Magnetic circular dichroism studies on acid and alkaline forms of horseradish peroxidase.

The heme vicinities of the acid and alkaline forms of native (Fd(III)) horseradish peroxidase were investigated in terms of the magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of the acid form of native horseradish peroxidase was characteristic of a ferric high spin heme group. The resemblance in the MCD spectrum between the acid form and acetato-iron (III)protoporphyrin IX dimethyl ester suggests that the heme iron of the acid form has the electronic structure similar to that in a pentocoordinated heme complex. The MCD spectra of native horseradish peroxidase did not shown any substantial pH dependence in the pH range from 5.20 to 9.00. The MCD spectral change indicated the pK value for the equilibrium between the acid and alkaline forms to be 11.0 which agrees with the results from other methods. The alkaline form of native horseradish peroxidase at pH 12.01 exhibited the MCD spectrum of a low spin complex. The near infrared MCD spectrum suggests that the alkaline form of native horseradish peroxidase has a 6th ligand somehow different from a normal nitrogen ligand such as histidine or lysine. It implicates that the alkaline form has an overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide.     

Interaction of spin-labeled tryptophan with sickle hemoglobin: probing the microenvironment of the contact sites by spin-probe--spin-label techniques

The spin-labeled tryptophan was used as a structural probe of hemoglobin contact sites. The ESR spectral data indicated that the probe exhibits weak binding to hemoglobin with a dissociation constant of 3.2.10(-5) and 4.0 mol bound per hemoglobin tetramer. The spectrum suggested that the bound tryptophan was 'partially immobilized' with a correlation time reflecting the environment of the tryptophan binding site of 8.2 ns. The topology of the contact sites was investigated by using dual spin-label methodology in which spin-labeled tryptophan and (2H,15N) substituted and deuterated maleimide spin label [2H-15N]MSL covalently-bound to Cys-beta 93 residue were used. The ESR spectral data suggested that the tryptophan binding sites were located within 8-10 A of the nitroxide free radical of spin-labeled hemoglobin. The environment of the contact sites is discussed.     

NH2-terminal spin labeling of human hemoglobin--spin states and temperature dependence

In order to explore fully how ligand- and temperature-induced alterations in the spin states of heme iron are related to protein readjustments, the spin label 4-isothiocyanate (I) was covalently attached at beta-93 cysteines and at NH2-terminal valines of various heme-iron ligand forms of human hemoglobin. It was found that the mobility of NH2-terminally bound spin labels depends on the magnetic moment of the heme iron. There is a an approximately linear relationship between the magnetic moment of the heme iron and the mobility of NH2-terminally bound spin labels. In accordance with our previous results, the temperature dependence of ESR spectra of spin-labeled hemoglobin suggests the temperature-induced protein conformational change in those heme-iron ligand forms that are characterized by the equilibrium of the spin states of the heme iron. The conformational change was sensed at both spin-label-binding sites: at beta-93 cysteines and at NH2-terminal valines.     

Saturation transfer electron spin resonance of Ca2(+)-ATPase covalently spin-labeled with beta-substituted vinyl ketone- and maleimide-nitroxide derivatives. Effects of segmental motion and labeling levels.

The Ca2(+)-ATPase in native sarcoplasmic reticulum membranes was selectively spin-labeled for saturation transfer electron spin resonance (ESR) studies by prelabeling with N-ethylmaleimide and by using low label/protein ratios. Results with the nitroxide derivative of the standard sulphydryl-modifying reagent, maleimide, were compared with a series of six novel nitroxide beta-substituted vinyl aryl ketone derivatives which differed (with two exceptions) in the substituent at the ketone position. The two exceptions had a different electron withdrawing group at the alpha-carbon, to enhance further the electrophilic character of the beta-carbon. Although differing in their reactivity, all the conjugated unsaturated ketone nitroxide derivatives displayed saturation transfer ESR spectra indicative of much slower motion than did the maleimide derivative. The saturation transfer ESR spectra of maleimide-labeled Ca2(+)-ATPase therefore most likely contain substantial contributions from segmental motion of the labeled group. The effects of the level of spin labeling were also investigated. With increasing degree of spin label incorporation, the linewidths of the conventional ESR spectrum progressively increased and the intensity of the saturation transfer spectrum dropped dramatically, as a result of increasing spin-spin interactions. The hyperfine splittings of the conventional spectrum and the outer lineheight ratios of the saturation transfer spectrum remained relatively unchanged. Extrapolation back to zero labeling level yielded comparable values for the effective rotational correlation times deduced from the saturation transfer spectrum intensities and from the lineheight ratios, for the vinyl ketone label. For the maleimide label the extrapolated values from the integral are significantly lower than those from the lineheight ratios, probably because of the segmental motion. Comparison is made of the effective rotational correlation time for the vinyl ketone label with the predictions of hydrodynamic models for the protein diffusion, in a discussion of the aggregation state of the Ca2(+)-ATPase in the native sarcoplasmic reticulum membrane. The implications for the study of protein rotational diffusion and segmental motion, and of the proximity relationships between labeled groups, using saturation transfer ESR spectroscopy are discussed.     

