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E I Solomon

Publications and source records attributed to E I Solomon.

At least 37 records · Page 2Linked to original sources
PubMed1999

Investigation of the anomalous spectroscopic features of the copper sites in chicken ceruloplasmin: comparison to human ceruloplasmin.

Chicken ceruloplasmin has been previously reported to display a number of key differences relative to human ceruloplasmin: a lower copper content and a lack of a type 2 copper signal by electron paramagnetic resonance (EPR) spectroscopy. We have studied the copper sites of chicken ceruloplasmin in order to probe the origin of these differences, focusing on two forms of the enzyme: "resting" (as isolated by a fast, one-step procedure) and "peroxide-oxidized". From X-ray absorption, EPR, and UV/visible absorption spectroscopies, we have shown that all of the copper sites are oxidized in peroxide-oxidized chicken ceruloplasmin and that none of the type 1 copper sites display the EPR features typical for type 1 copper sites that lack an axial methionine. In the resting form, the type 2 copper center is reduced. Upon oxidation, it does not appear in the EPR spectrum at 77 K, but it can be observed by using magnetic susceptibility, EPR at approximately 8 K, and magnetic circular dichroism spectroscopy. It displays unusually fast relaxation, indicative of coupling with the adjacent type 3 copper pair of the trinuclear copper cluster. From reductive titrations, we have found that the reduction potential of the type 2 center is higher than those of the other copper sites, thus explaining why it is reduced in the resting form. These results provide new insight into the nature of the additional type 1 copper sites and the redox distribution among copper sites in the different ceruloplasmins relative to other multicopper oxidases.

Animals↗
PubMed1999

Targeted mutations in a Trametes villosa laccase. Axial perturbations of the T1 copper.

Trametes villosa laccase was mutated on a tetrapeptide segment near the type 1 site. The mutations F463M and F463L were at the position corresponding to the type 1 copper axial methionine (M517) ligand in Zucchini ascorbate oxidase. The mutations E460S and A461E were near the T1 copper site. The mutated Trametes laccases were expressed in an Aspergillus oryzae host and characterized. The E460S mutation failed to produce a transformant with meaningful expression. The F463L and A461E mutations did not significantly alter the molecular and enzymological properties of the laccase. In contrast, the F463M mutation resulted in a type 1 copper site with an EPR signal intermediate between that of the wild type laccase and plastocyanin, an altered UV-visible spectrum, and a decreased redox potential (by 0.1 V). In oxidizing phenolic substrate, the mutation led to a more basic optimal pH as well as an increase in kcat and Km. These effects are attributed to a significant perturbation of the T1 copper center caused by the coordination of the axial methionine (M463) ligand.

Base Sequence↗
PubMed1998

Site-directed mutations in fungal laccase: effect on redox potential, activity and pH profile.

A Myceliophthora thermophila laccase and a Rhizoctonia solani laccase were mutated on a pentapeptide segment believed to be near the type-1 Cu site. The mutation L513F in Myceliophthora laccase and the mutation L470F in Rhizoctonia laccase took place at a position corresponding to the type-1 Cu axial methionine (M517) ligand in Zucchini ascorbate oxidase. The triple mutations V509L,S510E,G511A in Myceliophthora laccase and L466V,E467S,A468G in Rhizoctonia laccase involved a sequence segment whose homologue in ascorbate oxidase is flanked by the M517 and a type-1 Cu-ligating histidine (H512). The single mutation did not yield significant changes in the enzymic properties (including any significant increase in the redox potential of the type-1 Cu). In contrast, the triple mutation resulted in several significant changes. In comparison with the wild type, the Rhizoctonia and Myceliophthora laccase triple mutants had a phenol-oxidase activity whose pH optimum shifted 1 unit lower and higher, respectively. Although the redox potentials were not significantly altered, the Km, kcat and fluoride inhibition of the laccases were greatly changed by the mutations. The observed effects are interpreted as possible mutation-induced structural perturbations on the molecular recognition between the reducing substrate and laccase and on the electron transfer from the substrate to the type-1 Cu centre.

Amino Acid Sequence↗
PubMed1998

Spectroscopic and magnetic studies of human ceruloplasmin: identification of a redox-inactive reduced Type 1 copper site.

