Electron nuclear double resonance and electron paramagnetic resonance study on the structure of the NO-ligated heme alpha 3 in cytochrome c oxidase

The techniques of EPR and electron nuclear double resonance (ENDOR) were used to probe structure and electronic distribution at the nitric oxide (NO)-ligated heme alpha 3 in the nitrosylferrocytochrome alpha 3 moiety of fully reduced cytochrome c oxidase. Hyperfine and quadrupole couplings to NO (in both 15NO and 14NO forms), to histidine nitrogens, and to protons near the heme site were obtained. Parallel studies were also performed on NO-ligated myoglobin and model NO-heme-imidazole systems. The major findings and interpretations on nitrosylferrocytochrome alpha 3 were: 1) compared to other NO-heme-imidazole systems, the nitrosylferrocytochrome alpha3 gave better resolution of EPR and ENDOR signals; 2) at the maximal g value (gx = 2.09), particularly well resolved NO nitrogen hyperfine and quadrupole couplings and mesoproton hyperfine couplings were seen. These hyperfine and quadrupole couplings gave information on the electronic distribution on the NO, on the orientation of the g tensor with respect to the heme, and possibly on the orientation of the FeNO plane; 3) a combination of experimental EPR-ENDOR results and EPR spectral simulations evidenced a rotation of the NO hyperfine tensor with respect to the electronic g tensor; this implied a bent Fe-NO bond; 4) ENDOR showed a unique proton not seen in the other NO heme systems studied. The magnitude of this proton's hyperfine coupling was consistent with this proton being part of a nearby protein side chain that perturbs an axial ligand like NO or O2.     

Electron-spin resonance of nitrosyl haemoglobins: normal alpha and beta chains and mutants Hb M Iwate and Hb Zürich.

At 77 K the electron spin resonance (ESR) spectra of the NO derivatives of the mutant haemoglobins Hb M Iwate and Hb Zurich as well as of the isolated chains of normal haemoglobin were studied. Two types of ESR spectra differing in the g-value and the hyperfine splitting at gzz were observed. The type II spectrum is characterized by a hyperfine structure at gzz = 2.005 with a splitting constant of deltaH = 23 G (14NO) or 32 G (15NO), respectively. In the type I spectrum the splitting constant of the hyperfine structure at gzz = 2.009 amounts to deltaH = 18 G (14NO) or 23 G (15NO), respectively. In some cases this hyperfine structure is coincident with another one at gxx = 2.064 with nearly identical splitting constant. In addition, the type I spectrum is characterized by an increased ESR absorption at gxx = 2.064. At neutral pH the NO derivatives of the isolated chains as well as of the mutant haemoglobins give rise to a type II spectrum. In correspondence with previous results gained with normal NO haemoglobin, the ESR spectra of the NO-alpha chains and NO-Hb Zurich show a transition to type I in the acid region. This transition is favoured by binding of 2,3-bisphosphoglycerate. On the other hand, the ESR spectra of the NO-beta chains and NO-Hb M Iwate are of the type II also at acid pH. The NO-beta chains show a transition of the ESR spectrum from type II to type I only at alkaline pH. These results indicate that in the tetrameric NO haemoglobin only the alpha chains are responsible for the transition of the ESR spectrum from type II to type I in the acid region. The two types of ESR spectra are interpreted in terms of two kinds of haem-NO complexes differing in the iron-NO and iron-imidazole distances. The type II spectrum is attributed to a complex with a relatively short iron-imidazole distance which is responsible for a weakened sigma-bond in trans position. The type I spectrum arises then from a complex with a larger iron-imidazole bond leading to an approach of the NO molecule to the iron. The influence of the protein conformation upon the iron-imidazole bond length is discussed with regard to the ESR spectra of the mutant NO haemoglobins and considering the influence of agents modifying the protein structure.     

Dinitrosyl-iron complexes with thiol-containing ligands: spatial and electronic structures.

