Electron spin resonance and electron nuclear double resonance studies of cation radicals derived from tocopherol model compounds

Electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) measurements were performed for the cation radicals obtained from the model compounds of α-, β-, γ- and δ-tocopherol (vitamin E) by oxidizing the tocopherol precursors in an AlCl₃-CH₂Cl₂ solution. The proton hyperfine coupling constants g-values were precisely determined. The ENDOR spectra of the cation radicals of α-, β-, γ- and δ-tocopherol models in CH₂Cl₂ at ?100°C clearly show 10, 6, 6 and 12 different proton hyperfine couplings, respectively. By varying the temperature, the ESR spectra of the α- and δ-tocopherol model cations exhibit line-width alternation phenomena characteristic of the hindered rotation of the OH group. However, neither the β- nor the γ-tocopherol model cation radical ESR spectra show any sign of an alternative line-width effect. These results are interpreted by assuming that the β- and γ-tocopherol model cations are stabilized in the trans and cis conformations, respectively. On tocopherol model cations are stabilized in the trans and cis conformations, respectively. On the other hand, both the α- and δ-tocopherol model cations exist as cis and trans isomers.     

Low temperature electron paramagnetic resonance studies on iron-sulfur centers in cardiac NADH dehydrogenase

EPR signals arising from at least seven iron-sulfur centers were resolved in both reconstitutively active and inactive NADH dehydrogenases, as well as in particulate NADH-UQ reductase (Complex I). EPR lineshapes of individual iron-sulfur centers in the active dehydrogenase are almost unchanged from that in Complex I. Iron-sulfur centers in the inactive dehydrogenase give broadened EPR spectra, suggesting that modification of iron-sulfur active centers is associated with loss of the reconstitutive activity of the dehydrogenase. With the reconstitutively active dehydrogenase, the E_m8.0 value of Center N-2 (iron-sulfur centers associated with NADH dehydrogenase are designated with prefix N) was shifted to a more negative value than in Complex I and restored to the original value on reconstitution of the enzyme with purified phospholipids.     

Low temperature electron paramagnetic resonance studies on two iron-sulfur centers in cardiac succinate dehydrogenase

At temperatures below 20°K, EPR signals from a new iron-sulfur center (designated here as Center S-2 or (Fe-S)_S-2) in addition to the classical “g = 1.94 signal” (designated as Center S-1 or (Fe-S)_S-1) were detected in purified, soluble succinate dehydrogenase, particulate succinate ubiquinone reductase (Complex II) and particulate succinate cytochrome $\text{c}$ reductase from bovine heart. The measured half-reduction potential (E_m7.4) of Center S-1 was 0 ± 10 mV, while E_m7.4 of Center S-2 was ?260 ± 15 mV in the membrane bound preparations. Upon solubilization of succinate dehydrogenase, the EPR behavior of Center S-2 became extremely labile similar to the characteristics of the reconstitutive activity of succinate dehydrogenase toward the rest of the respiratory chain.     

Carbon-13 nuclear magnetic resonance studies on tobacco mosaic virus and its protein.

Fourier transform nuclear magnetic resonance studies on 12% 13C-enriched tobacco mosaic virus (TMV) and its rod-like protein oligomers in solution with molecular weights up to 42 X 10(6) are reported. In the virus approximately 17% of the carbons of the protein subunit have line widths of less than or equal to 300 Hz and T1 less than or equal to 1 s and are concluded to be mobile with more than one degree of freedom of internal rotation about a carbon--carbon bond. In the rodlike polymer of TMV protein at pH 5.3, 30% of the carbons are mobile, which implies rotational motions about carbon--carbon bonds and/or motions of the protein subunits within the polymer. The presence of internal mobility is supported by the observation that 20% of the carbons in the double disklike oligomer show decreasing line width upon increasing temperature; the remaining resonances have line widths which are temperature independent during the double disklike polymerization process. Since the molecular weight of TMV protein polymers increases with increasing temperature, this demonstrates that all nuclei within the double dislike oligomer are mobile. NMR and X-ray data on the double disklike polymer reveal differences with respect to internal mobility.     