Determining the motility of glycoprotein oligosaccharides from lymphocyte membranes using the spin label method

Splenocyte glycoproteins solubilized by papain were purified on lectyl-lectin Sepharose 4B. Glycoproteins eluted from lectin were spin-labeled at carbohydrates. Quantitative evaluation of ESR spectra of spin-labeled glycoproteins pointed to strong restriction of reorientation of the spin label bound to oligosaccharides. Calculated correlation time of relaxing volume tagged with the spin label was equal to the molecular weight about 6000-7000 dalton. This value suggests the existence of flexibility of the glycoproteins studied.     

Peroxidase-benzhydroxamic acid complexes: spectroscopic evidence that a Fe-H2O distance of 2.6 Å can correspond to hexa-coordinate high-spin heme

Resonance Raman (RR) spectra have been obtained for single-crystal horseradish peroxidase isozyme C complexed with benzhydroxamic acid (BHA). The data are compared with those obtained in solution by both RR and electronic absorption spectroscopies at room and low (12-80 K) temperatures. Moreover, the analysis has been extended to Coprinus cinereus peroxidase complexed with BHA. The results obtained for the two complexes are very similar and are consistent with the presence of an aqua six-coordinate high-spin heme. Therefore it can be concluded that despite the rather long Fe-H2O distance of 2.6-2.7 A found by X-ray crystallography in both complexes, the distal water molecule can still coordinate to the heme iron.     

Interaction of spin-labeled tryptophan with sickle hemoglobin: probing the microenvironment of the contact sites by spin-probe-spin-label techniques

The spin-labeled tryptophan was used as a structural probe of hemoglobin contact sites. The ESR spectral data indicated that the probe exhibits weak binding to hemoglobin with a dissociation constant of 3.2 · 10⁻⁵ and 4.0 mol bound per hemoglobin tetramer. The spectrum suggested that the bound tryptophan was ‘partially immobilized’ with a correlation time reflecting the environment of the tryptophan binding site of 8.2 ns. The topology of the contact sites was investigated by using dual spin-label methodology in which spin-labeled tryptophan and (²H,¹⁵N) substituted and deuterated maleimide spin label [²H-¹⁵N]MSL covalently-bound to Cys-ß93 residue were used. The ESR spectral data suggested that the tryptophan binding sites were located within 8–10Åof the nitroxide free radical of spin-labeled hemoglobin. The environment of the contact sites is discussed.     

Electron spin resonance and nuclear relaxation studies on spin-labeled glutamate dehydrogenase.

The reaction of glutamate dehydrogenase with two different stable nitroxides (spin labels) is reported. The two compounds contain a carbonyl and an iodoacetamide group as their reactive parts. The carbonyl compound inactivates the enzyme by the formation of a 1:1 covalent complex after NaBH4 reduction of an intermediate Schiff's base. Evidence indicates that the enzyme is modified at lysine-126 in the active site. The electron spin resonance (ESR) spectrum of spin-labeled enzyme indicates a high degree of immobilization of the nitroxide. The binding of reduced coenzyme NADPH is reflected by a change (immobilization) of the ESR spectrum. Nuclear relaxation of bound substrate, oxidized coenzyme, and inhibitor by the paramagnetic group is observed. This shows the existence of a binding site for these compounds close to the active site. The distances of selected protons of the binding ligands to the nitroxide are calculated. The iodoacetamide spin label reacts with several groups, one of which is not a sulfhydryl. The reaction of this particular group causes inactivation of the enzyme. Protection against this inactivation could be achieved with certain ligands. Only enzyme that was spin labeled without such protection caused paramagnetic relaxation of bound substrate and coenzyme.     