Parameters of the EPR signals of monomeric dinitrosyl-iron complexes with 1H-1,2,4-triazole-3-thiol (DNIC-MT), obtained by treating MT+ferrous iron in DMSO solution with gaseous NO, have been compared with those of the crystalline monomeric DNIC-MT with tetrahedral structure. Dissolved DNIC-MT were characterized by the isotropic EPR signal centered at g=2.03 with half-width of 0.7 mT and quintet hyperfine structure when recorded at ambient temperature or the anisotropic EPR signal with g( perpendicular)=2.045, g( parallel)=2.014 from frozen solution at 77 kappa, Cyrillic. DNIC-MT in crystalline state showed the structure-less symmetrical singlet EPR signal centered at g=2.03 and half-width of 1.7 mT at both room and liquid nitrogen temperature. The Lorentz shape of this signal indicates the strong exchange interaction between these complexes in the DNIC-MT crystal. Being dissolved in DMSO the crystalline sample of DNIC-MT demonstrated the EPR signal typical for DNIC-MT, obtained by treating MT+ferrous iron in DMSO solution with gaseous NO. Low spin (S=1/2) d(9) electron configuration of DNIC-MT with tetrahedral structure (formula [(MT-S(.))(2)Fe(-1)(NO(+))(2)](+)) was suggested to be responsible for the signal of DNIC-MT in crystalline state. Dissolving of the crystals of DNIC-MT may result in the change of their spatial and electronic structure, namely, tetrahedral structure of the complexes characterized by low spin d(9) electronic configuration transforms into a plane-square structure with d(7) electronic configuration and low spin S=1/2 state (formula [(MT- S(-))(2)Fe(+)(NO(+))(2)](+)). The latter was suggested to be characteristic of other DNICs with various thiol-containing ligands in the solutions. The proposed mechanism of these DNICs formation from ferrous iron, thiol and NO shows that the process could be accompanied by the ionization of NO molecules to NO(+) and NO(-) ions in the complexes. Detailed analysis of the shape of the EPR signals of these complexes provided additional information about the exchange interaction typical for DNIC-MT in crystals.     

Powder and single-crystal electron paramagnetic resonance studies of yeast cytochrome c peroxidase and its peroxide and its peroxide compound, Compound ES

Electron paramagnetic resonance absorption spectrum of ferric cytochrome c peroxidase exhibited a mixture of high- and low-spin compounds. The principal values and the eigenvectors of the g-tensor for the low-spin species were determined by single-crystal EPR spectroscopy at 77 K. The powder EPR spectra of the peroxide compound, Compound ES, were measured at S-, X-, and Q-band microwave frequencies. Careful examination at 77 K showed a narrow free radical-like signal at g = 2.004 with hyperfine structures accompanied by a broad signal spreading on both low- and high-field sides. Single-crystal EPR analyses of Compound ES clearly demonstrated that there exist at least two different radical species: one is isotropic with hyperfine structure at g = 2.004 and the other exhibits an axially symmetric signal at 5 K and broad signal centered at g = 2.004 at 77 K, respectively. The principal values and the eigenvectors of the g-tensor for the axially symmetric signal were determined: g(parallel) = 2.034 and g(perpendicular) = 2.006, 1.999. The orientation of the unique axis (g(parallel)) was found to be identical to that of the heme normal. A new radical signal with complicated hyperfine structures in the g = 2.004 region was observed upon illumination of Compound ES at both 5 and 77 K. The photoinduced species grew effectively by the illumination light around 500 nm. On warming to -80 degrees C, the photoinduced signal was reversibly brought back to the original radical species of Compound ES via an intermediate species. From these results, we have proposed the possible sites for the free radical centers in Compound ES.     

Analysis of powder and single crystal electron paramagnetic resonance spectra of deoxy myoglobins containing cobalt (II) proto-and mesoporphyrins IX at various microwave frequencies

Artificial myoglobins (Mbs) substituted for protoheme with Co(II) proto-and mesoporphyrins IX (proto-and meso-CoMbs, respectively) were prepared. The principal values and eigenvectors of g tensors and the hyperfine coupling tensors of the paramagnetic Co(II) centers of their deoxy forms have been determined by single crystal EPR spectroscopy at 77 K in order to elucidate orientation and electronic structure of the prosthetic group in myoglobin. The orientation of the porphyrin plane of deoxy meso-CoMb were found to be identical to that of deoxy proto-CoMb. However, the in-plane hyperfine coupling constants of deoxy meso-CoMb were more anisotropic and larger than those of deoxy proto-CoMb, suggesting an increase in the electron spin density on the Co(II) ion upon the exchange of protoporphyrin IX with mesoprophyrin IX. Powder EPR spectra of these CoMbs, which were measured at S- and L-band microwave frequencies, exhibited well resolved 59Co hyperfine splittings and can be clearly interpreted by the use of the EPR parameters obtained from single crystal EPR measurements.     

