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Biomedical subjects

I Bertini

Publications and source records attributed to I Bertini.

At least 127 records · Page 7Linked to original sources
PubMed1994

X-ray absorption studies on catechol 2,3-dioxygenase from Pseudomonas putida mt2.

X-ray absorption spectroscopy has been utilized to investigate the structure of the active site of iron(II) catechol 2,3-dioxygenase from Pseudomonas putida mt2 both in the native and the 2-chlorophenol inhibited forms. XANES (X-ray absorption near edge structure) and EXAFS (extended X-ray absorption fine structure) results allow us to discuss the coordination number and geometry of the ferrous ion in the native enzyme. The metal geometry is not significantly affected by the binding of the inhibitor. The EXAFS spectrum is consistent with an iron(II) bound to six N/O atoms at an average distance of 2.05 A or to five N/O at an average distance of 2.04 A. The stimulation of the experimental data is greatly enhanced by considering the iron ligands divided in two different shells. Analysis of the outer shells performed using multiple scattering theory shows that there are histidines in the coordination sphere. The best fitting is obtained assuming the presence of two of them. Similar results are obtained for the inhibited enzyme, which, however, are indicative of a slight shortening of the average metal-donor bond distances. The direct binding of inhibitors to the metal center is confirmed by 1H NMR data.

Bacterial Proteins↗
PubMed1994

Substrate, substrate analogue, and inhibitor interactions with the ferrous active site of catechol 2,3-dioxygenase monitored through XAS studies.

The interactions of catechol (substrate), 2-hydroxy-pyridine-N-oxide (substrate analogue), and 2-bromophenol (inhibitor) with the extradiol cleaving catechol-2,3-dioxygenase from Pseudomonas putida mt-2 have been monitored through X-ray absorption spectroscopy (XAS). The analysis of the data provides details about the mode of coordination of the substrate and of the inhibitors to the active site of the enzyme.

Bacterial Proteins↗
PubMed1994

X-ray, NMR and molecular dynamics studies on reduced bovine superoxide dismutase: implications for the mechanism.

Single crystals of the reduced form of Cu, Zn superoxide dismutase (space group P2(1)2(1)2(1), one dimer per asymmetric unit) have been obtained and their X-ray structure refined at 1.9 A resolution. The structure shows that the imidazolate bridge is maintained in the present crystalline form. It is confirmed that in solution the bridge is broken and the involved histidine is protonated on the side of copper. Based on the NOE constraints, and with the aid of molecular dynamics calculations, a structural model is proposed for the molecule in solution. Both structures are considered significant as far as the enzymatic mechanism is concerned.

Animals↗
PubMed1994

Active site coordination chemistry of the cytochrome c peroxidase Asp235Ala variant: spectroscopic and functional characterization.

Asp235 in yeast cytochrome c peroxidase forms a hydrogen bond with His175, the proximal histidyl residue, that has been suggested to be important in determining the electronic properties of the heme iron and that may be involved in stabilizing the higher oxidation states of the peroxidase that form transiently during catalysis. The current study employs 1H and 15N-NMR spectroscopy to study the electronic properties of and the effects of pH on the active site of the Asp235Ala variant. This variant exhibits three spectroscopic species between pH 5 and 9: a high-spin species that forms at low pH and two low-spin species that form successively at higher pH. Nevertheless, the activity of the variant exhibits a pH dependence virtually identical to that of the wild-type protein, though the activity of the variant is 3 orders of magnitude lower at all values of pH between pH 5 and 8.5. These findings suggest that the spin state and coordination environment of the heme iron in cytochrome c peroxidase do not dictate the rate of substrate (cytochrome c) oxidation. Binding of cyanide to the variant enzyme results in formation of a single species as detected by NMR spectroscopy. Analysis of high-resolution 1D and 2D 1H-NMR and 15N-NMR spectra of the cyanide adduct has permitted characterization of the properties of this derivative and the strength of the proximal ligand bond to the heme iron. Disruption of the hydrogen bond between the proximal histidine and Asp235 that exists in the wild-type enzyme dramatically reduces the strength of the interaction between the proximal ligand and the iron; this effect combined with concurrent changes in the distal heme-binding pocket accounts for the increase in reduction potential reported for the Fe3+/Fe2+ couple. The catalytic consequences of the structural and electronic properties of the variant elucidated in this study are discussed.

