Electron paramagnetic resonance studies of iron-sulfur centers in mitochondria prepared from three Morris hepatomas with different growth rates

Iron-sulfur centers in mitochondria prepared from Morris hepatomas with different growth rates were compared with those in host liver and nontumor-bearing rat liver mitochondria by EPR measurements (< 77° K). In the slow growing hepatoma 16, EPR signals from iron-sulfur centers located in the NADH dehydrogenase region were specifically diminished. In the rapidly growing hepatoma 7777, EPR signals of all the iron-sulfur centers showed considerably diminished intensity. In hepatoma 7800 having an intermediate growth rate, all iron-sulfur centers showed no change. Those changes in iron-sulfur centers correlated with observed respiratory activities of Morris hepatoma mitochondria. No general correlation was obtained between these parameters and the growth rate of the tumors.     

Formate dehydrogenase from Methanobacterium formicicum. Electron paramagnetic resonance spectroscopy of the molybdenum and iron-sulfur centers

Formate dehydrogenase from Methanobacterium formicicum was examined by electron paramagnetic resonance spectroscopy. Although oxidized enzyme yielded no EPR signals over the temperature range 8-200 K, dithionite reduction resulted in generation of two paramagnetic components. The first, a nearly isotropic signal visible at temperatures below 200 K with g1 = 2.018, g2 = 2.003, and g3 = 1.994, exhibited nuclear hyperfine interaction with two equivalent protons (A1 = 0.45, A2 = 0.6, and A3 = 0.55 milliTeslas). EPR spectra of partially reduced 95Mo-enriched formate dehydrogenase exhibited additional 3-4 milliTeslas splittings, due to spin interaction with the 95Mo nucleus. Thus, this signal is due to a Mo center. This is the first reported example of a Mo center with gav greater than 2.0 in a biological system. The second species, a rhombic signal visible below 40 K with g values of g1 = 2.0465, g2 = 1.9482, and g3 = 1.9111 showed no hyperfine coupling and was assigned to reduced Fe/S. Both paramagnetic species could be detected in samples of M. formicicum whole cells anaerobically reduced with sodium formate. The Mo(V) signal was altered following addition of cyanide (g1 = 1.996, g2 = 1.988, and g3 = 1.980). Growth of bacteria in the presence of 1 mM WO4(2-) resulted in abolition of the Mo(V) EPR signal and formate dehydrogenase activity. Em, 7.7 was -330 mV for Mo(VI)/Mo(V) and -470 mV for Mo(V)/Mo(IV).     

Electron paramagnetic resonance studies of zinc-substituted reaction centers from Rhodopseudomonas viridis.

The primary quinone acceptor radical anion Q(A)(-)(*) (a menaquinone-9) is studied in reaction centers (RCs) of Rhodopseudomonas viridis in which the high-spin non-heme Fe(2+) is replaced by diamagnetic Zn(2+). The procedure for the iron substitution, which follows the work of Debus et al. [Debus, R. J., Feher, G., and Okamura, M. Y. (1986) Biochemistry 25, 2276-2287], is described. In Rps. viridisan exchange rate of the iron of approximately 50% +/- 10% is achieved. Time-resolved optical spectroscopy shows that the ZnRCs are fully competent in charge separation and that the charge recombination times are similar to those of native RCs. The g tensor of Q(A)(-)(*) in the ZnRCs is determined by a simulation of the EPR at 34 GHz yielding g(x) = 2.00597 (5), g(y) = 2.00492 (5), and g(z) = 2.00216 (5). Comparison with a menaquinone anion radical (MQ(4)(-)(*)) dissolved in 2-propanol identifies Q(A)(-)(*) as a naphthoquinone and shows that only one tensor component (g(x)) is predominantly changed in the RC. This is attributed to interaction with the protein environment. Electron-nuclear double resonance (ENDOR) experiments at 9 GHz reveal a shift of the spin density distribution of Q(A)(-)(*) in the RC as compared with MQ(4)(-)(*) in alcoholic solution. This is ascribed to an asymmetry of the Q(A) binding site. Furthermore, a hyperfine coupling constant from an exchangeable proton is deduced and assigned to a proton in a hydrogen bond between the quinone oxygen and surrounding amino acid residues. By electron spin-echo envelope modulation (ESEEM) techniques performed on Q(A)(-)(*) in the ZnRCs, two (14)N nuclear quadrupole tensors are determined that arise from the surrounding amino acids. One nitrogen coupling is assigned to a N(delta)((1))-H of a histidine and the other to a polypeptide backbone N-H by comparison with the nuclear quadrupole couplings of respective model systems. Inspection of the X-ray structure of Rps. viridis RCs shows that His(M217) and Ala(M258) are likely candidates for the respective amino acids. The quinone should therefore be bound by two H bonds to the protein that could, however, be of different strength. An asymmetric H-bond situation has also been found for Q(A)(-)(*) in the RC of Rhodobacter sphaeroides. Time-resolved electron paramagnetic resonance (EPR) experiments are performed on the radical pair state P(960)(+) (*)Q(A)(-)(*) in ZnRCs of Rps. viridis that were treated with o-phenanthroline to block electron transfer to Q(B). The orientations of the two radicals in the radical pair obtained from transient EPR and their distance deduced from pulsed EPR (out-of-phase ESEEM) are very similar to the geometry observed for the ground state P(960)Q(A) in the X-ray structure [Lancaster, R., Michel, H. (1997) Structure 5, 1339].     

