Atomic structures of two nitroxide spin labels complexed with human thrombin: comparison with solution studies

Crystal structures of thrombin complexed with two spin labels called para-V, 4-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl)-p-(fluorosulfonyl) benzamidine, and meta-V, 3-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl)-m-(fluorosulfonyl) benzamidine, have been completed at 2.0 and 3.0 Å resolution, respectively. Previous electron spin resonance studies with these labels gave rise to a low-resolution topography map of thrombin's extended active site. These labels monitor two distinct areas of the thrombin active site: (1) an apolar binding site which manifests itself in an biphasic activation/inhibition effect on thrombin activity and (2) a region sensitive to -thrombin autoproteolytic cleavage(s) to -thrombin (Arg75-Tyr76 and/or Arg77A-Asn78, and Lys149E-Gly150, chymotrypsin numbering). Para-V was found to bind along the substrate binding cleft, while meta-V was found to bind both at the substrate primary specificity pocket and at a site which interacts with the -cleavage loop. These studies reaffirm that accurate information may be gained from solution studies and indicates the complementarity of solid-state studies.     

Structural dependence of nitroxide spin labels and nitroxide spin adducts on their reducibility by ascorbate ion

Summary

The reducibility of a series of nitroxides (aminoxyls) by ascorbate was tested by measuring the nitroxide decay rates with a stopped-flow electron paramagnetic resonance technique in aqueous phosphate buffer solution. The dependence of reactivity on the structures and pH of the medium was found for both cyclic nitroxides and nitroxide adducts of phenyl N-tert butyl nitrone (PBN). In cyclic nitroxides, the ring size is a dominant factor in determining reaction rates but substituents have additional effects on the rate depending on their electronegativity. For alkyl and hydroxyalkyl adducts of PBN, at fixed ascorbate concentration, half-lives increase with lengthening of the substituent, suggesting that a long chain in the substituent sterically protects the nitroxide group and thus prevents its reduction by ascorbate.     

Exchange and shuttling of electrons by nitroxide spin labels

The ability of nitroxide spin labels to act as oxidizers of reduced nitroxides (hydroxylamines) in biological and model systems was demonstrated. All of the nitroxides tested were able to act as oxidizing agents with respect to hydroxylamine derivatives of nitroxides. The rates of these reactions were first order with respect to nitroxide concentration and with respect to hydroxylamine concentration, making the reaction second order overall. The second-order rate constants are reported for a number of these reactions. These reactions proceeded to an equilibrium state and the equilibrium constants for several combinations of reactants are presented. Both the rate constants and the equilibrium constants were found to be dependent on the ring structure of the nitroxide and hydroxylamine, with piperidines being reduced more easily and pyrrolidines and oxazolidines being oxidized more easily. All of the hydroxylamine derivatives were oxidized by air to their respective nitroxides, with the rate of this oxidation greater for pyrrolidines than for piperidines. Furthermore, hydroxylamines that are permeable to lipid bilayers were able to act as shuttles of reducing equivalents to liposome-encapsulated nitroxides that were otherwise inaccessible to reducing agents. This mechanism of shuttling of electrons was able to explain the relatively rapid reduction by cells of a nonpermeable nitroxide in the presence of a permeable nitroxide.     

Computer modeling of nitroxide spin labels on proteins

Electron paramagnetic resonance using site‐directed spin labeling can be used as an approach for determination of protein structures that are difficult to solve by other methods. One important aspect of this approach is the measurement of interlabel distances using the double electron–electron resonance (DEER) method. Interpretation of experimental data could be facilitated by a computational approach to calculation of interlabel distances. We describe an algorithm, PRONOX, for rapid computation of interlabel distances based on calculation of spin label conformer distributions at any site of a protein. The program incorporates features of the label distribution established experimentally, including weighting of favorable conformers of the label. Distances calculated by PRONOX were compared with new DEER distances for amphiphysin and annexin B12 and with published data for FCHo2 (F‐BAR), endophilin, and α‐synuclein, a total of 44 interlabel distances. The program reproduced these distances accurately (r² = 0.94, slope = 0.98). For 9 of the 11 distances for amphiphysin, PRONOX reproduced the experimental data to within 2.5 Å. The speed and accuracy of PRONOX suggest that the algorithm can be used for fitting to DEER data for determination of protein tertiary structure. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 35–44, 2012.     

