A spectral investigation of the copper(II) complex of the antitumor compound bleomycin

A thorough spectral investigation of the copper(II) complex of the antitumor compound, bleomycin, has been carried out in solution employing optical, difference optical, electron spin resonance, and circular dichroism techniques. The optical spectrum of a pH = 7 solution of the 1:1 complex between copper(II) and bleomycin is characterized by a broad weak band in the visible region (λ_max = 610 nm) that cannot be resolved and intense ultraviolet bands at 317 (? = 2800), 327 (shoulder), 250 (? = 4700), and 257 nm (shoulder). The circular dichroism spectrum in the visible region shows the broad and weak visible absorption band contains at least three components (558, 675, and 880 nm) that are likely to be “d-d” in origin. The electron spin resonance spectrum is characteristic of a tetragonal d⁹ copper(II) system showing no rhombic distoritions at X-band frequencies (g_x = g_y ± 0.002). The spin Hamiltonian parameters for the pH = 7.0 solution corrected for second order effects are A_∥ = 177 × 10^?4 cm^?1, A_⊥ ? 15 × 10^?4 cm^?1, g_∥ = 2.214, g_⊥ = 2.039. Most interesting was the observation of extra hyperfine splitting due to endogenous nitrogen coordination in a 30% glycerol glass (A^N = 12.0 × 10^?4 cm^?1). That pattern is best interpreted as a seven-line sequence associated with three liganded nitrogens. A dramatic change in all spectral properties occurs when the pH of the copper(II)-bleomycin complex is lowered to 2.5. All these data taken together suggest a CuN₃O coordination complex in solution. Details and justifications as well as a discussion of the limitations of the interpretations are presented.     

pH dependent copper binding properties of a CuA azurin variant with both bridging cysteines replaced with serines

A double mutant of Cu_A azurin was prepared in which both bridging cysteine thiolate ligands of the binuclear Cu_A center were replaced by serine. The copper binding properties of this protein were investigated, and shown to be pH dependent. At lower pH (5.2 ± 0.1), the protein binds one copper per protein molecule as demonstrated by electrospray ionization mass spectrometry. Copper titrations resulted in electronic absorptions at 730 nm (peak) and ca. 330 nm (shoulder) in the UV-Vis spectrum. EPR data show a four line pattern with hyperfine A_∥ = 150 G and g_∥ and g_⊥ values 2.32 and 2.03, characteristic of a type II (T2) copper. Superhyperfines to two nitrogen atoms were also observed. At higher pH (8.5 ± 0.1), the protein binds upto two copper atoms per protein molecule, and copper titrations exhibit a blue transition at 595 nm in the UV-Vis spectrum. The EPR data are consistent with two monomeric sites very similar to one another having hyperfines A_∥ = 182 and 150 G, g_∥ = 2.24 and 2.22 and a similar g_⊥ value of 2.01. These results indicate that both bridging cysteines play a critical role in the Cu_A center, and replacing them with serines is not enough to maintain the symmetrical diamond core structure or the characteristic electronic and functional properties of the Cu_A center.     

Optical and EPR spectra of the thionitrosyl complex [Cr(OH2)5(NS)]

The green thionitrosyl complex [Cr(OH₂)₅(NS)]²⁺ was isolated in solution by the hydrolysis of [Cr(NCCH₃)₅(NS)]²⁺. The optical absorption spectra of both compounds are dominated by a band with vibrational progression around 600 nm assigned as a {d_yz,zx, π^∗(NS)} → {π^∗(NS), d_yz,zx}^∗ transition. The optical data indicate that the NS ligand is a weaker π-acceptor than the NO ligand. The EPR parameters of [Cr(OH₂)₅(NS)]²⁺ were determined: g_iso, g_∥ and g_⊥: 1.96515, 1.92686(5) and 1.986860(8); A_iso(⁵³Cr), A_∥(⁵³Cr) and A_⊥(⁵³Cr): 25.3 × 10⁻⁴, 38 × 10⁻⁴ and 18.5 × 10⁻⁴ cm⁻¹; A_iso(¹⁴N), A_∥(¹⁴N) and A_⊥(¹⁴N): 6.5 × 10⁻⁴, 2.81 × 10⁻⁴ and 8.346(12) × 10⁻⁴ cm⁻¹.     