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.     

Purification and characterization of the complex I from the respiratory chain of Rhodothermus marinus

The rotenone sensitive NADH: menaquinone oxidoreductase (NDH-I or complex I) from the thermohalophilic bacterium Rhodothermus marinus has been purified and characterized. Three of its subunits react with antibodies against 78, 51, and 21.3c kDa subunits of Neurospora crassa complex I. The optimum conditions for NADH dehydrogenase activity are 50°C and pH 8.1, and the enzyme presents a K _M of 9 M for NADH. The enzyme also displays NADH:quinone oxidoreductase activity with two menaquinone analogs, 1,4-naphtoquinone (NQ) and 2,3-dimethyl-1,4-naphtoquinone (DMN), being the last one rotenone sensitive, indicating the complex integrity as purified. When incorporated in liposomes, a stimulation of the NADH:DMN oxidoreductase activity is observed by dissipation of the membrane potential, upon addition of CCCP. The purified enzyme contains 13.5 ± 3.5 iron atoms and 3.7 menaquinone per FMN. At least five iron—sulfur centers are observed by EPR spectroscopy: two [2Fe–2S]^2+/1+ and three [4Fe–4S]^2+/1+ centers. By fluorescence spectroscopy a still unidentified chromophore was detected in R. marinus complex I.     

Electron paramagnetic resonance studies on the reduction of the components of complex I and transhydrogenase-inhibited complex I by NADH and NADPH.

NADH treatment of complex I at pH 7–8 results in the appearance of electron paramagnetic resonance (epr) signals at x band due to reduced ironsulfur centers 1, 2, 3 and 4, while NADPH treatment gives rise to the appearance of signals due to centers 2 and 3. Similar results are obtained with complex I preparations in which transhydrogenase activity from NADPH to NAD has been >95% inhibited by treatment of the complex with trypsin. At pH 6.5 and in the presence of rotenone, addition of NADPH to complex I or transhydrogenase-inhibited complex I results in partial reduction of iron-sulfur center 1 as well. These and other experiments with reduced 3-acetylpyridine adenine dinucleotide and NADPH + NAD as substrates have suggested that the differences in the reduction of complex I iron-sulfur centers by the above nucleotides are essentially quantitative and related to (a) the dehydrogenation rate of the nucleotides, and (b) autoxidation of complex I components under the epr experimental conditions.     

Electron paramagnetic resonance studies of spectrin-phospholipid associations

The interaction of spectrin, a peripheral cytoplasmic protein of the erythrocyte membrane, with synthetic phospholipids was characterized by density gradient centrifugation, electron microscopy, and the paramagnetic resonance of nitroxide spin labels. The organic solvent 2-chloroethanol, which favors the stability of hydrophobic surfaces on proteins, was utilized in the formation of the protein-lipid systems. Spectrin, upon dialysis to remove 2-chloroethanol, was found to associate into extensive network-like aggregates and in the presence of dipalmitoylphosphatidylcholine, the spectrin aggregates were found to associate with liposomes formed during dialysis. This interaction, which was significantly enhanced by the presence of dipalmitoylphosphatidylethanolamine, was found to reduce the mobility of fatty acid spin labels incorporated into the lipid regions of the lipid-protein associations. Evidence was found which suggests that spectrin tends to stabilize the phospholipid vesicles against fusion and decrease lipid mobility, particularly near the polar bilayer surfaces.     

