to multiple Ni(III) species. It is proposed that Ni is an important functional unit in this hydrogenase.     

Studies on avian heart pyruvate kinase during development.

The cytokinin-active nucleoside 6-(4-hydroxy-3-methyl-$\text{cis}$-2-butenylamino)-9-β-$\text{D}$-ribofuranosylpurine, i.e. ribosyl-$\text{cis}$-zeatin, has been isolated from an hydrolysate of tRNA from $\text{Corynebacterium fascians}$. The identification of ribosyl-$\text{cis}$-zeatin is based on biological activity, liquid chromatographic mobility and uv spectrum of the purified material as well as the mass spectrum and gas chromatographic mobility of its trimethylsilylated derivative.     

The isolation and properties of porcine ferritin and apoferritin.

Porcine ferritin and apoferritin were purified to a greater degree of homogeneity than has been reported previously. Porcine ferritin was insoluble in the absence of a reducing agent, possessed a high content of iron, with an average $\text{Fe}\text{N}$ ratio of ~2.5, and contained almost no detectable endogenous apoferritin. The amino acid composition, ultraviolet-absorption spectrum, and ultraviolet-circular dichroism spectrum of porcine apoferritin are very similar to the respective parameters of equine apoferritin. The native and subunit molecular weights of porcine apoferritin are 503,000 and 20,000, respectively.     

Resonance Raman spectroscopy of amicyanin, a blue copper protein from Paracoccus denitrificans

The copper binding site of amicyanin from Paracoccus denitrificans has been examined by resonance Raman spectroscopy. The pattern of vibrational modes is clearly similar to those of the blue copper proteins azurin and plastocyanin. Intense resonance-enhanced peaks are observed at 377, 392, and 430 cm-1 as well as weaker overtones and combination bands in the high frequency region. Most of the peaks below 500 cm-1 shift 0.5-1.5 cm-1 to lower energy when the protein is exposed to D2O. Based on the pattern of conserved amino acids, the axial type EPR spectrum, and the resonance Raman spectrum, it is proposed that the copper binding site in amicyanin contains a Cu(II) ion in a distorted trigonal planar geometry with one cysteine and two histidine ligands and an axial methionine ligand at a considerably longer distance. Furthermore, the presence of multiple intense Raman peaks in the 400 cm-1 region which are sensitive to deuterium substitution leads to the conclusion that the Cu-S stretch is coupled with internal ligand vibrational modes and that the sulfur of the cysteine ligand is likely to be hydrogen-bonded to the polypeptide backbone.     

An immune complex assay for SV40 T antigen.

The two forms of ferredoxin from $\text{Desulfovibrio gigas}$, Fd I and Fd II, are studied by differential pulse polarography 1. Fd I and Fd II give one well defined peak corresponding to $\text{E}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= ?0.33}\text{and}\text{?0.35}$ V (vs. the hydrogen electrode) respectively, at c > 5 μM. The influence of the concentration on the peak potentials Ep and the peak heights i_p is examined. The denaturation of the two forms of ferredoxin is studied by polarography in conjunction with UV spectrophotometry. Two new peaks at negative potentials before the reduction of the solvent are observed in denaturated proteins.     

EPR Spectrum at 4, 9 and 35 GHz of hydrogenase from Chromatium vinosum. Direct evidence for spin-spin interaction between Ni(III) and the ironsulphur cluster

EPR spectra at 4, 9 and 35 GHz of hydrogenase isolated from Chromatium vinosum have been compared. The spectra at 4 and 35 GHz confirmed our earlier conclusions, made from observations at 9 GHz (Albracht, S.P.J., Kalkman, M.L. and Slater, E.C. (1983) Biochim. Biophys. Acta 724, 309–316), that the irreversibly inactivated enzyme molecules in the preparation give rise to two EPR signals due to the independent non-interacting $\text{S =}\text{1}\text{2}$ systems of Ni(III) and a |3Fe-xS| cluster. It was observed that intact enzyme molecules show a complex EPR spectrum caused by a spin-coupled pair of Ni(III) and a |4Fe-4S|³⁺ cluster. The interaction energy is so weak (approx. 0.01 cm^?1) that the 35 GHz spectra of both the Ni(III) and the |4Fe-4S|³⁺ cluster have the appearance of rather normal $\text{S =}\text{1}\text{2}$ spectra with additional splittings as a result of the spin-spin interaction. At lower microwave frequencies, the spectra become increasingly complex but phenomenologically they behave as expected for an exchange-coupled pair of dissimilar ions. The distance between the two spin systems is estimated to be at the most 1.2 nm. The spin-relaxation rate of the Ni(III) ion is dramatically enhanced as a result of the coupling to the rapidly relaxing Fe-S cluster. The g values and so presumably also the ligand fields of Ni in intact and irreversibly inactivated enzyme molecules are identical. This suggests that the specific coordination of the nickel in the enzyme is not the only requirement for activity with artificial electron donors or acceptors, and that the presence of a nearby, intact |4Fe-4S|^3+(3+,2+) cluster might be another essential factor. From the g values and the probable function of Ni in the enzyme we propose, as a working hypothesis, that the nickel ion has five ligands provided by the protein in a square-pyramidal coordination.     

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.     

Identification of cysteines in subunit II as ligands to the redox centers of bovine cytochrome c oxidase

The cysteine residues in beef cytochrome $\text{c}$ oxidase (E.C.1.9.3.1) which act as ligands to a redox site have been located in the C-terminal portion of subunit II.     

Associative properties of the Escherichiacoli galactose binding protein and maltose binding protein

The periplasmic galactose binding protein and maltose binding protein of $\text{Escherichia}\text{coli}$ are recovered mostly in dimeric form when purified, from osmotically-shocked bacteria, in the presence of protease inhibitors and 2-mercaptoethanol without dialysis and concentration of the shock fluid. The specific ligands, galactose (but not glucose) for galactose binding protein, and maltose for maltose binding protein, provoque the monomerisation of the dimeric native forms. These results are discussed in relation to the function of both binding proteins in transport and chemotaxis.