Mechanisms of the protective action of diethyldithiocarbamate-iron complex on acute cardiac allograft rejection

In this study, we examined the actions of diethyldithiocarbamate-iron (DETC-Fe) complex in acute graft rejection heterotopically transplanted rat hearts. Chronic treatment with DETC-Fe inhibited the increase in plasma nitric oxide (NO) metabolites and nitrosylation of myocardial heme protein as determined by electron paramagnetic resonance (EPR) spectroscopy. Pulse injection with DETC-Fe normalized NO metabolites. We verified intragraft trapping of NO in vivo by pulse injection with DETC-Fe by the detection within allografts of an anisotropic triplet EPR signal for DETC-Fe-NO adduct with resonance positions (g tensor factors for perpendicular and parallel components, respectively g( perpendicular ) = 2.038 and g( parallel ) = 2.02; hyperfine coupling of 12.5 G). DETC-Fe prolonged graft survival and decreased histological rejection scores. DNA binding activity for nuclear factor (NF)-kappaB and activator protein-1 was increased in allografts and prevented by DETC-Fe. Abrogation of the activation of NF-kappaB by DETC-Fe was associated with increased IkappaBalpha inhibitory protein. Western blotting and RT-PCR analysis revealed that DETC-Fe inhibited inducible NO synthase protein and gene expression. Gene expression for the proinflammatory cytokine interferon-gamma was also decreased by DETC-Fe. Thus DETC-Fe limits NF-kappaB-dependent gene expression and possesses significant immunosuppressive properties.     

Chain equivalence in reaction of nitric oxide with hemoglobin.

Mixtures of nitric oxide and hemoglobin were prepared in a rapid freeze apparatus and analyzed by EPR spectroscopy. Spectra from samples at various degrees of saturation showed that the two subunits bound NO at equal rates. Identical results were observed in 0.1 M phosphate at pH 6.5 and 0.1 M 2,2'-bis(hydroxymethyl)-2,2',2'-nitrilotriethanol, 0.1 M NaCl at pH 7.0, both in the presence and absence of inositol hexaphosphate at either buffer condition. At subsaturating levels of NO (less than 60%), or at all levels of saturation in the presence of inositol hexaphosphate, it was found that the EPR spectrum of nitrosylhemoglobin varied with the length of time before freezing. This change was characterized by the development of a hyperfine structure at g = 2.01 which appeared with a half-time of approximately 0.4 s. Maxwell and Caughey (Maxwell, J. C., and Caughey, W. S. (1976) Biochemistry 15, 388-395) have attributed this three-line EPR hyperfine structure to the formation of a pentacoordinate ferroheme-NO complex. Corresponding slow changes were observed in the visible absorption spectrum following the binding of low levels of NO to deoxyhemoglobin or inositol hexaphosphate to fully saturated nitrosylhemoglobin. Thus it appears that NO binding to the alpha and beta subunits of deoxyhemoglobin takes place at equal rates and, under conditions favoring the T quaternary state (low saturation, presence of inositol hexaphosphate), a further slow structural change takes place, resulting in the cleavage of the iron--proximal histidine bond.     

M?ssbauer and EPR studies of the photoactivation of nitrile hydratase.

The alphabeta dimer of active nitrile hydratase from Rhodococcus sp. R312 contains one low-spin ferric ion that is coordinated by three Cys residues, two N-amide groups from the protein backbone, and one OH(-). The enzyme isolated from bacteria grown in the dark is inactive and contains the iron site as a six-coordinate diamagnetic Fe-nitrosyl complex, called NH(dark). The active state can be obtained from the dark state by photolysis of the Fe-NO bond at room temperature. Activation is accompanied by the conversion of NH(dark) to a low-spin ferric complex, NH(light), exhibiting an S = (1)/(2) EPR signal with g values of 2.27, 2.13, and 1.97. We have characterized both NH(dark) and NH(light) with M?ssbauer spectroscopy. The z-axis of the 57Fe magnetic hyperfine tensor, A, of NH(light) was found to be rotated by approximately 45 degrees relative to the z-axis of the g tensor (g(z) = 1.97). Comparison of the A tensor of NH(light) with the A tensors of low-spin ferric hemes indicates a substantially larger degree of covalency for nitrile hydratase. We have also performed photolysis experiments between 2 and 20 K and characterized the photolyzed products by EPR and M?ssbauer spectroscopy. Photolysis at 4.2 K in the M?ssbauer spectrometer yielded a five-coordinate low-spin ferric species, NH(A), which converted back into NH(dark) when the sample was briefly warmed to 77 K. We also describe preliminary EPR photolysis studies that have yielded new intermediates.     

