Electron spin echo envelope modulation studies of the Cu(II)-substituted derivative of isopenicillin N synthase: a structural and spectroscopic model.

Electron spin echo envelope modulation spectroscopy (ESEEM) was used to study the active site structure of isopenicillin N synthase (IPNS) from Cephalosporium acremonium with Cu(II) as a spectroscopic probe. Fourier transform of the stimulated electron spin-echo envelope for the Cu(II)-substituted enzyme, Cu(II)IPNS, revealed two nearly magnetically equivalent, equatorially coordinated His imidazoles. The superhyperfine coupling constant, Aiso, for the remote 14N of each imidazole was 1.65 MHz. The binding of substrate to the enzyme altered the magnetic coupling so that Aiso is 1.30 MHz for one nitrogen and 2.16 MHz for the other. From a comparison of the ESEEM of Cu(II)IPNS in D2O and H2O, it is suggested that water is a ligand of Cu(II) and this is displaced upon the addition of substrate.     

Structure of the Glycyl-L-histidyl-L-lysine--copper(II) complex in solution

Optical, electron paramagnetic resonance, and electron spin-echo envelope spectroscopies were used to examine the structure of the Cu(II) complex of glycyl-L-histidyl-L-lysine (GHL) in solution. At neutral pH, GHL forms a mononuclear 1:1 Cu(II) compound having an EPR spectrum resembling that of Cu(II) equatorially coordinated by two or three nitrogen atoms. Electron spin-echo studies demonstrate that one of these is located in the histidyl imidazole ring. A pH titration of Cu(II)-GHL shows three optical transitions with apparent pKs of 3.6, 9.2 and 11.4 and molecularities, with respect to protons, of 2, 2, and 1, respectively. At the lowest pK, GHL binds Cu(II), forming the species present at physiological pH. At elevated pH, spectroscopic experiments suggest that an alteration of the Cu(II) structure occurs, yet the bound imidazole is retained. These solution studies are consistent with nitrogen coordination of Cu(II) in Cu(II)-GHL, but the solid-state polymeric structure, with oxygen-bridged Cu(II) pairs as previously determined by X-ray crystallographic analysis [Pickart, L., Freedman, J. H., Loker, W. J., Peisach, J., Perkins, C. M., Steinkamp, R. E., & Weinstein, B. (1980) Nature (London) 288, 715-717; C. M. Perkins, N. J. Rose, R. E. Steinkamp, L. H. Jensen, B. Weinstein, and L. Pickart, unpublished results], does not exist in solution.     

X-ray absorption spectroscopic study of the active copper sites in dopamine beta-hydroxylase

X-ray absorption spectroscopy has been used to investigate the local environment of the copper sites in bovine dopamine beta-hydroylase, the enzyme that catalyzes the conversion of dopamine to norepinephrine in the adrenal medulla and noradrenergic nerve cells. The marked similarity of the x-ray absorption edge features of the oxidized and ascorbate-reduced forms of the enzyme with those of the corresponding Cu(imidazole)4 complexes suggests that the ligation in both cases is very similar. Furthermore, this similarity is found for the extended x-ray absorption fine structure data, and analysis shows only nitrogen (or oxygen) ligation for both enzyme forms. Thus, four nitrogen atoms provide the best fit to the data at an average distance of 1.97 +/- 0.02 A for the oxidized enzyme and four nitrogen atoms at 2.05 +/- 0.02 A for the ascorbate-reduced form. The present data analysis also indicates that there is little change in the average copper ligand environment upon reduction of the enzyme-bound copper from Cu(II) to the Cu(I). The data for the oxidized form of the enzyme are in agreement with previous spin-echo EPR experiments that show three to four imidazole nitrogen ligands for each copper (McCracken, J., Desai, P. R., Papadopoulos, N. J., Villafranca, J. J., and Peisach, J. (1988) Biochemistry 27, 4133-4137). In addition, the data do not indicate the presence of any heavy atom (sulfur or chlorine) ligation to the ascorbate-reduced form of the enzyme as reported by Scott et al. (Scott, R. A., Sullivan, R. J., DeWolf, W. E., Jr., Dolle, R. E., and Kruse, L. I. (1988) Biochemistry 27, 5411-5417).     