The tryptic digestion of spin-labelled heavy meromyosin

The S₁ thiol groups of heavy meromyosin (HMM) have been selectively spin labeled with a paramagnetic analog of iodoacetamide (10) and the effects of tryptic digestion on the ESR spectrum and ATPase activity have been studied. The loss of ATPase activity on tryptic digestion occurs at the same rate with spin-labeled or unlabeled HMM suggesting that spin labeling produces no major change in the conformation of HMM. ESR spectra indicate that spin labels bound to S₁ groups of HMM are strongly immobilized; spectra of subfragment-1 isolated from tryptic digests of spin-labeled HMM are the same as those of labeled HMM. ESR spectra of S₁-spin-labeled peptides produced by tryptic digestion of HMM indicate essentially no immobilization of labels, the spectra being similar to that of a solution of free labels. The ESR spectrum of an unfractionated digest of HMM exhibits a peak attributable to strongly immobilized labels on HMM and subfragment-1 and a peak attributable to weakly immobilized labels bound to peptides. The rate at which spin-labeled peptides are released on tryptic digestion can be measured on the unfractionated digest by the decrease in the ESR peak corresponding to HMM and subfragment-1. The appearance of peptides containing spin-labeled S₁ groups parallels the loss of ATPase activity. No evidence has been found for the existence of an enzymatically active subfragment-1 lacking S₁ thiol groups.     

Spin-labeling studies of beef-liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase was spin labeled with a nitroxide derivative of parachloromercuribenzoate. The ESR spectrum was of the immobilized type and the labeling yield 0.6 mole of spin label bound per mole of protomer under standard conditions. The specific activity of the labeled enzyme was not modified but the activation by ADP abolished. Inhibition by GTP was not altered but the ESR spectrum showed that the bound spin label was further immobilized in the presence of GTP and NADPH. In the presence of the coenzyme NADPH, the labeling yield decreased to half its initial value. Such a protection effect was observed neither with NADH nor with ADP.     

Conformational change in thrombospondin induced by removal of bound Ca2+. A spin label approach

The effect of removal of Ca2+ bound to thrombospondin (TSP) on the protein structure in solution has been investigated using ESR spin-label techniques. A maleimide spin label was selectively attached to the free thiol group presumably near the carboxyl-terminal domain in which Ca2+-binding sites are situated. The ESR spectra of spin-labeled TSP showed that the bound label undergoes a relatively fast rotational motion with an effective rotational correlation time in the nano-second time regimes. Removal of bound Ca2+ in TSP by dialyzing spin-labeled TSP from a Ca2+-containing buffer into an EDTA-containing buffer resulted in an increase in the mobility of the bound label by a factor of 2.3. The data suggest that EDTA chelation of bound Ca2+ in TSP induces a conformational change of TSP at least near the site of spin labeling.     

Nucleic acid interaction with VERO cells. A temperature barrier in the interaction pattern.

The interaction of VERO cell monolayers with spin (nitroxide)-(labeled polynucleotides (1(N)n) was examined by electron spin resonance (ESR) spectroscopy at various temperatures. Nitroxide labels covalently linked to (A)n, (dUfl)n, (U)n and (A)n . (U)n were used to monitor the interaction. The VERO cells were grown on small quartz plates with a cell viability of 95% or better and then used directly for the ESR studies. The ESR results indicated that the interaction between VERO cells and spin-labeled nucleic acids is temperature dependent. No temperature dependence was found when VERO cells were in contact with nitroxide radicals which were free in solution or covalently bound to Sepharose 4B. The temperature dependence established with nitroxide-labeled nucleic acids indicates that a temperature barrier must exist between 20 and 26 degrees C for the interaction between nucleic acids and VERO cells; namely, at 26 degrees C or above spin-labeled nucleic acids interact significantly with a VERO cell surface; whereas, at 20 degrees C the ESR signal reports no interaction. It is concluded that a temperature-dependent phase transition of membrane components or cell surface products active at 26 degrees C or above play a key role in the nucleic acid cell surface interaction process.     

Spatial arrangement of coenzyme and substrates bound to L-3-hydroxyacyl-CoA dehydrogenase as studied by spin-labeled analogues of NAD+ and CoA.

The synthesis of nitroxide spin-labeled derivatives of S-acetoacetyl-CoA, S-acetoacetylpantetheine, and S-acetoacetylcysteamine is described. These compounds are active substrates of L-3-hydroxyacyl-CoA dehydrogenase [(S)-3-hydroxyacyl-CoA:NAD+ oxidoreductase, EC 1.1.1.35] exhibiting vmax values from 20% to 70% of S-acetoacetyl-CoA itself. S-Acetoacetylpantetheine and S-acetoacetylcysteamine form binary complexes with the enzyme and exhibit ESR spectra typical for immobilized nitroxides. In the case of spin-labeled pantetheine, the radical is more mobile. When spin-labeled substrates are bound simultaneously to each active site of this dimeric enzyme, spin-spin interactions differentiate between two alternate orientations of the substrate [Birktoft, J.J., Holden, H.M., Hamlin, R., Xuong, N.H., & Banaszak, L.J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8262-8266]. The fatty acid moiety is thought to be located in a cleft between two domains whereas a large part of the CoA moiety probably extends into the solution. NAD+, spin-labeled at N6 of the adenine ring, is an active coenzyme of L-3-hydroxyacyl-CoA dehydrogenase (60% vmax). Complexes with the enzyme exhibit ESR spectra typical of highly immobilized nitroxides. Binding of coenzyme NAD+ causes conformational changes of the binary enzyme/substrate complex as revealed by changes in the ESR spectrum of spin-labeled S-acetoacetylpantetheine.     