Copper(II) complexes as superoxide dismutase mimics: Synthesis, characterization, crystal structure and bioactivity of copper(II) complexes

Two new copper(II) complexes of the type [Cu(L)X₂), where L = (E)-N-phenyl-2-[phenyl (pyridine-2-yl)methylene]hydrazinecarboxamide X = Cl/Br have been synthesized and characterized by elemental analyses, FAB (fast atomic bombardment) magnetic measurements, electronic absorption, conductivity measurements cyclic voltammetry (CV) and Electron paramagnetic resonance (epr) spectroscopy. The structures of these complexes determined by single crystal X-ray crystallography show a distorted square based pyramidal (DSBP) geometry around copper(II) metal center. The distorted CuN₂OX (X = Cl/Br) basal plane in them is comprised of two nitrogen and one oxygen atoms of the meridionally coordinated ligand and a chloride or bromide ion and axial position is occupied by other halide ion. The epr spectra of these complexes in frozen solutions of DMSO showed a signal at g ca. 2. The trend in g-value (g_|| > g_⊥ > 2.00) suggest that the unpaired electron on copper(II) has d_x²-y² character. Biological activities in terms of superoxide dismutase (SOD) and antimicrobial properties of copper(II) complexes have also been measured. The superoxide dismutase activity reveals that these two complexes catalyze the fast disproportionation of superoxide in DMSO solution.     

Copper complexes with new oxaaza-pendant-armed macrocyclic ligands: X-ray crystal structure of a macrocyclic copper(II) complex

The synthesis of new oxaaza macrocyclic ligands (2-4) derived from O¹,O⁷-bis(2-formylphenyl)-1,4,7-trioxaheptane and functionalized tris(2-aminoethyl)amine are described. Mononuclear copper(II) complexes were isolated in the reaction of the corresponding macrocyclic ligand and copper(II) perchlorate. The structure of the [Cu(2)](ClO₄)₂ complex was determined by X-ray diffraction analysis. The copper(II) ion is five-coordinated by all N₅ donor atoms, efficiently encapsulated by the amine terminal pendant-arm, with a trigonal-bipyramidal geometry. The complexes are further characterized by UV-Vis, IR and EPR studies. The electronic reflectance spectra evidence that the coordination geometry for the Cu(II) complexes is trigonal-bipyramidal with the ligands 1 and 2 or distorted square-pyramidal with the ligands 3 and 4. The electronic spectra in MeCN solutions are different from those in the solid state, which suggest that some structural modification may occur in solution. The EPR spectrum of powder samples of the copper complex with 2 presents axial symmetry with hyperfine split at g_// with the copper nuclei (I = 3/2), which is characteristic of weakly exchange coupled extended systems. The EPR parameters (g_// = 2.230, A_// = 156 × 10⁻⁴ cm⁻¹ and g_⊥ = 2.085) indicate a d_x²-y² ground state. The EPR spectra of the complexes with ligands 3 and 4 show EPR spectra with a poorly resolved hyperfine structure at g_//. In contrast, the complex with ligand 2 shows no hyperfine split and a line shape which was simulated assuming rhombic g-tensor (g₁ = 2.030, g₂ = 2.115 and g₃ = 2.190).     

Characterization of the copper(II) binding site in the pink copper binding protein CusF by electron paramagnetic resonance spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy has been used to structurally characterize the copper-binding site in CusF protein from Escherichia coli. The EPR spectra indicate a single type II copper center with parameters typical for nitrogen and oxygen ligands (A~200 G, g~2.186, g~2.051). The pulsed EPR data show that one of the ligands to Cu²⁺ is an imidazole ring of a histidine residue. The remote amino nitrogen of this imidazole ring is readily observed by electron spin-echo envelope modulation spectroscopy, while the imino nitrogen that is directly coordinated to the Cu²⁺ ion is observed by pulsed electron–nuclear double resonance (ENDOR). In addition, the ENDOR spectra reveal the presence of one more nitrogen ligand that was assigned to be a deprotonated peptide nitrogen. Apart from the two nitrogen ligands, it has been established that there are two nearby hydroxyl protons, although whether these belong to a single equatorial water ligand or two equatorial hydroxide ligands is not known.