Herbicide-degrading alpha-keto acid-dependent enzyme TfdA: metal coordination environment and mechanistic insights

TfdA is a non-heme iron enzyme which catalyzes the first step in the oxidative degradation of the widely used herbicide (2, 4-dichlorophenoxy)acetate (2,4-D). Like other alpha-keto acid-dependent enzymes, TfdA utilizes a mononuclear Fe(II) center to activate O(2) and oxidize substrate concomitant with the oxidative decarboxylation of alpha-ketoglutarate (alpha-KG). Spectroscopic analyses of various Cu(II)-substituted and Fe(II)-reconstituted TfdA complexes via electron paramagnetic resonance (EPR), electron spin-echo envelope modulation (ESEEM), and UV-vis spectroscopies have greatly expanded our knowledge of the enzyme's active site. The metal center is coordinated to two histidine residues as indicated by the presence of a five-line pattern in the Cu(II) EPR signal, for which superhyperfine splitting is attributed to two equivalent nitrogen donor atoms from two imidazoles. Furthermore, a comparison of the ESEEM spectra obtained in H(2)O and D(2)O demonstrates that the metal maintains several solvent-accessible sites, a conclusion corroborated by the increase in multiplicity in the EPR superhyperfine splitting observed in the presence of imidazole. Addition of alpha-KG to the Cu-containing enzyme leads to displacement of an equatorial water on copper, as determined by ESEEM analysis. Subsequent addition of 2,4-D leads to the loss of a second water molecule, with retention of a third, axially bound water. In contrast to these results, in Fe(II)-reconstituted TfdA, the cosubstrate alpha-KG chelates to the metal via a C-1 carboxylate oxygen and the alpha-keto oxygen as revealed by characteristic absorption features in the optical spectrum of Fe-TfdA. This binding mode is maintained in the presence of substrate, although the addition of 2,4-D does alter the metal coordination environment, perhaps by creating an O(2)-binding site via solvent displacement. Indeed, loss of solvent to generate an open binding site upon the addition of substrate has also been suggested for the alpha-keto acid-dependent enzyme clavaminate synthase 2 [Zhou et al. (1998) J. Am. Chem. Soc. 120, 13539-13540]. Nitrosyl adducts of various Fe-TfdA complexes have also been investigated by optical and EPR spectroscopy. Of special interest is the tightly bound NO complex of Fe-TfdA.(alpha-KG).(2,4-D), which may represent an accurate model of the initial oxygen-bound species.     

X-ray absorption spectroscopic analysis of Fe(II) and Cu(II) forms of a herbicide-degrading α-ketoglutarate dioxygenase

 The first step in the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by Ralstonia eutropha JMP134 is catalyzed by the α-ketoglutarate (α-KG)-dependent dioxygenase TfdA. Previously, EPR and ESEEM studies on inactive Cu(II)-substituted TfdA suggested a mixture of nitrogen/oxygen coordination with two imidazole-like ligands. Differences between the spectra for Cu TfdA and α-KG- and 2,4-D-treated samples were interpreted as a rearrangement of the g–tensor principal axis system. Herein, we report the use of X-ray absorption spectroscopy (XAS) to further characterize the metal coordination environment of Cu TfdA as well as that in the active, wild-type Fe(II) enzyme. The EXAFS data are interpreted in terms of four N/O ligands (two imidazole-like) in the Cu TfdA sample and six N/O ligands (one or two imidazole-like) in the Fe TfdA sample. Addition of α-KG results in no significant structural change in coordination for Cu or Fe TfdA. However, addition of 2,4-D results in a decrease in the number of imidazole ligands in both Cu and Fe TfdA. Since this change is seen both in the Fe and Cu EXAFS, loss of one histidine ligand upon 2,4-D addition best describes the phenomenon. These XAS data clearly demonstrate that changes occur in the atomic environment of the metallocenter upon substrate binding. Received: 3 July 1998 / Accepted: 13 October 1998     