Ceruloplasmin is unique among the multicopper oxidases in that in addition to the usual copper stoichiometry of one Type 1 copper site and a Type 2/Type 3 trinuclear copper cluster, it contains two other Type 1 sites. This assignment of copper sites, based on copper quantitation, sequence alignment, and crystallography, is difficult to reconcile with the observed spectroscopy. Furthermore, some chemical or spectroscopic differences in ceruloplasmin have been reported depending on the method of purification. We have studied the resting (as isolated by a fast, one-step procedure) and peroxide-oxidized forms of human ceruloplasmin. Using a combination of X-ray absorption spectroscopy, a chemical assay, magnetic susceptibility, electron paramagnetic resonance spectroscopy, and absorption spectroscopy, we have determined that peroxide-oxidized ceruloplasmin contains one permanently reduced Type 1 site. This site is shown to have a reduction potential of approximately 1.0 V. Thus, one of the additional Type 1 sites in ceruloplasmin cannot be catalytically relevant in the form of the enzyme studied. Furthermore, the resting form of the enzyme contains an additional reducing equivalent, which is distributed among the remaining five copper sites as expected from their relative potentials. This may indicate that the resting form of ceruloplasmin in plasma under aerobic conditions is a four-electron oxidized form, which is consistent with its function in the four-electron reduction of dioxygen to water.

Binding Sites↗
PubMed1996

A trinuclear intermediate in the copper-mediated reduction of O2: four electrons from three coppers.

The reaction of metal complexes with dioxygen (O2) generally proceeds in 1:1, 21, or 41 (metal:O2) stoichiometry. A discrete, structurally characterized 31 product is presented. This mixed-valence trinuclear copper cluster, which contains copper in the highly oxidized trivalent oxidation state, exhibits O2 bond scission and intriguing structural, spectroscopic, and redox properties. The relevance of this synthetic complex to the reduction of O2 at the trinuclear active sites of multicopper oxidases is discussed.

Copper↗
PubMed1996

A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox potential, substrate specificity, and stability.

A series of fungal laccases (Polyporus pinsitus, Rhizoctonia solani, Myceliophthora thermophila, Scytalidium thermophilum) and one bilirubin oxidase (Myrothecium verrucaria) have been studied to determine their redox potential, specificity, and stability. Polyporus and Rhizoctonia laccases possess potentials near 0.7-0.8 V (vs. NHE), while other oxidases have potentials near 0.5 V. It is observed that higher redox potential correlates with higher activity. By EPR, no significant change in the geometry of type 1 copper (II) site is observed over this series. At the optimal pH, the two substrates studied, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine, show Km values ranging form 10 to 120 and from 1 to 45 microM; and kcat values ranging from 50 to 16 000 and 200 to 3000 per min, respectively. The enzymes are more stable in the neutral-alkaline pH range. The thermal stability is in the order of bilirubin oxidase equivalent to Myceliophthora laccase equivalent to Scytalidium laccase > Polyporus laccase > Rhizoctonia laccase. Based on these results and the sequence alignments made against Zucchini ascorbate oxidase it is speculated that structural differences in the substrate-activation site (a 'blue', type 1 copper center) control the redox potential range as well as substrate specificity, and the cystine content contributes to stability.

Electron Spin Resonance Spectroscopy↗
PubMed1994

Magnetic circular dichroism studies of exogenous ligand and substrate binding to the non-heme ferrous active site in phthalate dioxygenase.

BACKGROUND: Mononuclear non-heme iron centers are found in the active sites of a variety of enzymes that require molecular oxygen for catalysis. The mononuclear non-heme iron is believed to be the active site for catalysis, and is presumed to bind and activate molecular oxygen. The mechanism of this reaction is not understood. Phthalate dioxygenase is one such enzyme. Because it also contains a second iron site, the Rieske site, it is difficult to obtain information on the structure of the active site. We therefore used magnetic circular dichroism (MCD) spectroscopy to probe the mononuclear, non-heme Fe2+ site in this biodegradative enzyme. RESULTS: The MCD spectrum of the resting enzyme shows features indicative of one six-coordinate Fe2+ site; substrate binding converts the site to two different five-coordinate species, opening up a coordination position for O2 binding. MCD spectra of the corresponding apoenzyme have been subtracted to account for temperature-independent contributions from the Rieske site. Azide binds both to the resting enzyme to produce a new six-coordinate species, showing that one of the ferrous ligands is exchangeable, and also to the enzyme-substrate complex to form a ternary species. The low azide binding constant for the substrate-enzyme species relative to the resting enzyme indicates steric interaction and close proximity between exogenous ligand and the substrate. CONCLUSIONS: We have been able to provide some detailed structural insight into exogenous ligand and substrate binding to the non-heme Fe2+ site, even in the presence of the enzyme's [2Fe-2S] Rieske center. Further mechanistic studies are now required to maximize the molecular-level detail available from these spectroscopic studies.