Statine and its derivatives. Conformational studies using nuclear magnetic resonance spectrometry and energy calculations

The conformations of derivatives of 3(S)-hydroxy-4(s)-amino-6-methylheptanoic acid (statine) and its analogs have been studied by n.m.r. in chloroform and in dimethyl sulfoxide, and by molecular mechanics calculations. The data obtained from these studies indicate that: 1) the coupling constant between NH and C4H is large, suggesting that the dihedral angle (theta) is near 165 degrees or 0 degree; 2) the coupling constant between C4H-C3H is small, indicating a vicinal bond angle of approximately 90 degrees; 3) the hydrogen deuterium exchange rate of statine amide protons is slow; however, the rate is dependent upon the electron withdrawing substituents adjacent to the amide NH's; 4) intramolecular hydrogen bonds involving the NH of the statine amide group do not stabilize conformations of single amino acid derivatives. Based on the n.m.r. results, four possible conformations of Boc-statine-OMe in solution are possible. MM1 calculations indicate one conformation is especially likely.     

Characterization by electron paramagnetic resonance and studies on subunit location and assembly of the iron-sulfur clusters of Bacillus subtilis succinate dehydrogenase

Succinate dehydrogenase is a conserved membrane-bound enzyme consisting of two nonidentical subunits: a flavo iron-sulfur protein (Fp) subunit, containing a covalently bound flavin, and an iron-sulfur protein (Ip) subunit. Bacillus subtilis succinate dehydrogenase in wild type bacteria and 12 well characterized succinate dehydrogenase-defective mutants were examined by low temperature EPR spectroscopy to characterize the enzyme and study subunit location and biosynthesis of its iron-sulfur clusters. The wild type B. subtilis enzyme contains iron-sulfur clusters which are analogous to clusters S-1 and S-3 of bovine heart succinate dehydrogenase but with slightly different EPR characteristics. Spins from cluster S-2 were not detectable as in the case of the intact form of bovine heart succinate dehydrogenase. However, dithionite reduction of the B. subtilis enzyme greatly enhanced spin relaxation of the ferredoxin-type cluster S-1, indicating the presence of the cluster S-2. Iron-sulfur cluster S-1 was found to be assembled in soluble succinate dehydrogenase subunits in the cytoplasm, but only if full-length Fp polypeptides and relatively large fragments of Ip polypeptides were present. Cluster S-1 was not detected in mutants with soluble mutated Fp polypeptides or in a mutant totally lacking Ip subunit polypeptide. Iron-sulfur clusters S-1, S-2, and S-3 were assembled also when the covalently bound flavin in the Fp subunit was absent. Clusters S-1 and S-3 in the membrane-bound flavin-deficient succinate dehydrogenase were not reduced by succinate but could be reduced by electron transfer from NADH dehydrogenase via the menaquinone pool.     

Effect of substrate on the diiron(III) site in stearoyl acyl carrier protein delta 9-desaturase as disclosed by cryoreduction electron paramagnetic resonance/electron nuclear double resonance spectroscopy

The diiron center in stearoyl-acyl carrier protein (ACP) desaturase (DS) from castor plant Ricinus communis catalyzes the dioxygen- and NADPH-dependent introduction of a cis double bond between C9 and C10 of stearoyl-ACP. Radiolytic reduction of diferric DS at 77 K produces an electron paramagnetic resonance (EPR)-detectable mixed-valence center (or [DS(ox)](mv)) that is trapped in the conformation of the diferric precursor and thus provides a sensitive EPR/electron nuclear double resonance (ENDOR) probe of the structure of the diamagnetic diiron(III) state. The cryoreduced DS shows two distinct EPR signals, suggesting the presence of two diiron(III) states: the mu-oxo (major)- and mu-hydroxo (minor)-bridged diiron centers. ENDOR studies show that in the dominant oxo-bridged diferric state each iron(III) coordinates a histidine and a water along with other ligands. Samples containing stoichiometric amounts of stearoyl-ACP show pronounced changes in the EPR and (1)H ENDOR spectra of cryoreduced DS. EPR spectra of the cryoreduced DS-substrate complex reveal two distinct substates of the parent. EPR and ENDOR studies show that both major conformers of the diferric cluster have a mu-oxo bridge. ENDOR shows that the major conformer has a histidine and a water bound to both Fe ions. In the minor conformer, one of the irons has lost the terminal water ligand. The structure of the trapped [DS(ox)](mv) state relaxes upon annealing to 170 K: the mu-oxo bridge in the major cryoreduced DS species protonates on annealing to 170 K; this does not occur for the major DS-substrate complex, even upon annealing to 230 K. The relaxed Fe(II)Fe(III) center in cryoreduced DS and DS-substrate show much less intense and resolved (14)N ENDOR spectra than those of the structurally similar cryoreduced diiron center in ribonucleotide reductase (RNR) protein R2. This difference may reflect some differences in His-Fe bonds. The alterations in the diferric site of DS induced by substrate are suggested to be mediated by conformational changes in the polypeptide chain produced by substrate binding. These structural alterations may provide DS with an additional mechanism for tuning the redox potential of the diferric site. The mixed-valence states of DS are unstable at temperatures above 230 K.     