Model studies on the coordination of copper in biological systems. The deprotonated peptide nitrogen as a potential binding site for copper(II)

1. A large number of potentially bidentate and tridentate amides, X-Y-CONH-Z, were used as model ligands to investigate the complex formation of Cu(II) with the deprotonated peptide nitrogen in biological molecules. A combination of potentiometric titration, spectrophotometry and electron paramagnetic resonance was applied to analyse the structure of the Cu(II) chelates formed at neurtal and basic pH. 2. By systematic variation of the primary binding function X, the ring size of the chelate, and the spatial properties of the C-terminal and N-terminal substituents, three classes of amide ligands could be established with reference to their capacity for Cu(II)-induced deprotonation of NHCO and metal binding. 3. Under physiological conditions of pH, peptide (class A) chelates are only formed by those bidentate amide ligands with X being an imidazole (sp2) nitrogen or a terminal (sp3) amino nitrogen. Mercaptide sulfur must also be considered to belong in this group of strong copper(II)-binding sites, but in our low-molecular-weight model ligands the redox equilibrium 2 Cu(II) + 2 RSH in equilibrium or formed from 2 CU(II) + RSSR + 2 H+ interferes, yielding insoluble Cu(I)-S polymers above pH 4. In addition to the unidentate binding strength of X, entropy effects play an important role. Depending on whether X is an imidazole or amino nitrogen, only five-membered or six-membered monocyclic chelate structures respectively cause coordination of the deprotonated peptide function. 4. Biuret (class B) Cu(II) chelates are only formed under non-physiological conditions at pH > 11.5 producing the well known violet chromophores CuIIN4(-). In general these complexes, which also include the Cu(II) biguanides, show a clearly resolved electron paramagnetic resonance spectrum with nitrogen superhyperfine structure. 5. A third class of peptide model ligands (class C) consists of those amides where the CuII-X bond does not provide enough thermodynamic stability. The binding site of these class C amides includes functional groups such as carboxylate (COO-), methionine sulfur (RSR'), aliphatic or aromatic hydroxyl (OH) and amide nitrogen (NHCO) itself. When X is a pyridine (sp2) nitrogen or an amino (sp3) nitrogen, NHCO deprotonation is only promoted in five-membered but not six-membered ring chelates. On the other hand, a combination of COO- and NH2, as in asparagine, will allow deprotonation of NHCO in the presence of Cu(II). And third, despite a pronounced unidentate affinity of the imidazole nitrogen for Cu(II), N-acetylhistamine acts as a class C amine, in contrast to imidazolylacetamide, which forms a stable Cu(II) peptide chelate. This difference in Cu binding is explained on the basis of space-filling models. These clearly demonstrate that in the case of the 2:1 complex of Cu(II) with N-acetylhistamine, the planarity of the ionised peptide function can not be retained in a square planar arrangement of the two amide ligands around the copper center.     

Cu XAS shows a change in the ligation of CuB upon reduction of cytochrome bo3 from Escherichia coli

Copper X-ray absorption spectroscopy (XAS) has been used to examine the structures of the Cu(II) and Cu(I) forms of the cytochrome bo3 quinol oxidase from Escherichia coli. Cytochrome bo3 is a member of the superfamily of heme-copper respiratory oxidases. Of particular interest is the fact that these enzymes function as redox-linked proton pumps, resulting in the net translocation of one H+ per electron across the membrane. The molecular mechanism of how this pump operates and the manner by which it is linked to the oxygen chemistry at the active site of the enzyme are unknown. Several proposals have featured changes in the coordination of CuB during enzyme turnover that would result in sequential protonation or deprotonation events that are key to the functioning proton pump. This would imply lability of the ligands to CuB. In this work, the structure of the protein in the immediate vicinity of CuB, in both the fully oxidized and fully reduced forms of the enzyme, has been examined by Cu XAS, a technique that is particularly sensitive to changes in metal coordination. The results show that in the oxidized enzyme, CuB(II) is four-coordinate, consistent with three imidazoles and one hydroxyl (or water). Upon reduction of the enzyme, the coordination of CuB(I) is significantly altered, consistent with the loss of one of the histidine imidazole ligands in at least a substantial fraction of the population. These data add to the credibility that changes in the ligation of CuB might occur during catalytic turnover of the enzyme and, therefore, could, in principle, be part of the mechanism of proton pumping.     