Nonequivalence of subunits in [15N]nitrosylhemoglobin Kansas. A single crystal electron paramagnetic resonance investigation.

EPR spectra of Hb15NO crystals of mutant Kansas (Asn G4(102) beta leads to Thr) have been recorded at every 5' intervals and in three orthogonal planes. The nitrosylhemes are nonequivalent for the alpha and beta subunits, their assignments are made possible by comparison with the powder EPR specrtra of Hb15NO of mutant Iwate (His F8(87)alpha leads to Tyr) (Trittelvitz, E., Gersonde, K., and Winterhalter, K.H. (1975) Eur. J. Biochem. 51, 33-42). The EPR parameters for the beta-nitrosylhemes of Hb Kansas are: gxx=2.094 gyy=2.031, gzz=2.00, Azetazeta=11 G, Azetazeta=32.5 G, Aetaeta=12.5 G; the Fe-N-O bond angle is about 105 degrees. The paramters for the alpha-nitrosyl hemes are: gxx=2.058, gyy=2.021, gzz=1.977, Azetazeta=24.5 G, Azetazeta less than or equal to 5G, Aetaeta=23 G; the Fe-N-O bond angle is about 167 degrees. Hyperfine splittings of 7 to 8 gauss with 14Nepsilon atom of His(F8) were observed for the beta-nitrosylhemes; none was resolved for the alpha-nitrosylhemes. The results were interpreted to mean that the tension on the iron of the beta subunits is not large in the unliganded state and this tension was not greatly increased by the binding of nitric oxide in the strongly bent configuration. The tension at the iron in the deoxyhemoglobin is dominant at the alpha subunits. Binding of nitric oxide in this case causing either the breaking or great weakening of the Fe-His(F8) bond. The nitrosyl is in a nearly linear configuration. The unpaired electron densities at the nitrogen atom of the bound nitric oxide is about 63% for the beta-nitrosylheme and 37% for the alpha-nitrosylhemes.     

Single-crystal EPR study of novel azide complex of cyanogen bromide-modified myoglobin. Influence of specific modification of the heme distal histidyl residue on the ligand-binding structure

Stable azide complex of cyanogen bromide-modified met-myoglobin (metMb) was prepared and crystallized. The principal values and eigen vectors of g-tensor were determined by single-crystal EPR spectroscopy at 77 K: gxx = 1.50, gyy = 2.32, and gzz = 2.91. These g values were similar to those of tetrazole derivative rather than azide derivative of native metMbs, suggesting that tetrazole derivative might be formed from N-cyanoimidazole of distal histidyl residue via nucleophilic attack of azide ion by 1,3-dipolar cycloaddition reaction. The orientation of the maximal g value (gzz) of the novel product was found to deviate about 13 degrees from the heme normal of native aquometMb. Thus, the orientation of the heme plane might be altered in passing from native metMb to cyanogen bromide-mediated metmyoglobin. The present EPR results demonstrated that the modification of the histidyl residue at the heme distal side causes the changes in the stereochemical and electronic natures of the ligand binding to the heme.     

Structural studies of agar-gelatin complex coacervates by small angle neutron scattering, rheology and differential scanning calorimetry

Agar-gelatin complex coacervates are studied by small angle neutron scattering (SANS), rheology (in both flow and temperature scan modes) and differential scanning calorimetry (DSC) in order to probe the microscopic structure of this dense protein-polysaccharide-rich phase. DSC and isochronal temperature sweep (rheology) experiments yielded a characteristic temperature at approximately 35+/-2 degrees C. Rheology data revealed a second characteristic temperature at approximately 75+/-5 degrees C which was absent in DSC thermograms. In the flow mode, shear viscosity (eta) was found to scale with (Carreau model) applied shear rate (gamma ) as: eta(gamma ) approximately (gamma )(-k) with k=1.2+/-0.2 indicating non-Newtonian and shear-thinning features independent of ionic strength. The static structure factor S(q) deduced from SANS data in the low wave vector (0.018 A(-1)相似文献   

The reaction of nitrogen monoxide and of nitrite with deoxyhaemocyanin and methaemocyanin of Helix pomatia.