Interaction of nitroxide spin labels with chloroplasts

Chloroplasts isolated from oats eliminated the electron spin resonance (ESR) signals from spin labels in white light and partially restored them in far-red light. Only the white light-mediated reaction was blocked by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). In contrast, oat (Avena sativa L. cv. Garry and Park) leaf mesophyll protoplasts oxidized the spin labels in both white and far-red light, with and without DCMU. Light had no obvious effect on spin label motion within chloroplast membranes. The results suggest that, in isolated chloroplasts, nitroxide spin labels may be reduced by photosystem I within the thylakoid bilayer resulting in loss of the ESR signals. The reduced forms may be reoxidized by an element of the photosynthetic electron transport chain which operates between the DCMU block and the photosystem I reaction center. In addition, a light-mediated destruction of the spin labels occurs in both chloroplasts and protoplasts. The reduced form of the nitroxide (i.e. the hydroxylamine) may be resistant to this destruction.     

Synthesis and properties of chlorophyll-derived nitroxide spin labels

A general synthesis of chlorophyll-derived spin labels is reported. The starting point is chlorophyll a and a long chain spin-labeled alcohol. The four-step synthetic route yields new spin labels in quantities practical for membrane spin-labeling studies. Two examples prepared are 12′-proxyltridecyl pyropheophorbide a and 12′-proxylhexadecyl pyropheophorbide a. The spin labels were purified and characterized by thin-layer chromatography, high-pressure liquid chromatography, mass spectroscopy, nmr and visible spectroscopy, and the number of unpaired spins per molecule. ESR spectral parameters are reported. The chlorophyll-derived spin labels intercalate into fluid phospholipid bilayers and are observed in both the bilayer phase and bound to membrane proteins in chromatophores of the purple photosynthetic bacterium, Rhodopseudomonas sphaeroides.     

Effects of oxygen on the metabolism of nitroxide spin labels in cells

The products of the reduction of nitroxides in cells are the corresponding hydroxylamines, which cells can oxidize back to the nitroxides in the presence of oxygen. Both the reduction of nitroxides and the oxidation of hydroxylamines are enzyme-mediated processes. For lipid-soluble nitroxides, the rates of reduction are strongly dependent on the intracellular concentration of oxygen; severely hypoxic cells reduce nitroxides more rapidly than cells supplied with oxygen. In contrast, the rates of oxidation of hydroxylamines increase smoothly with increasing intracellular oxygen concentration up to 150 microM. In order to separate the effects on the rates of metabolism of nitroxides due directly to oxygen from effects due to the redox state of enzymes, we studied the cells under conditions in which each of these variables could be changed independently. Oxygen affects the metabolism of these nitroxides primarily by interacting with cytochrome c oxidase to change the redox state of the enzymes in the respiratory chain. Our results are consistent with the conclusions that in these cells reduction of lipophilic nitroxides occurs at the level of ubiquinone in the respiratory chain in mitochondria, and oxidation of the corresponding hydroxylamines occurs at the level of cytochrome c oxidase.     

Metabolism of nitroxide spin labels in subcellular fraction of rat liver: I. Reduction by microsomes

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is locted Important variables that were studied included adding NADH, adding, induction of enzymed by intake of phenobarbital and the effects of oxygen. Reduction of nonparamagnetic derivatives and oxidation to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added NADh or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital and paralleled the levels of NADPH cytochrome c reductase, cytochrome P-450, cytochrome b₅ and NADH cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylmines. We conclude that there are significant NADH- and NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondrial metabolism seems to play an even more important role in intact cells.     