Redox-active daunomycin-spin-labeled nucleic acid complexes

Interaction studies between daunomycin (DM) and enzymatically spin-labeled nucleic acid duplexes reveal two modes of binding by electron spin resonance (ESR) spectroscopy. At a low drug/nucleotide (D/N) ratio, the drug binds in the intercalative mode with only a slight reduction in base mobility. Saturation in the intercalative mode is achieved at a lower D/N ratio for B' DNA than for B DNA. After full intercalation, further addition of DM seems to destabilize the helix and to allow the formation of redox-active DM stacks complexed to the nucleic acid lattice. These stacks will irreversibly oxidize all the nitroxides covalently bound to the 4- or 5-position of the pyrimidine base. Interactions between DM and spin-labeled single-stranded nucleic acids lead directly to the formation of redox-active complexes, while mixing of the drug with spin-labeled nucleic acid building blocks not incorporated in a nucleic acid lattice causes no ESR signal change. Complete disappearance of the ESR signal of spin-labeled nucleic acids extrapolates to a D/N value which is a constant for a particular lattice system and is independent of spin-labeling content.     

Binding of spin-labeled fatty acids and lysophospholipids to hydrophobic region of calmodulin.

In the presence of bovine brain calmodulin activated by calcium, the sharp triplet electron spin resonance (ESR) lines of free doxyl stearic acids decreased, and the broad resonance lines increased concomitantly, suggesting that the doxyl stearic acids bound to calmodulin calcium-dependently. The bound molecules were displaced by a calmodulin inhibitor, W-7, whereas their nitroxide radicals were hardly reduced by ascorbic acid, suggesting that the spin-labeled fatty acids bind to hydrophobic regions of calmodulin, and consequently inhibit calmodulin-dependent phosphodiesterase activity. These binding characteristics to calmodulin were different from those to bovine serum albumin. Moreover, the ESR spectra of two spin-labeled derivatives of lysophospholipid having a spin-labeled acyl group or a spin-labeled polar head group showed that it is the acyl chain of lysophospholipid that interacts with the hydrophobic region of calmodulin. The interactions of fatty acids and lysophospholipids with calmodulin seem to be quite different from those of acidic phospholipids, described previously [Suzuki, T., Katoh, H., & Uchida, M.K. (1986) Biochim. Biophys. Acta, 873, 379-386]. Thus, from the results of ESR study, we can obtain information on the function of fatty acids and lysophospholipids on calmodulin. Instead of enzyme assay, ESR spectroscopy is a useful means to examine lipid-protein interaction.     

Effects of ouabain on the rotational dynamics of renal Na,K-ATPase studied by saturation-transfer EPR.

The interaction of a nitroxide spin-labeled derivative of ouabain with sheep kidney Na,K-ATPase and the motional behavior of the ouabain spin label-Na,K-ATPase complex have been studied by means of electron paramagnetic resonance (EPR) and saturation-transfer EPR (ST-EPR). Spin-labeled ouabain binds with high affinity to the Na,K-ATPase with concurrent inhibition of ATPase activity. Enzyme preparations retain 0.61 +/- 0.1 mol of bound ouabain spin label per mole of ATP-dependent phosphorylation sites, even after repeated centrifugation and resuspension of the purified ATPase-containing membrane fragments. The conventional EPR spectrum of the ouabain spin label bound to the ATPase consists almost entirely (greater than 99%) of a broad resonance at 0 degrees C, characteristic of a tightly bound spin label which is strongly immobilized by the protein backbone. Saturation-transfer EPR measurements of the spin-labeled ATPase preparations yield effective correlation times for the bound labels significantly longer than 100 microseconds at 0 degrees C. Since the conventional EPR measurements of the ouabain spin-labeled Na,K-ATPase indicated the label was strongly immobilized, these rotational correlation times most likely represent the motion of the protein itself rather than the independent motion of mobile spin probes relative to a slower moving protein. Additional ST-EPR measurements of ouabain spin-labeled Na,K-ATPase (a) cross-linked with glutaraldehyde and (b) crystallized in two-dimensional arrays indicated that the observed rotational correlation times predominantly represented the motion of large Na,K-ATPase-containing membrane fragments, as opposed to the motion of individual monomeric or dimeric polypeptides within the membrane fragment. The results suggest that the binding of spin-labeled ouabain to the ATPase induces the protein to form large aggregates, implying that cardiac glycoside induced enzyme aggregation may play a role in the mechanism of action of the cardiac glycosides in inhibiting the Na,K-ATPase.     