Deoxyhaemocyanin, treated with NO under strictly anaerobic conditions, yielded methaemocyanin and N2O in a fast reaction. In a further slow reaction this methaemocyanin lost its triplet electron paramagnetic resonance (EPR) signal at g = 4 and yielded a nitrosyl derivative with a characteristic g = 2 Cu(II) EPR signal, indicating the binding of a single NO per copper pair. Thus under strictly anaerobic conditions deoxyhaemocyanin and methaemocyanin, treated with NO, gave the same derivative as shown by circular dichroism and EPR spectra. Methaemocyanin yielded, moreover, reversibly a nitrite derivative, characterized by a triplet signal at g = 4 with 7 hyperfine lines.     

Ligand orientation of oxyproto- and oxymesocobalt porphyrin-substituted myoglobin by single crystal EPR spectroscopy

Single crystals of oxyproto- and oxymesocobalt myoglobin have been examined by electron paramagnetic resonance spectroscopy at ambient and cryogenic temperatures in order to determine the principal values and eigenvectors of g tensors and the hyperfine coupling tensors. The Co--O--O bond angle was determined to be 125 degrees +/- 5 degrees for oxyprotocobalt myoglobin, and 153 degrees +/- 5 degrees for oxymesocobalt myoglobin at ambient temperature. This result suggests that differences in stereochemical interactions of the modified 2,4-side chains of porphyrin with protein contribute to the ligand orientations as well as the altered ligand-binding behavior in these hemoproteins. Upon freezing, two unequivalent orientations of the O--O axis (species I and II) were found in both oxycobalt myoglobin single crystals. Shifts of the resonance spectra of these species were observed below the freezing point of the crystals. The signal intensities of two paramagnetic species in oxyprotocobalt myoglobin were approximately equivalent (I congruent to II), whereas those in oxymesocobalt myoglobin were quite different (I greater than II) at 77 K. The present electron paramagnetic resonance studies demonstrate that changes in the bonding structure of Co--O2 are induced upon freezing the biological macromolecule, including the movement of the residues of the heme environment.     

Crystal structures of ferrous horse heart myoglobin complexed with nitric oxide and nitrosoethane

The interactions of nitric oxide (NO) and organic nitroso compounds with heme proteins are biologically important, and adduct formation between NO-containing compounds and myoglobin (Mb) have served as prototypical systems for studies of these interactions. We have prepared crystals of horse heart (hh) MbNO from nitrosylation of aqua-metMb crystals, and we have determined the crystal structure of hh MbNO at a resolution of 1.9 A. The Fe-N-O angle of 147 degrees in hh MbNO is larger than the corresponding 112 degrees angle previously determined from the crystal structure of sperm whale MbNO (Brucker et al., Proteins 1998;30:352-356) but is similar to the 150 degrees angle determined from a MS XAFS study of a frozen solution of hh MbNO (Rich et al., J Am Chem Soc 1998;120:10827-10836). The Fe-N(O) bond length of 2.0 A (this work) is longer than the 1.75 A distance determined from the XAFS study and suggests distal pocket influences on FeNO geometry. The nitrosyl N atom is located 3.0 A from the imidazole N(epsilon) atom of the distal His64 residue, suggesting electrostatic stabilization of the FeNO moiety by His64. The crystal structure of the nitrosoethane adduct of ferrous hh Mb was determined at a resolution of 1.7 A. The nitroso O atom of the EtNO ligand is located 2.7 A from the imidazole N(epsilon) atom of His64, suggesting a hydrogen bond interaction between these groups. To the best of our knowledge, the crystal structure of hh Mb(EtNO) is the first such determination of a nitrosoalkane adduct of a heme protein.     

EPR and X-ray crystallographic characterization of the product-bound form of the MnII-loaded methionyl aminopeptidase from Pyrococcus furiosus