Oxidation of hydroxylamines to nitroxide spin labels in living cells

In the presence of oxygen, cells can oxidize hydroxylamines, which are the products of the reduction of nitroxides in cells, back to nitroxides. Lipid-soluble hydroxylamines are oxidized much more rapidly than water-soluble ones, and most of this oxidation is inactivated by heat or trichloroacetic acid, indicating that the principal mechanism is enzyme-linked. The rates of oxidation of some lipophilic hydroxylamines are comparable to the rates of reduction of the corresponding nitroxides. Hydroxylamines formed by reduction of aqueous soluble nitroxides are not oxidized by cells, except for slight oxidation of some pyrrolidine derivatives. The latter is due to autoxidation. The kinetics of oxidation of reduced lipid-soluble nitroxides are all first-order with respect to hydroxylamines, regardless of the position of the nitroxide group along the carbon backbone, indicating that the oxidation occurs within the membrane. The oxidation of hydroxylamines in cells in inhibited by cyanide but not by antimycin A or SKF-525A. We also describe an effective method to oxidize hydroxylamines and follow this reaction; the method is based on the use of perdeuterated [15N]Tempone.     

Binding of antibodies to nitroxide spin labels and to the corresponding hydroxylamines.

The affinities of rabbit antibodies directed against the spin-label nitroxide group have been found to be of the order of 10⁶ 1/mole for a number of low molecular weight water soluble haptens. It is shown that the same antibodies have almost equal binding affinities to corresponding hydroxylamines.     

Metabolism of nitroxide spin labels in subcellular fraction of rat liver. I. Reduction by microsomes

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is located. Important variables that were studied included adding NADH, adding NADPH, induction of enzymes by intake of phenobarbital and the effects of oxygen. Reduction to nonparamagnetic derivatives and oxidation back to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added NADH or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital, and paralleled the levels of NADPH cytochrome c reductase, cytochrome P-450, cytochrome b5 and NADH cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylamines. We conclude that there are significant NADH- and NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondrial metabolism seems to play an even more important role in intact cells.     

Kinetics of enzyme-mediated reduction of lipid soluble nitroxide spin labels by living cells

Nitroxide spin labels can be reduced to the corresponding hydroxylamines in cells. The selective action of inhibitors, and thermal and chemical inactivation demonstrate that the reduction of nitroxides in cells is an enzymatic or enzyme-mediated process. The kinetics of reduction of doxylstearates are affected by the position of the doxyl moiety along the stearic acid chain. The doxyl moiety of 5-doxylstearate is close to the membrane surface, and its reduction is first order with respect to the nitroxide, whereas the doxyl moieties of 10- and 12-doxylstearate are in the membrane hydrocarbon region and their reduction is a zero-order process. The reduction of 16-doxylstearate which usually has a mixture of first- and zero-order kinetics becomes zero order with addition of an extracellular broadening agent, potassium trioxalatochromiate(III). These results suggest that the rate of reduction of doxyl moieties is controlled by their accessibility to reducing equivalents, i.e., the rate-limiting step for the reduction of the doxyl moiety deep in the membrane is the diffusion of reducing equivalents within or into the membrane. The reduction of doxylstearates in cells is inhibited by rotenone but not antimycin A, cyanide, propyl gallate or SKF-525A. It appears that the reduction of doxylstearates takes place at the level of the ubiquinone in the respiratory chain in mitochondria in these cells.     

Synthesis of charged amphipathic nitroxide lipid spin labels and an example of their application in membrane studies

The synthesis of a series of amphipathic nitroxide lipid spin labels is reported. Thus, 12-proxylhexadecanol has been converted into the versatile fatty acid spin label 14-proxylstearic acid. This substance was used to prepare 14-proxylstearyltrimethylammonium methanesulfonate, a positively charged label, and 14-proxylstearylmethyl phosphate sodium salt, a negatively charged label. Also prepared in the doxyl series were quaternary ammonium salts derived from 16-doxyl- and 7-doxylstearic acid. The positively charged and negatively charged proxyl labels were used in a preliminary experiment to investigate the role of charge in their interaction with reconstituted cytochrome oxidase. The average binding affinity of the negatively charged label is approximately 2-fold higher than that of the positively charged label at pH 7.4. At pH 5.5 the average relative affinity for negatively charged label is about 3.5-fold higher than that of positively charged label, suggesting that the ionizable group(s) on the protein can interact with the lipid headgroup.     