Electron paramagnetic resonance properties and oxidation-reduction potentials of the molybdenum,flavin, and iron-sulfur centers of chicken liver xanthine dehydrogenase

The oxidation-reduction potentials of the various prosthetic groups in the native and desulfo forms of chicken liver xanthine dehydrogenase, determined by potentiometric titration in 0.05 m potassium phosphate buffer, pH 7.8, are: Mo(VI)/Mo(V) (native), ?357 mV; Mo(VI)/Mo(V) (desulfo), ?397 mV; Mo(V)/Mo(IV) (native), ?337 mV; Mo(V)/Mo(IV) (desulfo), ?433 mV; FAD/FADH · ?345 mV; FADH · FADH₂, ? 377 mV; (Fe/S)I_ox/(Fe/S)I_red, ?280 mV; (Fe/S)II_ox/(Fe/S)II_red, ? 275 mV. Titration at pH 6.8 revealed that the Mo and FAD centers but not the Fe/S centers are in prototropic equilibrium. Spectroscopic studies on the native and deflavinated enzymes show that environment of the flavin in xanthine dehydrogenase differs from that in bovine milk xanthine oxidase.     

Electron paramagnetic resonance studies of the low temperature photolytic behavior of oxidized hydrogenase I from Clostridium pasteurianum

The effects of CO and O2 on the EPR spectrum of oxidized Clostridium pasteurianum hydrogenase I have been investigated both before and after prolonged exposure to white light at 8 K and 30 K. Low concentrations of O2 were found to induce analogous changes in the EPR spectrum as CO, i.e. conversion of the rhombic signal with g approximately 2.10, 2.04, 2.00, a characteristic of the novel H2-activating center in oxidized Fe-hydrogenases, to an axial signal with g approximately 2.07, 2.01, 2.01. The results suggest a common binding site and mode of coordination for CO and O2 and permit rationalization of conflicting reports from different laboratories concerning the EPR properties of oxidized Fe-hydrogenases. The CO- and O2-induced axial EPR signals were found to be light-sensitive at low temperatures. Moreover, they exhibited indistinguishable and unusual photolysis behavior with the dominant photo-product being dependent on the temperature at which illumination was performed. At 8 K, photodissociation of CO or O2 occurs, resulting in an EPR signal identical with that of the oxidized enzyme in the absence of CO or O2. However, at 30 K, the dominant photoproduct is a rhombic EPR signal with g approximately 2.26, 2.12, 1.89. While the origin of this new EPR signal is uncertain, the g-value anisotropy and relaxation characteristics resemble those of a low spin Fe(III) center. These two photoproducts cannot be thermally or photolytically interconverted, but both are quantitatively reconverted to the original axial EPR signal on warming in the dark to 200 K. A tentative working hypothesis for the nature of the H2-activating center of Fe-hydrogenases is presented that is consistent with the available physiochemical data and permits rationalization of the novel photolysis behavior.     

A ferredoxin from the thermohalophilic bacterium Rhodothermus marinus

The substrate specificity of human sphingosine kinase was investigated using a bacterially expressed poly(His)-tagged protein. Only the D-erythro isomer of the sphingoid bases, sphinganine and sphingenine, was effectively phosphorylated. Long chain 1-alkanols, alkane-1,2-diols, 2-amino-1-alkanol or 1-amino-2-alkanol and short chain 2-amino-1,3-alkanediols were very poor substrates, indicating that the kinase is recognizing the chain length and the position of the amino and secondary hydroxy group. A free hydroxy group at carbon 3 is not a prerequisite, however, since 1-O-hexadecyl-2-desoxy-2-amino-sn-glycerol was an efficient substrate with an apparent K(m) value of 3.8 microM (versus 15.7 microM for sphingenine). This finding opens new perspectives to design sphingosine kinase inhibitors. It also calls for some caution since it cannot be excluded that this ether lipid analogue is formed from precursors that are frequently used in research on platelet activating factor or from phospholipid analogues which are less prone to degradation.     