Metallobleomycins: electron spin resonance study on nitrosyl complex of iron(II)-Bleomycin

The ESR spectrum of the bleomycin-Fe(II)NO complex shows rhombic symmetry with a triplet hyperfine interaction in the g_z signal, and its ESR parameters have been compared with those of the ferrous-NO complexes of hemoproteins. The substitution of ₁₄NO by ₁₅NO gives the transition from a triplet to a doublet in the g_z absorption with a concomitant change in the nitrogen hyperfine constant. The addition of DNA to the ferrous-NO complex of bleomycin induces the greater separation of the g_x and g_y absorptions in comparison with the original ESR spectrum. The present three-line g_z signal for the bleomycin-Fe(II)-NO complex is indicative of weakened fifth axial nitrogen ligand-to-iron bonding with concomitantly stronger NO-to-iron bonding. On the other hand, the ESR feature of the bleomycin-Fe(III) complex is typical of the rhombic low-spin type, and no stable ferric-NO complex of bleomycin is formed.     

14N-ENDOR evidence for imidazole coordination in copper proteins

¹⁴N-ENDOR studies of simple nitrogen-coordinated copper(II) complexes in frozen aqueous solutions show that the nitrogen hyperfine constants, A_″ and A_⊥, of imidazole are much more isotropic ($\text{R =}\text{A}{}_{\mathrm{″}}\text{A}{}_{\mathrm{\perp }}\text{= 1.05}$) than those of the other biologically-related ligand nitrogens. From this result, combined with ¹⁴N-ENDOR results of some copper proteins containing imidazoles as ligands, it is concluded that R < 1.10 for nitrogen hyperfine constants can be employed as an empirical criterion for demonstration of the existence of imidazole coordination in copper proteins.     

Spectroscopic and electrochemical properties of a new nitrosyl-complex of Ru(II): trans-[Ru(NO){(CH3CH2)2PCH2CH2P(CH2CH3)2}2Cl](PF6)2

A new ruthenium nitric oxide complex with the bidentate phosphine, 1,2-bis(diethylphosphino)ethane (depe), has been synthesized and characterized by UV-Vis, infrared, EPR, NMR, electrochemical techniques and X-ray structure determination. The electronic spectrum showed a typical band of dπ→pπ* charge-transfer (CT) transition, assigned to Ru(II)NO transition, and the vibrational spectrum exhibited a peak of nitrosyl ligand at (ν_NO=1851 cm⁻¹). A model structure for this complex has been proposed based on ¹H, ¹H{³¹P}, ³¹P{¹H}, ¹³C{¹H}, COSY ¹H¹H{³¹P}, J-Resolved, HSQC, HMBC, HSQC ¹H¹³C{³¹P} and ¹H¹³C HSQC/¹H¹H TOCSY spectral data, and confirmed by X-ray diffraction. The nitrosonium character for the NO ligand become evident through both electron paramagnetic resonance and X-ray data (angle RuNO=177.4(3)°). The reversible monoeletronic process at E_1/2=0.040 V versus SHE was assigned to the ligand NO⁺/NO redox couple. Under treatment with Cd(Hg) solutions containing the [Ru(NO)(depe)₂Cl](PF₆)₂ yields a signal in the EPR spectrum (g_⊥=1.99 and g_//=1.88) which fitted quite well with the simulated spectra of coordinated NO species.     