Replacement of non-heme Fe(II) with Cu(II) in the alpha-ketoglutarate dependent DNA repair enzyme AlkB: spectroscopic characterization of the active site

The bacterial DNA repair enzyme AlkB is an alpha-ketoglutarate (alphaKG) dependent non-heme Fe(II) containing dioxygenase. Here we describe, for the first time, the preparation of a Cu(II)-reconstituted form of AlkB in various complexes. Spectroscopic characterization showed correct AlkB folding upon incorporation of Cu(II) in the active site. The Cu site was classified as a type 2 site by EPR spectroscopy. The accessibility of the active site metal was studied using imidazole as a probe. Although addition of imidazole did not change the EPR spectrum of the AlkB-Cu-alphaKG complex, the spectrum of the AlkB-Cu-succinate complex clearly changed, indicating binding of imidazole at the Cu site. Binding of substrate (methylated DNA) to the AlkB-Cu-alphaKG complex did not induce changes in the EPR spectrum, demonstrating that the substrate does not bind in the immediate vicinity of the metal centre. This work provides a basis for advanced EPR approaches aimed at studying the interactions and dynamics of AlkB complexes in solution.     

EPR of Cu+2 binding to apo-yeast enolase

We have studied the electron paramagnetic resonance (epr) spectra of complexes of apo-yeast enolase with 65Cu+2 in the presence and absence of substrate and magnesium ion. An unusual epr spectrum with large g parallel, large g and A rhombicity and very narrow line-widths (10 G) is seen for the first two 65Cu+2 bound in the presence of substrate 2-phosphoglycerate (2PGA). the epr parameters, consistent with rhombic and tetragonal distortion of an octahedral geometry of the coordination sphere of the Cu+2 are g = (2.123, 2.042, 2.405) and A = (2.58, 4.19, 12.0) mK. The high g parallel and absence of super-hyperfine splitting are strong evidence for absence of nitrogen ligands. In the presence of Mg+2 and 2PGA, the Cu+2-enolase solutions exhibit a complex epr spectrum reflecting exchange and dipolar interaction between the first two Cu+2 ions bound. The spectra of Cu+2 plus enolase in the presence and absence of Mg+2 without 2PGA are distinct but not unambiguous, each reflecting at least two inequivalent binding sites. In addition to providing information on the geometry and location of the divalent cation binding sites, the data show unequivocally that imidazole residues, previously found to have a role in catalysis, do not participate in Cu+2 binding. Although Cu+2 does not activate the enzyme, direct binding measurements show that Cu+2 competes stoichiometrically with the activating ion, Mg+2. A reinterpretation of earlier Mn+2 enolase studies is proposed to reconcile the Cu+2 and Mn+2 data.     

Inhibitory copper binding site on the spinach cytochrome b6f complex: implications for Qo site catalysis