Binding Sites↗
PubMed1993

Electronic structure contributions to function in bioinorganic chemistry.

Many metalloenzymes exhibit distinctive spectral features that are now becoming well understood. These reflect active site electronic structures that can make significant contributions to catalysis. Copper proteins provide well-characterized examples in which the unusual electronic structures of their active sites contribute to rapid, long-range electron transfer reactivity, oxygen binding and activation, and the multielectron reduction of dioxygen to water.

Amino Acid Sequence↗
PubMed1993

Electronic absorption spectroscopy of copper proteins.

We have seen from the previous discussion that absorption spectral studies in the ligand field region probe the energy splittings of the d orbitals and that this relates to the geometry of the metal center. The energies and intensities of ligand-to-metal charge transfer transitions sensitively probe bonding interactions of the ligand with the metal center. Charge transfer transitions can be used both qualitatively to observe ligand binding to a metal center, owing to the requirement of orbital overlap for significant charge transfer intensity, and quantitatively to define the electron donor ability of that ligand and experimentally evaluate the results of electronic structure calculations. Studies of the intensities of peaks at the ligand K edge can define the covalent interaction of the ligand with the metal valence orbitals, whereas copper K-edge spectroscopy is a powerful probe of metal ion oxidation state and the ligand field geometry of d10 cuprous sites that are inaccessible through other spectroscopic methods. Absorption spectral studies in all regions are strongly complemented by CD, variable temperature MCD, and single-crystal polarized absorption spectroscopies, which should also be pursued whenever possible to obtain detailed electronic structural insight of relevance to catalysis.

Animals↗
PubMed1987

Electron paramagnetic resonance studies of the tungsten-containing formate dehydrogenase from Clostridium thermoaceticum.

The redox centers in the tungsten-containing formate dehydrogenase from Clostridium thermoaceticum were examined by potentiometric titration and electron paramagnetic resonance spectroscopy. At low temperature two overlapping iron-sulfur signals which correlated with enzymatic activity were observed with formal potentials near -400 mV vs. SHE. Based on their temperature dependences, one signal is assigned to a reduced Fe2S2 cluster and one to a reduced Fe4S4 cluster. Quantitation of signal intensity suggests two Fe2S2 and two Fe4S4 clusters per formate dehydrogenase molecule. Another signal (g = 2.101, 1.980, 1.950) present in low concentrations at more negative potentials was observable up to 200 degrees K and is not attributed to any iron-sulfur cluster. The possible origin of this signal is analyzed using ligand field theory, and the redox behavior is considered with respect to possible ligation at the active site.

Aldehyde Oxidoreductases↗
PubMed1987

Allosteric interactions in sipunculid and brachiopod hemerythrins.

Chemical and spectroscopic consequences of allosteric interactions for ligand binding to sipunculid (Phascolopsis gouldii) and brachiopod (Lingula reevii) hemerythrins (Hrs) have been investigated. Possible allosteric effectors for homotropic effects in sipunculid Hrs have been examined, but only reduction in ligand affinity is observed without cooperativity. In contrast to sipunculid Hr, L. reevii Hr binds O2 cooperatively in the pH range 7-8 and exhibits a Bohr effect. Spectroscopic comparisons of the sipunculid and brachiopod Hrs show no significant differences in the active site structures; therefore, modulation of oxygen affinity is attributable to effects linking the site to quaternary structural changes in the octamer. Oxygen equilibria can be fit with a conformational model incorporating a minimum of three states, tensed (T), relaxed (R), and an R-T hybrid. Resonance Raman spectra of L. reevii oxyHr show a shift in the peroxo stretching frequency when the pH is lowered from pH 7.7 (predominantly R oxyHr) to pH 6.3 (a mixture of R, T, and R-T hybrid), but P. gouldii Hr does not have a frequency shift under the same conditions. In contrast to hemoglobins, ligand binding to the deoxy and met forms is noncooperative for brachiopod (and sipunculid) Hrs. It is thus suggested that conformational changes in the protein are linked to the oxidation state change that accompanies oxygenation of the coupled binuclear iron site (deoxy [FeIIFeII]----oxy [FeIIIFeIII]). The total allosteric energy expended in oxygenation is about 1.4 kcal/mol, and such a shift is possible in the relaxed-tense conversion with relatively limited constraints of the iron coordination environment via the protein quaternary structure. The mechanism of cooperativity in the binuclear copper oxygen carrier hemocyanin is discussed in light of these results.