Responses of the a3 component of cytochrome c oxidase to substrate and ligand addition

We have previously described a transient high spin ferric heme species in cytochrome c oxidase (EC 1.9.3.1) which represents a³⁺₃ (Beinert, H. and Shaw, R.W. (1977) Biochim. Biophys. Acta 462, 121–130), and can be detected and quantitatively determined by EPR. We have now used our ability to generate this species to study reactions of a³⁺₃ with substrates and ligands and also responses to pH changes. This was accomplished by multiple rapid mixing and freezing techniques in conjunction with low temperature EPR and optical reflectance spectroscopies. The substrates used were O₂ and ferrocytochrome c and the ligands cyanide, sulfide, azide and carbon monoxide. Contrary to the oxidized, resting form of the enzyme, the transient high spin species of a³⁺₃ reacts within <10 ms stoichiometrically with cyanide and sulfide and at a slower rate with azide. The transient a³⁺₃ species responds to O₂ and CO by changes in signal size or shape, although no oxidoreduction is involved, indicating that a³⁺₃ registers the presence of these gases. The high spin signal of the transient species is readily abolished by ferrocytochrome c or on raising the pH. Decreasing the pH induces a shift from the rhombic towards the axial component of the signal. Since the responses to CO and pH are analogous for the rhombic transient species to those observed with the rhombic high spin ferric heme species produced on partial reduction, it is suggested that the rhombic signals represent a³⁺₃ in either case. In all these experiments, in which EPR detectable a³⁺₃ was observed in large yield, no extra signals for copper or correspondingly increased intensity in the copper signal at g = 2 were seen. The relationship is discussed of the obviously reactive transient species of a³⁺₃ to other ‘activated’ species that have been reported and to the oxidized resting form of the enzyme, which is known to react only slowly with ligands and to respond sluggishly to substrate.     

Cd(II) and Cu(II) complexes of polydentate Schiff base ligands: synthesis, characterization, properties and biological activity

We report the synthesis of the Schiff base ligands, 4-[(4-bromo-phenylimino)-methyl]-benzene-1,2,3-triol (A₁), 4-[(3,5-di-tert-butyl-4-hydroxy-phenylimino)-methyl]-benzene-1,2,3-triol (A₂), 3-(p-tolylimino-methyl)-benzene-1,2-diol (A₃), 3-[(4-bromo-phenylimino)-methyl]-benzene-1,2-diol (A₄), and 4-[(3,5-di-tert-butyl-4-hydroxy-phenylimino)-methyl]-benzene-1,3-diol (A₅), and their Cd(II) and Cu(II) metal complexes, stability constants and potentiometric studies. The structure of the ligands and their complexes was investigated using elemental analysis, FT-IR, UV-Vis, ¹H and ¹³C NMR, mass spectra, magnetic susceptibility and conductance measurements. In the complexes, all the ligands behave as bidentate ligands, the oxygen in the ortho position and azomethine nitrogen atoms of the ligands coordinate to the metal ions. The keto-enol tautomeric forms of the Schiff base ligands A₁-A₅ have been investigated in polar and non-polar organic solvents. Antimicrobial activity of the ligands and metal complexes were tested using the disc diffusion method and the strains Bacillus megaterium and Candida tropicalis.Protonation constants of the triol and diol Schiff bases and stability constants of their Cu²⁺ and Cd²⁺ complexes were determined by potentiometric titration method in 50% DMSO-water media at 25.00 ± 0.02 °C under nitrogen atmosphere and ionic strength of 0.1 M sodium perchlorate. It has been observed that all the Schiff base ligands titrated here have two protonation constants. The variation of protonation constant of these compounds was interpreted on the basis of structural effects associated with the substituents. The divalent metal ions of Cu²⁺ and Cd²⁺ form stable 1:2 complexes with Schiff bases.The Schiff base complexes of cadmium inhibit the intense chemiluminescence reaction in dimethylsulfoxide (DMSO) solution between luminol and dioxygen in the presence of a strong base. This effect is significantly correlated with the stability constants K_CdL of the complexes and the protonation constants K_OH of the ligands; it also has a nonsignificant association with antibacterial activity.     

An EPR Study of NO Bonding to Erythrocruorin from the Earthworm Octolasium Complanatum

The EPR properties of the nitric oxide derivative of Octolasium complanatum erythrocruorin have been investigated as a function of the concentration of protons and cations which are known to affect the oxygen-linked allosteric equilibrium. The EPR spectrum has a rhombic shape with g_x = 2.08, g_z = 2.005, and g_y = 1.99, and remains unchanged under all the experimental conditions used. A supernyperfine pattern consisting of nine equally spaced lines is present in the g_z region indicating an interaction with two nonequivalent nitrogen atoms, one contributed by the nitric oxide and the other by the proximal histidine. The constancy of the EPR spectrum suggests that changes in the allosteric equilibrium do not involve differences in the strain of the Fe(II)-histidine bond as in tetrameric hemoglobins.     