Classification of iron-sulfur cores in ferredoxins by 1H nuclear magnetic resonance spectroscopy

A 1H nuclear magnetic resonance (NMR) study was carried out on various ferredoxins which possess one of three types of iron-sulfur clusters, (2Fe-2S), (3Fe-3S), or (4Fe-4S). In the isolated form, (2Fe-2S) ferredoxins from spinach (Spinacea oleracia), pokeweed (Phytolacca americana), a blue-green alga (Spirulina platensis), and a halobacterium (Halobacterium halobium) exhibited two broad resonances common in chemical shift at the region downfield of 10 ppm. In their reduced forms, seven contact-shifted resonances appeared spread over 30 ppm. Although the positions of the contact-shifted resonances in the reduced state differed among the four, a common trend in the temperature dependence of their resonance positions was recognized. Two (4Fe-4S) ferredoxins from Bacillus stearothermophilus and Bacillus thermoproteolyticus exhibited almost indistinguishable spectral patterns in both the oxidized and reduced forms. The ferricyanide-treated ferredoxins of B. stearothermophilus and B. thermoproteolyticus showed characteristic contact-shifted resonances distinct from the spectra of the original (4Fe-4S) ferredoxins. This corresponds to the recent finding of the interconversion of (4Fe-4S) and (3Fe-3S) clusters with ferricyanide in the ferredoxin. Based on our data together with reported NMR data on other ferredoxins, contact-shift resonances of three types of clusters were tabulated. The reliability of NMR classification increases when we compare the NMR spectra of a ferredoxin with the classification standards at the two redox states. Moreover, not only the absolute values of the chemical shifts of contact-shifted resonances but also their temperature dependence give distinctive information applicable to iron core identification.     

Carbon-13 and proton nuclear magnetic resonance studies on methyl aldofuranosides and their O-alkyl derivatives

The effect of O-alkylation on the carbon-13 n.m.r. spectra of methyl pentofuranosides has been determined. O-Alkylation of an OH group displaced the signal of the appended.¹³C nucleus downfield, whereas the adjacent ¹³C nuclei were, in most instances, shifted upfield to a smaller extent. The effect of O-methylation was appreciably larger than O-isopropylation or O-glycosylation, but either O-methyl or O-isopropyl derivatives may be used as models for interpreting the spectra of furanoid oligo- and poly-saccharides, including the galactomannan of Penicillium charlesii. The signal displacements are, to a large extent, comparable to those observed in the conformationally more stable mannopyranose series, so that they are insensitive to effects of steric distortion and population (or both). These effects occurred on 3-O-alkylation of methyl pentofuranosides, as appreciable changes in J_1,2 values in their p.m.r. spectra were observed.     

Protein-tyrosyl radical interactions in photosystem II studied by electron spin resonance and electron nuclear double resonance spectroscopy: comparison with ribonucleotide reductase and in vitro tyrosine.

The stable tyrosine radical in photosystem II, YD*, has been studied by ESR and ENDOR spectroscopies to obtain proton hyperfine coupling constants from which the electron spin density distribution can be deduced. Simulations of six previously published ESR spectra of PSII (one at Q band; five at X band, of which two were after specific deuteration and two others were of oriented membranes) can be achieved by using a single set of magnetic parameters that includes anisotropic proton hyperfine tensors, an anisotropic g tensor, and noncoincident axis systems for the g and A tensors. From the spectral simulation of the oriented samples, the orientation of the phenol head group of YD* with respect to the membrane plane has been determined. A similar orientation for YZ*, the redox-active tyrosine in PSII that mediates electron transfer between P680 and the oxygen-evolving complex, is expected. ENDOR spectra of YD* in PSII preparations from spinach and Synechocystis support the set of hyperfine coupling constants but indicate that small differences between the two species exist. Comparison with the results of spectral simulations for tyrosyl radicals in ribonucleotide reductase from prokaryotes or eukaryotes and with in vitro radicals indicates that the spin density distribution remains that of an odd-alternant radical but that interactions with the protein can shift spin density within this basic pattern. The largest changes in spin density occur at the tyrosine phenol oxygen and at the ring carbon para to the oxygen, which indicates that mechanisms exist in the protein environment for fine-tuning the chemical and redox properties of the radical species.     