Allosteric Regulation↗
PubMed1985

Low-temperature magnetic circular dichroism studies of native laccase: spectroscopic evidence for exogenous ligand bridging at a trinuclear copper active site.

The detailed nature of N-3 binding at the multi-copper active site in native laccase is investigated through a combination of low-temperature magnetic circular dichroism (LTMCD) and absorption spectroscopies. This combination of techniques allows charge-transfer spectral features associated with N-3 binding to the paramagnetic type 2 Cu(II) to be differentiated from those associated with binding to the antiferromagnetically coupled, and therefore diamagnetic, binuclear type 3 Cu(II) site. Earlier absorption titration studies have indicated that N-3 binds with two different binding constants, yielding a high-affinity and a low-affinity form. The studies presented here are interpreted as strong evidence that low-affinity N-3 bridges the paramagnetic type 2 and diamagnetic type 3 binuclear Cu(II) sites in fully oxidized laccase. This assignment is further supported by features in the MCD spectrum whose intensity correlates with an EPR signal associated with uncoupled type 3 Cu(II) sites. In these sites, N-3 has displaced the endogenous bridge, thereby rendering the site paramagnetic and detectable by both LTMCD and EPR spectroscopy. High-affinity N-3 is found to bind to the paramagnetic type 2 Cu(II) site in a limited fraction of the protein molecules that contains reduced type 3 sites. Finally, the possible role of this trinuclear (type 2-type 3) Cu(II) active site in enabling the irreversible reduction of dioxygen to water is considered.

Azides↗
PubMed1984

Extended X-ray absorption fine structure study of the coupled binuclear copper active site of tyrosinase from Neurospora crassa.

Cu K-edge X-ray absorption spectra have been recorded for the enzyme tyrosinase from Neurospora crassa, in its oxy, resting (met-aquo), and inhibitor-bound (met-mimosine) forms. The K-edges proper resemble those of oxy- and met-hemocyanin, and confirm the presence of CuII. The forbidden 1s----3d transition is noticeably stronger for the 1-mimosine-bound enzyme, implying some distortion of the tetragonal Cu coordination group on inhibitor binding. The extended fine structure (EXAFS) beyond the K-edge has been analyzed. The first shell scattering is consistent with the presence of two N- and two O-ligand atoms, at 2.0 and 1.9 A, for all three forms of the enzyme; there is no evidence for heavy atom (S) scattering in the first shell. As in analogous hemocyanin derivatives, the outer shell scattering contains contributions from distant atoms of imidazole ligands, as well as from an addition scattering atom, at 3.4-3.6 A. For oxy-tyrosinase the additional scatterer is unambiguously a heavy atom (Cu), although a larger Debye-Waller factor suggests a somewhat less rigid binuclear site than in oxy-hemocyanin.

Binding Sites↗
PubMed1984

Activation of nit-1 nitrate reductase by W-formate dehydrogenase.

Formate dehydrogenase ( FDH ) from Clostridium thermoaceticum is a known tungsten enzyme. FDH was tested for the presence of nitrogenase-type cofactor and nitrate reductase-type cofactor by the Azotobacter vinelandii UW-45 and Neurospora crassa nit-1 reconstitution assays, respectively. Tungsten formate dehydrogenase (W- FDH ), containing only a small Mo impurity, activated the nit-1 nitrate reductase extracts when molybdate was also added, but not when tungstate was added. These results show W- FDH contains the cofactor common to all known Mo-enzymes except nitrogenase. The difference between the redox chemistries of W- FDH and W-substituted sulfite oxidase appears to relate to differences in tungsten ligation other than that donated by the cofactor or to variations in the protein environment surrounding the tungsten active site.

Aldehyde Oxidoreductases↗
PubMed1984

On the spectral features associated with peroxide reactivity of the coupled binuclear copper active site in type 2 depleted and native Rhus laccase.

We report herein an X-ray absorption spectroscopic (XAS) determination of the oxidation state of the copper sites in T2D and native Rhus laccase. The increase in intensity of the 330 nm absorption feature which results from peroxide titration of T2D laccase (T3: [Cu(I)Cu(I)], T1: [Cu(II)]) is found to correlate linearly with the percent of oxidation of the binuclear copper site (determined by XAS analysis). This indicates that peroxide oxidizes but does not bind to the T3 site. We have used this correlation to determine that native laccase, as isolated, contains approximately 25% reduced T3 sites and that all spectral changes observed upon peroxide addition to native laccase can be accounted for by oxidation of these reduced sites. The importance of this result to previous reports of peroxide binding at the laccase active site is discussed.

Binding Sites↗