Structural and electronic mimics of the active site of cobalt(II)-substituted zinc metalloenzymes

Complexes of cobalt(II) and zinc(II) which involve monodentate coordination of two alkyl carboxylate and two imidazole ligands in a slightly distorted tetrahedral fashion have visible and magnetic circular dichroism spectra remarkably similar to the cobalt(II)-substituted proteolytic enzymes thermolysin and carboxypeptidase A. Single crystal x-ray structure determinations on [Co(C₂H₅COO)₂Im₂], Im = imidazole, and its zinc counterpart reveal only minor structural differences between the cobalt and zinc species. Electron paramagnetic resonance spectra of cobalt(II) doped into zinc(II) complexes with known structures demonstrate the extreme sensitivity of the g-values to minor structural differences.     

Crossover of a high-spin (S=7/2, 3/2) to a low-spin (S=1/2) iron-selenium cluster in FA and FB of photosystem I on rebinding of PsaC onto P700-FX cores

 PsaC is a tightly bound ferredoxin in the Photosystem I (PS I) reaction center which contains two [4Fe-4S] clusters named F_A and F_B. We recently proposed that the mixed-ligand F_B cluster in C14D_PsaC and the mixed-ligand F_A cluster in C51D_PsaC exist in a spin state of S=3/2, and that a spin state crossover to S=1/2 occurs when the PsaC mutants are rebound onto P700-F_X cores. Since EPR signals from a highly rhombic S=3/2 spin state can be difficult to study, wild-type PsaC was reconstituted with iron and selenium to introduce an easily detected S=7/2 spin state similar to that shown for Clostridial ferredoxin. When the unbound [4Fe-4Se] PsaC was chemically reduced, a sharp derivative resonance was found at g=5.171 attributed to the excited ±3/2 doublet from an S=7/2 spin multiplet. An additional peak was found at g=5.616 attributed to the superimposed ±1/2 and ±3/2 doublets from a highly rhombic S=3/2 spin multiplet, and an axial set of resonances found around g=2.0 attributed, in part, to a classical S=1/2 spin state. When the [4Fe-4Se] PsaC was rebound onto P700-F_X cores, the spin population derived from the S=7/2 and 3/2 spin states was negligible. Illumination of the rebuilt PS I complex at 15 K resulted in two rhombic sets of resonances, one with g values of 2.043, 1.941 and 1.854, diagnostic of F_A, and the other with g values of 2.067, 1.941 and 1.878, diagnostic of F_B. Chemical reduction with sodium dithionite at pH 10.5 or photoaccumulation by freezing during illumination resulted in a set of resonances with g values of 2.046, 1.938, 1.920 and 1.883, characteristic of a spin-coupled F_A ^–/F_B ^– pair. The spin state crossover in this iron chalcogenide cluster is the first known to be induced by protein-protein association and reinforces the hypothesis that an S=3/2 to 1/2 crossover occurs in the PS I-rebound mutants C14D_PsaC and C51D_PsaC. Received: 6 August 1996 / Accepted: 28 December 1996     

Studies on the reaction of imidazole with cytochrome c3 from Desulfovibrio vulgaris