Characterization of iron-sulfur clusters in lysine 2,3-aminomutase by electron paramagnetic resonance spectroscopy.

Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of L-alpha-lysine with L-beta-lysine. The purified enzyme contains iron-sulfur ([Fe-S]) clusters, pyridoxal phosphate, and Co(II) [Petrovich, R. M., Ruzicka, F. J., Reed, G. H., & Frey, P. A. (1991) J. Biol. Chem. 266, 7656-7660]. Enzymatic activity depends upon the presence and integrity of these cofactors. In addition, the enzyme is activated by S-adenosylmethionine, which participates in the transfer of a substrate hydrogen atom between carbon-3 of lysine and carbon-2 of beta-lysine [Moss, M., & Frey, P. A. (1987) J. Biol. Chem. 262, 14859-14862]. This paper describes the electron paramagnetic resonance (EPR) properties of the [Fe-S] clusters. Purified samples of the enzyme also contain low and variable levels of a stable radical. The radical spectrum is centered at g = 2.006 and is subject to inhomogeneous broadening at 10 K, with a p1/2 value of 550 +/- 100 microW. The low-temperature EPR spectrum of the [Fe-S] cluster is centered at g = 2.007 and undergoes power saturation at 10 K in a homogeneous manner, with a p1/2 of 15 +/- 2 mW. The signals are consistent with the formulation [4Fe-4S] and are adequately simulated by a rhombic spectrum, in which gxx = 2.027, gyy = 2.007, and gzz = 1.99. Treatment of the enzyme with reducing agents converts the cluster into an EPR-silent form. Oxidation of the purified enzyme by air or ferricyanide converts the [Fe-S] complex into a species with an EPR spectrum that is consistent with the formulation [3Fe-4S].(ABSTRACT TRUNCATED AT 250 WORDS)     

The presence of the iron-sulfur protein (subunit V) of complex III in mitochondria of heme-deficient yeast cells lacking iron-sulfur clusters detectable by electron paramagnetic resonance

The presence of subunit V, the iron-sulfur protein, of complex III has been demonstrated in mitochondria from a mutant of Saccharomyces cerevisiae which lacks 5-aminolevulinic acid synthase and, hence, is devoid of heme. The mature form (24 K Da) of the iron-sulfur protein was observed in equal amounts in the heme-deficient and heme-sufficient cells with antiserum against subunit V and either the sensitive immuno-transfer technique or immunoprecipitation from dodecylsulfate-solubilized mitochondria. In addition, a slight shoulder with a molecular mass 1.5 kDa larger than the mature form was present in mitochondria from the heme-deficient cells. Electron paramagnetic resonance spectroscopy revealed the absence of iron-sulfur signals due to clusters S-1, S-2 and S-3 of succinate dehydrogenase or to Rieske's iron-sulfur cluster of complex III in mitochondria from the heme-deficient cells. The lack of iron-sulfur centers in these cells may be a consequence of the absence of sulfite reductase in the cells without heme.     

Probing protein dynamics by nuclear magnetic resonance

Proteins are dynamic molecules that often undergo conformational changes while performing their specific functions, such as target recognition, ligand binding and catalysis. NMR spectroscopy is uniquely suited to study protein dynamics, because site-specific information can be obtained for motions that span a broad range of time scales. The information obtained from NMR dynamics experiments has provided insights into specific structural changes or conformational energetics associated with molecular function. In the last decade, a number of new advancements in NMR methodologies have further extended our ability to characterize protein dynamics. Here, we present an overview of current NMR technology that is used to monitor the dynamic properties of proteins.     

The iron-oxygen reconstitution reaction in protein R2-Tyr-177 mutants of mouse ribonucleotide reductase. Epr and electron nuclear double resonance studies on a new transient tryptophan radical.