Electron spin resonance studies of the bound iron-sulfur centers in Photosystem I. Photoreduction of center A occurs in the absence of center B

The Photosystem I acceptor system of a subchloroplast particle from spinach was investigated by optical and electron spin resonance (ESR) spectroscopy following graduated inactivation of the bound iron-sulfur proteins by urea/ferricyanide solution. The chemical analysis of iron and sulfur and the ESR properties of centers A, B and X are consistent with the participation of three iron-sulfur centers in Photosystem I. A differential decrease in centers A, B and X is observed under conditions that induce S^2? →S⁰ conversion in the bound iron-sulfur proteins. Center B is shown to be the most susceptible, while center ‘X’ is the least susceptible component to oxidative denaturation. Stepwise inactivation experiments suggest that electron transport in Photosystem I does not occur sequentially from X→B→A, since there is quantitative photoreduction of center A in the absence of center B. We propose that center A is directly reduced by X; thus, X may serve as a branch point for parallel electron flow through centers A and B.     

Electron paramagnetic resonance studies of a ras p21-MnIIGDP complex in solution.

The number of water molecules bound to Mn2+ in the complex with a variant of Ha ras p21 and GDP has been determined by electron paramagnetic resonance (EPR) measurements in 17O-enriched water. A resolution enhancement method has been used to improve quantitation of the spectral data. These spectroscopic measurements show that Mn2+ has four water ligands in this complex, a result in agreement with the conclusions of a previous paper [Smithers, G. W., Poe, M., Latwesen, D. G., & Reed, G. H. (1990) Arch. Biochem. Biophys. 280, 416-420]. The resolution enhancement method has also been applied in a measurement of the 17O-Mn2+ superhyperfine coupling constant of 17O in the beta-phosphate of the GDP in the ras p21 complex. The intrinsically narrow EPR signals of Mn2+ in the complex with ras p21 and GDP in 2H2O respond to resolution enhancement such that the superhyperfine splitting from the 17O nuclear spin (I = 5/2) becomes visible in the EPR signals. An 17O-Mn2+ superhyperfine coupling constant is obtained from simulation of the resolution-enhanced EPR spectrum.     

Electron paramagnetic resonance characterization of membrane bound iron-sulfur clusters and aconitase in plant mitochondria

Electron paramagnetic resonance (EPR) characteristics of the iron-sulfur clusters of potato tuber mitochondria have been examined in various subfractions of the mitochondria. We confirm that EPR signals comparable to those of the iron-sulfur proteins of mammalian mitochondria respiratory complexes are also present in plant mitochondria. Two distinct iron-sulfur centers paramagnetic in the oxidized state exhibit signals which differ in their detailed line shape and field position. One of these which is present in the inner membrane corresponds to center S.3. The EPR spectrum of the soluble fraction revealed the presence of another center with a low field maximum at g = 2.03 and is associated with aconitase. The EPR signal observed in the mitochondrial matrix from potato tuber and characteristic of 3Fe cluster is significantly changed in shape after addition of citrate and differs clearly from the spectrum of pig heart mitochondrial aconitase. The aconitase in plant mitochondria differs from that of mammalian mitochondria by several features.     

Electron paramagnetic resonance studies of Pseudomonas cytochrome c peroxidase

The EPR spectrum at 15 K of Pseudomonas cytochrome c peroxidase, which contains two hemes per molecule, is in the totally ferric form characteristic of low-spin heme giving two sets of g-values with gz 3.26 and 2.94. These values indicate an imidazole-nitrogen : heme-iron : methionine-sulfur and an imidazole-nitrogen : heme-iron : imidazole-nitrogen hemochrome structure, respectively. The spectrum is essentially identical at pH 6.0 and 4.6 and shows only a very small amount of high-spin heme iron (g 5--6) also at 77 K. Interaction between the two hemes is shown to exist by experiments in which one heme is reduced. This induces a change of the EPR signal of the other (to gz 2.83, gy 2.35 and gx 1.54), indicative of the removal of a histidine proton from that heme, which is axially coordinated to two histidine residues. If hydrogen peroxide is added to the partially reduced protein, its EPR signal is replaced by still other signals (gz 3.5 and 3.15). Only a very small free radical peak could be observed consistent with earlier mechanistic proposals. Contrary to the EPR spectra recorded at low temperature, the optical absorption spectra of both totally oxidized and partially reduced enzyme reveal the presence of high-spin heme at room temperature. It seems that a transition of one of the heme c moieties from an essentially high-spin to a low-spin form takes place on cooling the enzyme from 298 to 15 K.