The isolated cytochrome (cyt) b(6)f complex from spinach is inhibited by Cu(2+) with a K(D) of about 1 microM at pH 7.6 in the presence of 1.6 microM decyl-plastoquinol (C(10)-PQH(2)) as a substrate. Inhibition was competitive with respect to C(10)-PQH(2) but noncompetitive with respect to horse heart cyt c or plastocyanin (PC). Inhibition was also pH-sensitive, with an apparent pK at about 7, above which inhibition was stronger, suggesting that binding occurred at or near a protonatable amino acid residue. Equilibrium binding titrations revealed ca. 1.4 tight Cu(2+) binding sites with a K(D) of about 0.5 microM and multiple (>8) weak (K(D) > 50 microM) binding sites per complex. Pulsed electron paramagnetic resonance (EPR) techniques were used to identify probable binding sites for inhibitory Cu(2+). A distinct enhancement of the relaxation time constant for the EPR signal from bound Cu(2+) was observed when the cyt f was paramagnetic. The magnitude and temperature-dependence of this relaxation enhancement were consistent with a dipole interaction between Cu(2+) and the cyt f (Fe(3+)) heme at a distance of between 30 and 54 A, depending upon the relative orientations of Cu(2+) and cyt f heme g-tensors. Two-pulse electron spin-echo envelope modulation (ESEEM) and 4-pulse 2-dimensional hyperfine sublevel correlation (2D HYSCORE) measurements of Cu(2+) bound to isolated cyt b(6)f complex indicated the presence of a weakly coupled nitrogen nucleus. The nuclear quadrupole interaction (NQI) and the hyperfine interaction (HFI) parameters identified one Cu(2+) ligand as an imidazole nitrogen of a His residue, and electron-nuclear double resonance (ENDOR) confirmed the presence of a directly coordinated nitrogen. A model of the 3-dimensional structure of the cytochrome b(6)f complex was constructed on the basis of sequences and structural similarities with the mitochondrial cyt bc(1) complex, for which X-ray structures have been solved. This model indicated three possible His residues as ligands to inhibitory Cu(2+). Two of these are located on the "Rieske" iron-sulfur protein protein (ISP) while the third is found on the cyt f protein. None of these potential ligands appear to interact directly with the quinol oxidase (Q(o)) binding pocket. A model is thus proposed wherein Cu(2+) interferes with the interaction of the ISP protein with the Q(o) site, preventing the binding and subsequent oxidation of plastoquinonol. Implications for the involvement of ISP "domain movement" in Q(o) site catalysis are discussed.     

Pulsed EPR studies of the type 2 copper binding site in the mercury derivative of laccase.

The nuclear modulation effect in pulsed EPR spectroscopy was used to study the type 2 copper binding site in the mercury derivative of laccase (MDL) in which the type 1 copper is substituted by Hg(II). By comparing the three-pulse electron spin-echo modulations and Fourier transform spectra of MDL and several model compounds, we conclude that the imidazole groups of two histidyl amino acid residues are equatorially coordinated to Cu(II) in the type 2 site. Computer simulations of these data suggest that the remote nonbonding nitrogens of the two imidazoles possess nuclear quadrupole parameters e2qQ = 1.47 MHz and eta = 0.83. A(iso) values of these two nitrogens are not identical, being 1.5 and 2.0 MHz. We have also used samples of the enzyme exchanged with D2O to examine the coordination of the water to the type 2 copper site. The deuterium modulation that is resolved by taking the ratio of the time domain ESEEM data from native and D2O-exchanged enzyme indicates that there is an equatorial water ligand, and further data show that this water is displaced by azide.     

Coordination environment for the type 3 copper center of tree laccase and CuB of cytochrome c oxidase as determined by electron nuclear double resonance

A new rhombic EPR signal was recently discovered in the partially reduced type 2 copper-depleted Rhus vernicifera laccase (Reinhammar, B. (1983) J. Inorg. Biochem., in press). The signal originates from one of the type 3 Cu(II) ions that becomes EPR-detectable as a result of the selective reduction of the other copper ion in the exchange-coupled Cu(II)-Cu(II) pair. The 14N and 1H and 63,65Cu electron nuclear double resonance (ENDOR) of this uncoupled Cu(II) now have been collected and represent the first ENDOR measurements of a type 3 copper site. The data indicate that the copper is coordinated by at least three nitrogenous ligands, at least one of which is an imidazole. H/D exchange suggests a nearby H2O or OH-, perhaps as a fourth ligand. A similar EPR signal is seen for CuB of reduced cytochrome c oxidase under turnover conditions. The 14N ENDOR, and, therefore, the structure, of this site corresponds extremely closely to that of the laccase type 3 (Cu(II).     