The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a mu-oxo-bridged diferric iron center, both necessary for enzyme activity. We have studied the reconstitution reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse RNR to investigate if other residues at the site of the radical forming Tyr-177 can harbor free radicals. In Y177W we observed for the first time the formation of a tryptophan radical in protein R2 of mouse RNR with a lifetime of several minutes at room temperature. We assign it to an oxidized neutral tryptophan radical on Trp-177, based on selective deuteration and EPR and electron nuclear double resonance spectroscopy in H2O and D2O solution. The reconstitution reaction at 22 degrees C in both Y177F and Y177C leads to the formation of a so-called intermediate X which has previously been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster. Surprisingly, in both mutants that do not have successor radicals as Trp. in Y177W, this cluster exists on a much longer time scale (several seconds) at room temperature than has been reported for X in E. coli Y122F or native mouse protein R2. All three mouse R2 mutants were enzymatically inactive, indicating that only a tyrosyl radical at position 177 has the capability to take part in the reduction of substrates.     

Effect of succinylphosphatidylcholine on phosphatidylcholine vesicles. Structural studies by gel chromatography,electron microscopy and nuclear magnetic resonance

The effect of an aqueous dispersion of succinylphosphatidylcholine on an aqueous suspension of phosphatidylcholine vesicles was studied by gel chromatography, freeze-fracture electron microscopy and proton nuclear magnetic resonance with Mn²⁺ (broadening paramagnetic reagent). Total phospholipid concentrations were in the range 10–20 mM.Succinylphosphatidylcholine is in micellar form and behaves as a detergent. The structures obtained depend on the molar percentage of succinylphosphatidylcholine.Above a succinylphosphatidylcholine molar percentage of 60%, mixed micelles are formed, assumed to be essentially spherical.Below a succinylphosphatidylcholine molar percentage of 30%, principally mixed vesicles are observed, with an external diameter of 215–240 Å, and an almost constant internal volume.Between 30 and 60% of succinylphosphatidylcholine, a mixture of these structures is obtained; rod-shaped profiles are also observed in electron microscopy, which may correspond to sections of leaky vesicles or to a new kind of cylindrical micelle.     

Direct probing of copper active site and free radical formed during bicarbonate-dependent peroxidase activity of bovine and human copper, zinc-superoxide dismutases. Low-temperature electron paramagnetic resonance and electron nuclear double resonance studies

Using X-band electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopy at liquid helium temperatures, the Cu(II) coordination geometry at the active site of bovine and human copper,zinc-superoxide dismutases (bSOD1 and hSOD1) treated with H(2)O(2) and bicarbonate (HCO(3)(-)) was examined. The time course EPR of wild type human SOD1 (WT hSOD1), W32F hSOD1 mutant (tryptophan 32 substituted with phenylalanine), and bSOD1 treated with H(2)O(2) and HCO(3)(-) shows an initial reduction of active site Cu(II) to Cu(I) followed by its oxidation back to Cu(II) in the presence of H(2)O(2). However, HCO(3)(-) induced a Trp-32-derived radical from WT hSOD1 but not from bSOD1. The mutation of Trp-32 by phenylalanine totally eliminated the Trp-32 radical signal generated from W32F hSOD1 treated with HCO(3)(-) and H(2)O(2). Further characterization of the free radical was performed by UV irradiation of WT hSOD1 and bSOD1 that generated tryptophanyl and tyrosyl radicals. Both proton ((1)H) and nitrogen ((14)N) ENDOR studies of bSOD1 and hSOD1 in the presence of H(2)O(2) revealed a change in the geometry of His-46 (or His-44) and His-48 (or His-46) coordinated to Cu(II) at the active site of WT hSOD1 and bSOD1, respectively. However, in the presence of HCO(3)(-) and H(2)O(2), both (1)H and (14)N ENDOR spectra were almost identical to those derived from native bSOD1. We conclude that HCO(3)(-)-derived oxidant does not alter significantly the Cu(II) active site geometry and histidine coordination to Cu(II) in SOD1 as does H(2)O(2) alone; however, the oxidant derived from HCO(3)(-) (i.e. carbonate anion radical) reacts with surface-associated Trp-32 in hSOD1 to form the corresponding radical.