Alanine↗
PubMed1994

Aliphatic and aromatic inhibitors binding to the active site of catechol 2,3-dioxygenase from Pseudomonas putida mt-2.

The interaction of different classes of inhibitors with the extradiol cleaving catechol 2,3-dioxygenase from Pseudomonas putida mt-2 was monitored by longitudinal and transverse proton relaxation measurements as well as by kinetic activity studies in order to characterize the type of interaction of such inhibitors with the active site of the enzyme. The average distances of the inhibitors from the catalytic iron(II) ion have been estimated from the 1H longitudinal relaxation rates. Phenols and aliphatic ketones appear to be coordinated to the iron(II) ion in the active site.

Alcohols↗
PubMed1994

1H one-dimensional and two-dimensional NMR studies of the ferricytochrome c 551 from Rhodocyclus gelatinosus.

1H two-dimensional NMR spectroscopy has been applied to the oxidized form of cytochrome c 551 from Rhodocyclus gelatinosus, which is paramagnetic with S = 1/2. The investigation has allowed a complete and unambiguous assignment of the heme protons and some residues around the heme. We have learned that: the conformation of the axial methionine is equal to that of horse heart cytochrome c and different from two isoenzymes of the same cytochrome c 551 from a different strain; pKa of 6.6 +/- 0.3 has been detected through the shift variations of seventh propionate protons. The detailed differences with other cytochromes c in the hyperfine shifts are discussed.

Amino Acid Sequence↗
PubMed1994

The enzymatic mechanism of carboxypeptidase: a molecular dynamics study.

An MD simulation of the system carboxypeptidase A (CPA) with the tetrapeptide Val-Leu-Phe-Phe has been performed in order to learn about the substrate disposition just prior to nucleophilic attack. We have explored the model in which the substrate does not substitute the zinc-coordinated water (the "water" mechanism). The simulations do suggest as feasible that the Zn-OH2 group performs a nucleophilic attack on the Phe-Phe peptidic bond. We have also investigated the model in which the carbonyl oxygen displaces the zinc-coordinated water. In this case the substrate and Glu-270 orient themselves to allow an anhydride intermediate during the peptidic bond cleavage (the "anhydride" mechanism). Based on the results of the simulations, both "water" and "anhydride" mechanisms are structurally feasible, although the former model seems more probable on chemical grounds.

Amino Acid Sequence↗
PubMed1994

Strategies of signal assignments in paramagnetic metalloproteins. An NMR investigation of the thiocyanate adduct of the cobalt (II)-substituted human carbonic anhydrase II.

The title protein with MW 30,000 containing high-spin cobalt (II) has been thoroughly investigated through 1H NMR spectroscopy with the aid of selectively deuterated amino acids and 15N enrichment. The aim is that of showing the potentiality of the approach when local information by NMR is needed and the X-ray structure is available. The potential use of the pseudocontact shift is discussed; 90, 200, and 600 MHz spectrometers are used to investigate a spherical region at various distances from the metal ion. More than 35 signals of protons around cobalt (II) have been assigned.

Carbonic Anhydrases↗
PubMed1994

A spectroscopic characterization of a monomeric analog of copper, zinc superoxide dismutase.

A mutated protein of human Cu(II)2Zn(II)2 SOD in which residues Phe50 and Gly51 at the dimer interface were substituted by Glu's, thus producing a monomeric species, has been characterized by electronic absorption spectroscopy, EPR, relaxivity and 1H NMR techniques. Such substitutions and/or accompanying remodeling and exposure of the dimer interface to solvent, alter the geometry of the active site: increases in the axiality of the copper chromophore and the Cu-OH2 distance have been observed. The affinity of both metal binding sites for Co(II) is also altered. The observed NMR parameters of the Co(II) substituted derivative have been interpreted as a function of the decrease of rotational correlation time as a consequence of the lower molecular weight of the mutated protein. Sharper NMR signals are also obtained for the reduced diamagnetic enzyme. Results are consistent with an active site structure similar to that observed for the dimeric analog. Thr137Ile characterized elsewhere. An observed proportional decrease in enzymatic activity and affinity for the N3-anion suggests the importance of electrostatic forces during substrate docking and catalysis.