Methionine aminopeptidases (MetAPs) are ubiquitous metallohydrolases that remove the N-terminal methionine from nascent polypeptide chains. Although various crystal structures of MetAP in the presence of inhibitors have been solved, the structural aspects of the product-bound step has received little attention. Both perpendicular- and parallel-mode electron paramagnetic resonance (EPR) spectra were recorded for the Mn(II)-loaded forms of the type-I (Escherichia coli) and type-II (Pyrococcus furiosus) MetAPs in the presence of the reaction product l-methionine (L-Met). In general, similar EPR features were observed for both [MnMn(EcMetAP-I)]-L-Met and [MnMn(PfMetAP-II)]-L-Met. The observed perpendicular-mode EPR spectra consisted of a six-line hyperfine pattern at g = 2.03 (A = 8.8 mT) with less intense signals with eleven-line splitting at g = 2.4 and 1.7 (A = 4.4 mT). The former feature results from mononuclear, magnetically isolated Mn(II) ions and this signal are 3-fold more intense in the [MnMn(PfMetAP-II)]-L-Met EPR spectrum than in the [MnMn(EcMetAP-I)]-L-Met spectrum. Inspection of the EPR spectra of both [MnMn(EcMetAP-I)]-L-Met and [MnMn(PfMetAP-II)]-L-Met at 40 K in the parallel mode reveals that the [Mn(EcMetAP-I)]-L-Met spectrum exhibits a well-resolved hyperfine split pattern at g = 7.6 with a hyperfine splitting constant of A = 4.4 mT. These data suggest the presence of a magnetically coupled dinuclear Mn(II) center. On the other hand, a similar feature was not observed for the [MnMn(PfMetAP-II)]-L-Met complex. Therefore, the EPR data suggest that L-Met binds to [MnMn(EcMetAP-I)] differently than [MnMn(PfMetAP-II)]. To confirm these data, the X-ray crystal structure of [MnMn(PfMetAP-II)]-L-Met was solved to 2.3 A resolution. Both Mn1 and Mn2 reside in a distorted trigonal bipyramidal geometry, but the bridging water molecule, observed in the [CoCo(PfMetAP-II)] structure, is absent. Therefore, L-Met binding displaces this water molecule, but the carboxylate oxygen atom of L-Met does not bridge between the two Mn(II) ions. Instead, a single carboxylate oxygen atom of L-Met interacts with only Mn1, while the N-terminal amine nitrogen atom binds to M2. This L-Met binding mode is different from that observed for L-Met binding [CoCo(EcMetAP-I)]. Therefore, the catalytic mechanisms of type-I MetAPs may differ somewhat from type-II enzymes when a dinuclear metalloactive site is present.     

Calculation of translational friction and intrinsic viscosity. I. General formulation for arbitrarily shaped particles.

A general method for calculating translational friction and intrinsic viscosity is developed through exploiting relations between hydrodynamics and electrostatics. An approximate relation xi = 6 pi eta 0C between the translational friction coefficient xi of a particle (eta 0: solvent viscosity) and its capacitance C was derived previously. This involved orientationally preaveraging the Oseen tensor, but the result was found to be very accurate. Based on preaveraging, we find that the intrinsic viscosity [eta] of a particle can be estimated from its polarizability alpha through [eta] = 3/4 alpha + 1/4 Vp, where Vp is the volume of the particle. Both the capacitance and the polarizability can be obtained in a single calculation using the boundary-element technique. An efficient approach is thus found for estimating [eta], a quantity that is very useful in practice because of its sensitivity to particle shape but is notoriously difficult to calculate. Illustrative calculations on ellipsoids, cylinders, and dumbbells demonstrate both the accuracy of the approximate relations and the efficiency of the present method.     

Multi-frequency EPR and high-resolution M?ssbauer spectroscopy of a putative [6Fe-6S] prismane-cluster-containing protein from Desulfovibrio vulgaris (Hildenborough). Characterization of a supercluster and superspin model protein.