Filipin fluorescence quenching by spin-labeled probes: studies in aqueous solution and in a membrane model system.

A detailed photophysical study of the fluorescence quenching (transient and steady state) of the macrolide antibiotic filipin by nitroxide-substituted fatty acids and a cholesterol derivative was carried out, aimed at determining its transverse position in a model system of membranes (multilamellar vesicles of dipalmitoylphosphatidylcholine). Filipin partitions efficiently into membranes (Kp = (5.0 +/- 1.0).10(3), 20 degrees C) and it was concluded that the antibiotic is buried in the membrane, away from the lipid-water interface. In addition, information on the organization of the quenchers was also obtained. The 5-nitroxide derivative of the fatty acid is essentially randomly distributed, while the 16-nitroxide is aggregated at concentrations higher than approximately 5% molar. For the cholesterol compound the results point to a phase separation at concentrations higher than 3% molar (below this limit concentration filipin associates with the derivatized sterol with KA = 20 M-1, assuming a 1:1 interaction). We propose that this phase separation and the aggregation state of filipin in the aqueous solution may be key processes in the antibiotic mode of action. A systematic and general approach to fluorescence quenching data analysis in complex (e.g., biochemical) systems is also presented.     

Metabolism of nitroxide spin labels in subcellular fractions of rat liver. II. Reduction in the cytosol

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of five nitroxides in cytosol derived from rat hepatocytes. The nitroxides were chosen to provide information on the effects of the type of charge and the ring on which the nitroxyl group is located. The rates of reduction were fastest for a six-membered positively charged nitroxide ('CAT-1') and slowest for an anionic five-membered ring nitroxide ('PCA'). Changing levels of glutathione, sulphydryl groups in general, NADPH or NADH had little or no effect on the rates of reduction, while the addition of ascorbate oxidase essentially abolished reduction of the nitroxides. The products of reduction by the cytosol were the corresponding hydroxylamines. The overall rates of reduction of neutral or anionic nitroxides were much slower than those observed with intact cells. We conclude that the primary source of metabolism of nitroxides by cytosol is reduction by ascorbate and that under most conditions reduction of nitroxides in the cytosol is not a major factor in the metabolism of nitroxides by cells.     

Dipolar coupling between nitroxide spin labels: the development and application of a tether-in-a-cone model

A tether-in-a-cone model is developed for the simulation of electron paramagnetic resonance spectra of dipolar coupled nitroxide spin labels attached to tethers statically disordered within cones of variable halfwidth. In this model, the nitroxides adopt a range of interprobe distances and orientations. The aim is to develop tools for determining both the distance distribution and the relative orientation of the labels from experimental spectra. Simulations demonstrate the sensitivity of electron paramagnetic resonance spectra to the orientation of the cones as a function of cone halfwidth and other parameters. For small cone halfwidths (< approximately 40 degrees ), simulated spectra are strongly dependent on the relative orientation of the cones. For larger cone halfwidths, spectra become independent of cone orientation. Tether-in-a-cone model simulations are analyzed using a convolution approach based on Fourier transforms. Spectra obtained by the Fourier convolution method more closely fit the tether-in-a-cone simulations as the halfwidth of the cone increases. The Fourier convolution method gives a reasonable estimate of the correct average distance, though the distance distribution obtained can be significantly distorted. Finally, the tether-in-a-cone model is successfully used to analyze experimental spectra from T4 lysozyme. These results demonstrate the utility of the model and highlight directions for further development.     

Rapid assessment of liposomal stability in blood by an aqueous nitroxide spin label

An electron spin resonance method using an aqueous nitroxide spin label, 2,2,6,-tetramethyl-piperidine-N-oxyl-4-trimethyl-ammonium, for rapid assessment of liposome stability in blood is presented. The retention of the nitroxide in liposomes is measured by its electron spin resonance signal intensity, a procedure which does not require separation of the sample from the blood. Any nitroxide that is released from the liposomes is reduced by external ascorbic acid which is added to the sample. The method permits kinetic studies on the integrity of liposomes in the presence of destabilizing factors such as detergent, blood, or alteration in temperature.