The metal site of stellacyanin: EXAFS studies of the Cu(II), Cu(I), Ni(II) and Co(II) derivatives

Extended X-ray absorption fine structure (EXAFS) studies of Cu(II) (oxidized), Cu(I) (reduced), Ni(II) and Co(II) stellacyanin from Rhus vernicifera are reported. For Cu(II) stellacyanin, the coordination by three close ligands, viz. 2 N and 1 S, with the presence of smaller shells pointing to imidazole coordination, indicates similarities with the coordination in other so-called type 1 or 'blue'-copper proteins. Upon reduction, slightly longer ligand distances and an additional sulphur ligand are found. Ni(II) and Co(II) stellacyanin resemble Cu(I) and Cu(II) stellacyanin, respectively, in ligand distances, but have a tendency for three rather than two N (or O) ligands in the first shell. The results are compared with the three-dimensional model derived from 1H-NMR relaxation measurements for Co(II) stellacyanin, and are consistent with the proposal that apart from the three close ligands found in all blue-copper proteins, a sulphur from a disulphide bridge and the amide oxygen from an asparagine residue come to within coordinating distance of the metal in stellacyanin.     

Mapping the copper ligands of Cu,Zn superoxide dismutase by nuclear Overhauser enhancement of the isotropically shifted 1H-NMR lines of the Cu,Co derivative

Transient nuclear Overhauser effect (NOE) experiments were made with bovine Cu,Co superoxide dismutase on the hyperfine shifted resonances of protons of the imidazole groups bound to the Cu,Zn cluster of the native enzyme. Assignments of lines based on the observed magnetization transfers were satisfactorily obtained only by altering the arrangement of the ligands around the catalytically active copper shown by X-ray crystallography.     

Binding of inhibitory metals to yeast enolase

Certain divalent cations can inhibit yeast enolase by binding at sites that are distinct from those metal binding sites normally associated with catalytic activity, i.e., the conformational and catalytic binding sites. By using a buffer that does not compete with metal ions (tetrapropylammonium borate) Zn, Co, Mn, Cu, Cd, and Ni are found to exhibit similar inhibitory characteristics. Inhibition by those metals is alleviated by the addition of imidazole or tris buffer and, for zinc, by a metal chelating agent (Calcein). Inhibition by zinc was examined in detail through binding studies and enzymatic activity measurement. In tetrapropylammonium buffers at pH 8.0, enolase binds up to four moles of zinc per mole of enzyme (two moles per subunit). An imidazole concentration of 0.05 M reduces the binding: in the absence of substrate, just two moles of zinc per enzyme are bound. The enzyme will bind two additional moles of zinc upon the addition of substrate in either buffer, but the enzyme in tetrapropylammonium buffer is nearly inactive. Inhibition is, therefore, correlated with the binding of two moles of zinc per mole of enzyme. Some additional metal ions, Ca, Tb, Hg, and Ag also caused inhibition of yeast enolase but not by binding to the inhibitory site described.     

Identification of two imidazole binding sites and key residues for substrate specificity in human primary amine oxidase AOC3

Human membrane primary amine oxidase (hAOC3; also known as vascular adhesion protein-1, VAP-1) is expressed upon inflammation in most tissues, where its enzymatic activity plays a crucial role in leukocyte trafficking. We have determined two new structures of a soluble, proteolytically cleaved form of hAOC3 (sAOC3), which was extracted from human plasma. In the 2.6 ? sAOC3 structure, an imidazole molecule is hydrogen bonded to the topaquinone (TPQ) cofactor, which is in an inactive on-copper conformation, while in the 2.95 ? structure, an imidazole molecule is covalently bound to the active off-copper conformation of TPQ. A second imidazole bound by Tyr394 and Thr212 was identified in the substrate channel. We furthermore demonstrated that imidazole has an inhibitory role at high concentrations used in crystallization. A triple mutant (Met211Val/Tyr394Asn/Leu469Gly) of hAOC3 was previously reported to change substrate preferences toward those of hAOC2, another human copper-containing monoamine oxidase. We now mutated these three residues and Thr212 individually to study their distinct role in the substrate specificity of hAOC3. Using enzyme activity assays, the effect of the four single mutations was tested with four different substrates (methylamine, benzylamine, 2-phenylethylamine, and p-tyramine), and their binding modes were predicted by docking studies. As a result, Met211 and Leu469 were shown to be key residues for substrate specificity. The native structures of sAOC3 and the mutational data presented in this study will aid the design of hAOC3 specific inhibitors.     