Circular Dichroism↗
PubMed1993

NMR investigation of isotopically labeled cyanide derivatives of lignin peroxidase and manganese peroxidase.

The 1H NMR spectroscopy was used to study lignin peroxidase (LiP) and manganese peroxidase (MnP) containing deuterated histidines. LiP and MnP were obtained from a histidine auxotroph of the fungus Phanerochaete chrysosporium grown in the presence of deuterated histidines. The derivatives with deuterated histidines have allowed a firm assignment of the protons of the distal and proximal histidines. We have also found that the LiP from this strain exhibits different orientations of the 2-vinyl group compared to the LiP from the strain previously studied. Mobility of the group has also been detected, thus explaining the apparent inconsistency between X-ray solid-state and NMR solution data. The 15N shift values of 15N-enriched CN- in the cyanide derivatives of LiP and MnP have also been measured. The shift patterns, both for 15N and for the proximal histidine protons of several peroxidases, are consistent with predominant contact shift contributions which reflect the bond strength of the metal-axial ligand. Finally, our results confirm a correlation between shift values of 15N and those of proximal histidine protons and the Fe3+/Fe2+ redox potentials.

Basidiomycota↗
PubMed1993

Electron self-exchange in high-potential iron-sulfur proteins. Characterization of protein I from Ectothiorhodospira vacuolata.

During previous research on oxidized and reduced high-potential iron-sulfur proteins (HiPIP hereafter), qualitative different electron self-exchange rates were noticed. We have now investigated this phenomenon in detail for HiPIP I and II from Ectothiorhodospira vacuolata, which differ significantly in total charge and in which the sequence homology is the largest among all known HiPIPs. We have also characterized the electronic structure of HiPIP I through 1H NMR and EPR spectroscopies to parallel the existing characterization of HiPIP II and other HiPIPs. This investigation has allowed us to propose a model, according to which the productive collisions for electron transfer occur through a hydrophobic patch near the cluster. The effects of total charge and redox potential are considered. The possible formation of dimers through the hydrophobic patch at liquid helium temperature is discussed in light of the EPR spectra.

Amino Acid Sequence↗
PubMed1993

Selective interaction of ferricyanide with cluster I of Clostridium pasteurianum 2[Fe4S4] ferredoxin.

Treatment of Clostridium pasteurianum ferredoxin (CpFd) with stoichiometric amounts of potassium ferricyanide results in the specific conversion of cluster I into a Fe3S4+ species while leaving cluster II unaltered. Ferricyanide-treated CpFd derivative has been purified and characterized through biochemical and spectroscopical techniques. The cluster conversion process is reversible and reconstitution of native CpFd has been afforded under appropriate conditions.

Amino Acid Sequence↗
PubMed1993

The electronic structure of [Fe4S4]3+ clusters in proteins. An investigation of the oxidized high-potential iron-sulfur protein II from Ectothiorhodospira vacuolata.

Within the framework of an investigation of the electronic structure of oxidized high-potential iron-sulfur proteins (HiPIP), we have studied the HiPIP II from Ectothiorhodospira vacuolata, which was known to have a peculiar temperature dependence of the 1H NMR isotropic hyperfine shifts. The signals of the cysteine ligand protons have been sequence specifically assigned through NOE, NOESY, and TOCSY experiments. Nine hyperfine-shifted signals are observed: seven in the downfield and two in the upfield region. They have been assigned to the eight beta-CH2 protons of the four coordinated cysteines and to one alpha-CH cysteine proton. The two most downfield-shifted signals belong to the beta-CH2 protons of Cys 63 (Chromatium vinosum numbering) and the two upfield protons to those of Cys 43. These two pairs of protons show a Curie-type temperature dependence of the hyperfine shifts. Among the remaining five downfield-shifted signals, three show a Curie-type temperature dependence and two have an anti-Curie temperature dependence. The former are assigned to the beta-CH2 and alpha-CH protons of Cys 77 and the latter to the beta-CH2 protons of Cys 46. The shift patterns are thus similar, in a sequence-specific sense, to those of the analogous proteins from C. vinosum and Rhodocyclus gelatinosus, whereas they differ from those of Rhodocyclus globiformis HiPIP and even more from those of Ectothiorhodospira halophila HiPIP II. Oxidized HiPIPs can be formally viewed as containing a cluster of four ferric ions plus one extra electron. We present here a model based on a chemical equilibrium, fast on the NMR time scale, between two species, both of which contain a pair of iron(III) ions and a mixed-valence pair but are differently oriented within the protein frame. The EPR data are also discussed in the light of the debate on the nature of the different species detected at low temperature. The interpretation of the whole set of data on HiPIPs in the light of the present model is compared with that based on previous models.