ESR spectral analysis of the molecular motion of spin labels in lipid bilayers and membranes based on a model in terms of two angular motional parameters and rotational correlation times

Electron spin resonance (ESR) spectral line shapes are calculated for a nitroxide spin-labeled molecule undergoing rapid restricted rotations (twisting) about its long molecular axis while simultaneously tumbling within a cone. Explicit expressions are derived for the hyperfine splittings and $\text{g-}\text{values}$, as well as for the secular contributions to the motionally modulated linewidths. The present model is useful for analyzing the restricted twisting and tumbling motions, and rotational correlation times, of spin-labeled molecules in bilayers. Simulated spectra compare well with experimental spectra of lecithin bilayers marked with cholestane spin label, over a wide temperature range.     

Synthesis and use of a new spin-labeled analogue of ADP with platelet-aggregating activity.

A new spin-labeled derivative of ADP, 2-(4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxyl)thioadenosine-5'-diphosphate, has been synthesized. The compound causes both the reversible and irreversible phases of aggregation of human blood platelets at concentrations similar to those required for similar phases of aggregation by ADP itself. The spin-labeled ADP also rivals ADP as a substrate for pyruvate kinase. The interaction of intact human blood platelets and of isolated platelet membranes with the platelet-aggregating spin-labeled derivatives of ADP has been studied. The dramatic decrease in the ESR signal of the spin label is primarily due to chemical reduction of the nitroxide, rather than immobilization of the label. When platelets and spin-labeled ADP are mixed, a rapid burst of nitroxide reduction occurs, followed by a much slower reduction similar in time course to that seen for other spin labels. The rapid burst of reduction, but not the slow reduction, is inhibited by adenosine, an inhibitor of ADP-induced platelet aggregation, and by sulfhydryl-blocking agents. Experiments conducted with Ellman's reagent and platelet membranes or washed platelets revealed a 10 to 30% increase in the number of reactive membrane sulfhydryl groups when ADP was present. These results indicate that there is an increase in the number of reactive sulfhydryl groups on the platelet surface when platelets or membranes are stimulated by ADP.     

Structural studies of human IgA1 and IgA2 immunoglobulins labeled with two different spin labels

The spin-label method was used for structural study of different subclasses of human immunoglobulin A. The spin label was incorporated into the protein part, as well as into carbohydrates of the IgA molecules. Well resolved outer wide extrema were characteristic of the ESR spectra of IgA spin-labeled at the protein moiety. ESR spectra of IgA tagged at carbohydrates reflected moderately immobilized rotation of the spin label. Dependencies of the parameters of ESR spectra of spin-labeled IgA1 and IgA2 upon viscosity at constant temperature have been investigated and a quantitative analysis of the isotherms was carried out. Spin-labeled oligosaccharide chains of IgA2 possessed great freedom of rotation. At least some of IgA1 oligosaccharides were closely attached to the protein moiety. Both proteins under study have shown flexible structure. The Fc fragment of IgA1 molecule appeared to have a rigid structure.     

Nuclear magnetic resonance studies on the spatial relationship of aromatic donor molecules to the heme iron of horseradish peroxidase

The geometry of hydrogen donor molecules bound to horseradish peroxidase was investigated using nuclear magnetic resonance techniques. Between resorcinol and 2-methoxy-4-methylphenol which showed different optical difference spectra, little difference was observed in the orientation of the molecules bound to horseradish peroxidase: the minimal distances between the enzyme iron and the protons of the phenol rings are in the range of 8.4-11.0 A. This situation was not greatly different for the third compound studied in this paper, benzhydroxamic acid, providing evidence against the view that its side chain coordinates to the heme iron. Furthermore, it was found that transferred nuclear Overhauser effect for the signals of these compounds was observable only when the heme peripheral 8-methyl proton signal was irradiated. These results, together with a hypothetical model of the enzyme structure obtained by computer-aided simulation procedures, suggest that the binding of these donor molecules and competitive inhibitors occur in the vicinity of the heme peripheral 8-methyl group, with hydrophobic interactions probably with Tyr-185 and with hydrogen bond with adjacent amino acid residues such as Arg-183.