EPR study of dimerization and conformational change of Cu(II)-cytochrome c and its undeca- and octapeptide

The EPR study of cytochrome c in which FE(III) ion is replaced with Cu(II) shows that there are two types of monomer (a: 4 less than pH less than 6, and b: 6 less than pH less than 11.5) and two types of dimer (A: pH less than 4 and B: pH less than 11.5) formed depending upon the pH value of the solution. Computer simulation of the EPR spectra of the dimers indicates that the structure of the dimer A has a larger lateral shift than in the dimer B. It is also shown that in monomer a, the imidazole nitrogen of 18-His is not bound to Cu(II), while it is bound in the monomer b. In the undeca- and octapeptide of Cu(II)-cytochrome c, polymers are formed in acidic solutions. As the pH is raised, depolymerization proceeds to yield the monomer and the dimer. The structure of the dimer in both peptides is found to be similar to that of the dimer B of Cu(II)-cytochrome c. In the monomer of the peptides, neither the imidazole of 18-His nor the imidazole added in excess is bound to Cu(II) in the entire pH range. It is also concluded that the dimerization in Cu(II)-porphyrins interferes with the apical coordination of basic ligand, or vice versa.     

Demonstration of nitrogen coordination in metal--bleomycin complexes by electron spin--echo envelope spectroscopy

We have studied the Cu(II), Co(II), and Fe(III) complexes of the antineoplastic drug bleomycin by using electron spin--echo envelope spectroscopy. For all three complexes, nitrogen coordination of the metal ions is demonstrated. For the Cu(II)-- and Co(II)--drug complexes, we have been able to identify imidazole as a metal ligand.     

The catalytic center in nitrous oxide reductase, CuZ, is a copper-sulfide cluster

The crystal structure of nitrous oxide reductase, the enzyme catalyzing the final step of bacterial denitrification in which nitrous oxide is reduced to dinitrogen, exhibits a novel catalytic site, called Cu(Z). This comprises a cluster of four copper ions bound by seven histidines and three other ligands modeled in the X-ray structure as OH(-) or H(2)O. However, elemental analyses and resonance Raman spectroscopy of isotopically labeled enzyme conclusively demonstrate that Cu(Z) has one acid-labile sulfur ligand. Thus, nitrous oxide reductase contains the first reported biological copper-sulfide cluster.     

Stable copper-nitrosyl formation by nitrite reductase in either oxidation state

Nitrite reductase (NiR) is an enzyme that uses type 1 and type 2 copper sites to reduce nitrite to nitric oxide during bacterial denitrification. A copper-nitrosyl intermediate is a proposed, yet poorly characterized feature of the NiR catalytic cycle. This intermediate is formally described as Cu(I)-NO+ and is proposed to be formed at the type 2 copper site after nitrite binding and electron transfer from the type 1 copper site. In this study, copper-nitrosyl complexes were formed by prolonged exposure of exogenous NO to crystals of wild-type and two variant forms of NiR from Alcaligenes faecalis (AfNiR), and the structures were determined to 1.8 A or better resolution. Exposing oxidized wild-type crystals to NO results in the reverse reaction and formation of nitrite that remains bound at the active site. In a type 1 copper site mutant (H145A) that is incapable of electron transfer to the type 2 site, the reverse reaction is not observed. Instead, in both oxidized and reduced H145A crystals, NO is observed bound in a side-on manner to the type 2 copper. In AfNiR, Asp98 forms hydrogen bonds to both substrate and product bound to the type 2 Cu. In the D98N variant, NO is bound side-on but is more disordered when observed for the wild-type enzyme. The solution EPR spectra of the crystallographically characterized NiR-NO complexes indicate the presence of an oxidized type 2 copper site and thus are interpreted as resulting from stable copper-nitrosyls and formally assigned as Cu(II)-NO-. A reaction scheme in which a second NO molecule is oxidized to nitrite can account for the formation of a Cu(II)-NO- species after exposure of the oxidized H145A variant to NO gas.