Bacteria↗
PubMed1993

1H-NMR study of reduced heme proteins myoglobin and cytochrome P450.

The 1H-NMR spectra of deoxymyoglobin and reduced cytochrome P450 are analyzed by NOE spectroscopy. Progress has been made in the assignment of the hyperfine-shifted signals of deoxymyoglobin. The nuclear longitudinal-relaxation-time values indicate short electron-relaxation times whereas Curie relaxation contributes significantly to the signals linewidths. For reduced cytochrome P450 the linewidths are larger due to the Curie-relaxation contribution in a large protein. Therefore, the spectral information is poor. The electron-relaxation rates are discussed in terms of possible electronic structure.

Animals↗
PubMed1993

Binding of horseradish, lignin, and manganese peroxidases to their respective substrates.

The present study utilizes 1H NMR spectroscopy to characterize the binding of substrate to heme active site of three different peroxidases, horseradish peroxidase, lignin peroxidase, and manganese peroxidase. Information has been obtained on the site of p-cresol binding to the active-site cavity of the cyanide derivative of horseradish peroxidase. This information was obtained by relaxation enhancements of the substrate protons and connectivities between the latter and heme 8-CH3 and a Phe residue. Manganese(II) is shown to bind to ferri-manganese peroxidase and its cyanide derivative in a specific site with a high-affinity constant (10(4) M-1). Manganese(II) binding exhibits a slow exchange rate with respect to the difference in T2(-)1 of the affected signals in the manganese(II)-containing and manganese(II)-free species. Manganese(II) affects the line width of certain heme methyl resonances and of certain one-proton intensity signals in manganese peroxidase and its cyanide derivative. The behavior of MnP toward manganese(II) is compared to that of the closely related peroxidase, lignin peroxidase (LiP), with its native substrate veratryl alcohol. LiP does not have a specific binding site for manganese(II).

Benzyl Alcohols↗
PubMed1993

Investigation of a new Cu,Zn superoxide dismutase mutant: the Thr-->Arg 137 derivative.

The preparation and biophysical characterization of a mutant of superoxide dismutase in which the native Thr 137 has been substituted with a positive Arg residue are reported. Thr 137 forms, together with Arg 143, a bottleneck at the entrance to the active-site Cu ion. The geometry of the Cu ligands shows only minor changes after the above substitution. However, the enzymatic activity of the Arg 137 mutant is smaller than that of the wild type at physiological ionic strength and approaches that of wild type in the limit of zero ionic strength. The binding constant of the anion N3-, which had previously been shown to be a good probe of the O2- substrate, is increased about 20-fold in the mutant with respect to the value found in the wild type. These results are discussed on the bases of the whole charge of the cavity and the possible change in the conformation of the active-site channel.

Amino Acid Sequence↗
PubMed1993

1H-NMR investigation of oxidized and reduced high-potential iron-sulfur protein from Rhodopseudomonas globiformis.

1H one-dimensional and two-dimensional NMR spectra have been recorded for the oxidized and reduced forms of the high-potential iron-sulfur protein (HiPIP) from Rhodopseudomonas globiformis which has the highest known reduction potential. The spectrum of the oxidized protein is similar to that of Chromatium vinosum and Rhodocyclus gelatinosus HiPIP but different from that of the HiPIP II from Ectothiorhodospira halophila. Surprisingly, site-specific assignment has shown that in the oxidized protein the distribution of oxidation numbers within the cluster is very similar to that found for E. halophila HiPIP II and different from that of the other two proteins. The spectrum of the reduced species is very similar to that of all other HiPIPs known to date, indicating very similar electronic and geometric structures for the reduced forms. These findings are discussed in terms of cluster structure in HiPIPs and of redox potentials.

Amino Acid Sequence↗