The putative [6Fe-6S] prismane cluster in the 6-Fe/S-containing protein from Desulfovibrio vulgaris, strain Hildenborough, has been enriched to 80% in 57Fe, and has been characterized in detail by S-, X-, P- and Q-band EPR spectroscopy, parallel-mode EPR spectroscopy and high-resolution 57Fe M?ssbauer spectroscopy. In EPR-monitored redox-equilibrium titrations, the cluster is found to be capable of three one-electron transitions with midpoint potentials at pH 7.5 of +285, +5 and -165 mV. As the fully reduced protein is assumed to carry the [6Fe-6S]3+ cluster, by spectroscopic analogy to prismane model compounds, four valency states are identified in the titration experiments: [6Fe-6S]3+, [6Fe-6S]4+, [6Fe-6S]5+, [6Fe-6S]6+. The fully oxidized 6+ state appears to be diamagnetic at low temperature. The prismane protein is aerobically isolated predominantly in the one-electron-reduced 5+ state. In this intermediate state, the cluster exists in two magnetic forms: 10% is low-spin S = 1/2; the remainder has an unusually high spin S = 9/2. The S = 1/2 EPR spectrum is significantly broadened by ligand (2.3 mT) and 57Fe (3.0 mT) hyperfine interaction, consistent with a delocalization of the unpaired electron over 6Fe and indicative of at least some nitrogen ligation. At 35 GHz, the g tensor is determined as 1.971, 1.951 and 1.898. EPR signals from the S = 9/2 multiplet have their maximal amplitude at a temperature of 12 K due to the axial zero-field splitting being negative, D approximately -0.86 cm-1. Effective g = 15.3, 5.75, 5.65 and 5.23 are observed, consistent with a rhombicity of [E/D] = 0.061. A second component has g = 9.7, 8.1 and 6.65 and [E/D] = 0.108. When the protein is reduced to the 4+ intermediate state, the cluster is silent in normal-mode EPR. An asymmetric feature with effective g approximately 16 is observed in parallel-mode EPR from an integer spin system with, presumably, S = 4. The fully reduced 3+ state consists of a mixture of two S = 1/2 ground state. The g tensor of the major component is 2.010, 1.825 and 1.32; the minor component has g = 1.941 and 1.79, with the third value undetermined. The sharp line at g = 2.010 exhibits significant convoluted hyperfine broadening from ligands (2.1 mT) and from 57Fe (4.6 mT). Zero-field high-temperature M?ssbauer spectra of the protein, isolated in the 5+ state, quantitatively account for the 0.8 fractional enrichment in 57Fe, as determined with inductively coupled plasma mass spectrometry.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gamma-irradiation potentiates L-arginine-dependent nitric oxide formation in mice.

Gamma-irradiation of mongrel mice at a sublethal dose (700 Roentgen) enhanced the formation of nitric oxide (NO) in the liver, intestine, lung, kidney, brain, spleen or heart of the animals. NO formation was determined by the increase in intensity of the EPR signal due to trapping of NO into mononitrosyl iron complexes (MNIC) with exogenous diethyldithiocarbamate (DETC) injected intraperitoneally. The EPR signal of these MNIC-DETC complexes was characterized by g-factor values at g perpendicular values at g perpendicular = 2.035 and g parallel = 2.02 and a triplet hyperfine structure at g perpendicular. The NO synthase inhibitor, NG-nitro-L-arginine, prevented MNIC-DETC complex formation both in liver and intestine, demonstrating the involvement of endogenous NO formed. Thus, gamma-irradiation may enhance endogenous NO biosynthesis in these tissues, presumably by facilitating the entry of Ca2+ ions into the membrane as well as the cytosol of NO-producing cells through irradiation-induced membrane lesions.     

Proton electron nuclear double resonance from nitrosyl horse heart myoglobin: the role of His-E7 and Val-E11

Electron nuclear double resonance (ENDOR) spectroscopy has been used to study protons in nitrosyl horse heart myoglobin (MbNO). (1)H ENDOR spectra were recorded for different settings of the magnetic field. Detailed analysis of the ENDOR powder spectra, using computer simulation, based on the "orientation-selection" principle, leads to the identification of the available protons in the heme pocket. We observe hyperfine interactions of the N(HisF8)-Fe(2+)-N(NO) complex with five protons in axial and with eight protons in the rhombic symmetry along different orientations, including those of the principal axes of the g-tensor. Protons from His-E7 and Val-E11 residues are identified in the two symmetries, rhombic and axial, exhibited by MbNO. Our results indicate that both residues are present inside the heme pocket and help to stabilize one particular conformation.     

A simple method for determination of rotational correlation times and separation of rotational and polarity effects from EPR spectra of spin-labeled biomolecules in a wide correlation time range

A method using nitroxide radical spin labels for determining both the isotropic rotational correlation time tau R and the environmental polarity of the label is described. By means of a least square fitting method, the values of an effective hyperfine tensor A' and of an effective g value tensor g' of randomly oriented spin labels are determined from X-band EPR spectra on the basis of an effective time-independent Hamiltonian. The traces of the tensors deliver the information about the environmental polarity of the label and are not dependent on the rotational correlation time tau R. A new averaging parameter S (tau R), calculated on the basis of the principal values of the tensor A', permits the evaluation of the rotational correlation time tau R in a very wide time range between 10(-10) and 10(-6) s. An application of this method to spin-labeled methemoglobin over a large temperature range and in environments of different polarity is discussed.