1. Cytochrome c₃, a unique hemoprotein with a negative redox potential and four heme groups bound to a single polypeptide chain, reacts with imidazole in the reduced state to form a low-spin ferro · imidazole complex which is spectrally characterized by a 3.1 nm blue shift in the α-peak (from 550.5 to 547.4 nm). The spectral imidazole · cytochrome c₃ complex is detectable at 77 but not at 298 K.2. Mammalian ferrocytochrome c did not undergo a spectral interaction with imidazole at either 77 or 298 K, indicating that the imidazole · cytochrome c₃ complex reflects a unique event for cytochrome c₃.3. Formation of the imidazole · cytochrome c₃ complex is strongly dependent on imidazole concentration (apparent K_d of approx. 50 mM), and is abolished in the presence of 100 mM phosphate. This latter effect is attributable to formation of an imidazole · phosphate complex. A pH titration of the imidazole · cytochrome c₃ spectral complex implicates ionization of an imidazole function (pK = 8.5).4. EPR studies at 8.5 K of ferricytochrome c₃ before and after one reduction-oxidation cycle indicate that at least two of the hemes undergo reaction with imidazole forming two different low-spin ferric heme · imidazole complexes, with significant shifts in the g values of two heme signals.5. The spectral and EPR results are consistent with formation as the primary event of a low-spin ferrocytochrome c₃ · imidazole complex in which increased hydrophobicity and protonation-deprotonation effects are contributary to the consequent lability of cytochrome c₃.     

Crystal structure, optical, magnetic, and photochemical properties of the complex pentakis(dimethyl sulfoxide)nitrosylchromium(2+) hexafluorophosphate

The nitrosyl complex [Cr(dmso)₅(NO)](PF₆)₂ (1) (dmso = dimethyl sulfoxide) has been prepared by the solvolysis of [Cr(NCCH₃)₅(NO)](PF₆)₂ in neat dmso. The optical absorption spectrum of 1 in dmso shows maxima at 734, 567, 450, 413, and 337 nm. Continuous photolysis of 1 with λ = 365-580 nm light in dmso solution results in a release of NO with quantum yield, Φ, in the range 0.034-0.108 mol Einstein⁻¹. Irradiation of a deoxygenated CH₃CN solution of [Cr(NCCH₃)₅(NO)](PF₆)₂ in the presence of excess of [Fe(S₂CNEt₂)₂] results in a transfer of NO to the iron centre as shown from the characteristic EPR spectrum of [Fe(S₂CNEt₂)₂(NO)] with A_iso(¹⁴N) = 12.2 × 10⁻⁴ cm⁻¹. The EPR parameters of 1 were determined: g_iso, g_‖ and g_⊥ : 1.96725, 1.91881(4) and 1.992763(2); A_iso(⁵³Cr), A_‖ (⁵³Cr) and A_⊥(⁵³Cr): 22.8 × 10⁻⁴, 39 × 10⁻⁴ and 15.8 × 10⁻⁴ cm⁻¹; A_iso(¹⁴N), A_‖ (¹⁴N) and A_⊥(¹⁴N): 5.9 × 10⁻⁴, 2 × 10⁻⁴ and 7.540(4) × 10⁻⁴ cm⁻¹.     

Low-temperature electron transfer suggests two types of QA in intact photosystem II

The correlation between the reduction of Q_A and the oxidation of Tyr_Z or Car/Chl_Z/Cyt_b559 in spinach PSII enriched membranes induced by visible light at 10 K is studied by using electron paramagnetic resonance spectroscopy. Similar g = 1.95-1.86 Q_A^-•EPR signals are observed in both Mn-depleted and intact samples, and both signals are long lived at low temperatures. The presence of PPBQ significantly diminished the light induced EPR signals from Q_A^-•, Car^+•/Chl^+• and oxidized Cyt_b559, while enhancing the amplitude of the S₁Tyr_Z• EPR signal in the intact PSII sample. The quantification and stability of the g = 1.95-1.86 EPR signal and signals arising from the oxidized Tyr_Z and the side-path electron donors, respectively, indicate that the EPR-detectable g = 1.95-1.86 Q_A^-• signal is only correlated to reaction centers undergoing oxidation of the side-path electron donors (Car/Chl_Z/Cyt_b559), but not of Tyr_Z. These results imply that two types of Q_A^-• probably exist in the intact PSII sample. The structural difference and possible function of the two types of Q_A are discussed.     

The metal-binding site of imidazole glycerol phosphate dehydratase; EPR and ENDOR studies of the oxo-vanadyl enzyme

 The apo protein of imidazole glycerol phosphate dehydratase (IGPD) from Saccharomyces cerevisiae combines stoichiometrically with certain specific divalent metal cations to assemble the catalytically active form comprising 24 protein subunits and tightly bound metal. VO²⁺ ions react similarly but, uniquely, result in a metallo-protein (VO-IGPD) with neither catalytic activity nor the ability to bind to the reaction intermediate analogue, 2-hydroxy-3-(1,2,4-triazol-1-yl) propylphosphonate. Since VO²⁺ apparently assembles the quaternary structure correctly, it is used in the present study as a spin probe to investigate the metal centre coordination environment by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopy. At neutral pH, the EPR spectrum of VO-IGPD reveals at least three distinct VO²⁺ sub-spectra with one predominant at low pH. The spin Hamiltonian parameters for some of the sub-spectra are consistent with ⁵¹V having nitrogen in the inner-sphere equatorial coordination environment from, most probably, multiple coordinating histidines. Further evidence for inner-sphere nitrogen ligands is obtained from ENDOR spectroscopy. The spectra of the low rf region show signals from interactions with ¹⁴N which are consistent with couplings to the imino nitrogen of coordinated histidine residues. In addition a number of proton ENDOR line pairs are resolved. Of the few that disappear upon exchange of the protein into D₂O, one most likely originates from the exchangeable proton of the N-H group of a coordinated histidine imidazole. ¹H-ENDOR line pairs from non-exchangeable protons with splittings of approximately 3 MHz can be attributed to imidazole carbon protons. Thus, most of the couplings observed by ENDOR are consistent with being from the imidazole heterocycle of one or more histidine ligands. Received: 27 June 1996 / Accepted: 14 March 1997     

Electron paramagnetic studies of the copper and iron containing soluble ammonia monooxygenase from <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis>

Soluble ammonia monooxygenase (AMO) from Nitrosomonas europaea was purified to homogeneity and metals in the active sites of the enzyme (Cu, Fe) were analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR spectra were obtained for a type 2 Cu(II) site with g_|| = 2.24, A_|| = 18.4 mT and g_⊥ = 2.057 as well as for heme and non heme iron present in purified soluble AMO from N. europaea. A second type 2 Cu(II) EPR signal with g_|| = 2.29, A_|| = 16.1 mT and g_⊥ = 2.03 appeared in the spectrum of the ferricyanide oxidized enzyme and was attributed to oxidation of cuprous sites. Comparison of EPR-detectable Cu²⁺ with total copper determined by inductively coupled plasma-mass spectrometry (ICP-MS) suggests that there are six paramagnetic Cu²⁺ and three diamagnetic Cu¹⁺ per heterotrimeric soluble AMO (two paramagnetic and one diamagnetic Cu per αβγ-protomer). A trigonal EPR signal at g = 6.01, caused by a high-spin iron, indicative for cytochrome bound iron, and a rhombic signal at g = 4.31, characteristic of specifically bound Fe³⁺ was detectable. The binding of nitric oxide in the presence of reductant resulted in a ferrous S = 3/2 signal, characteristic of a ferrous nitrosyl complex. Inactivation of soluble AMO with acetylene did neither diminish the ferrous signal nor the intensity of the Cu²⁺-EPR signal.     

Sterically demanding bi- and tridentate alkoxy-N-heterocyclic carbenes

In contrast to the one-pot, two step syntheses we recently reported for a large number of substituted bidentate alkoxy-carbene ligands derived from epoxides, the reaction of imidazole with 2-adamantyl epoxide readily affords the bis(ethoxyadamantyl) substituted imidazolium salt [1-C{(NR)CHCH(NRH)}] (RH = CH₂(2-adamantyl)OH), which has been isolated and structurally characterised as its iodide salt, [HC{(NRH)CH}₂]I. Treatment with group 1 bases results in the loss of one ethoxy arm, to afford the structurally characterised monosubstituted alcohol imidazole, [HC{(NR)CHCHN}], or the lithium carbene complex [LiC{(NR)CHCHN}], a carbene complex containing a singly-N-functionalised alkoxy carbene. Alternatively, the monosubstituted alcohol imidazole may ben requaternised at the nitrogen atom with iso-propyl iodide to form [HC{(NRH)CHCH(NPrⁱ)}]I, from which a more standard lithium alkoxycarbene complex [LiC{(NR)CHCHNPrⁱ}] may be generated.