The interaction between heme and protein in cytochrome c1

The optical spectrum of reduced bovine cytochrome c₁ at 77 K shows a fine splitting of the β-band, which is indicative of the native conformation of the protein. At room temperature, this conformation is reflected in an absorbance band at 530 nm. The exposure of the heme of ferrocytochrome c₁, investigated by means of solvent-perturbation spectroscopy, appears to be extremely sensitive to temperature and SH reagents bound to the oxidized protein. Addition of combinations of potential ligands to the isolated tryptic heme peptide of cytochrome c₁ reveals that only a mixture of methionine and cysteine (or their equivalents) generates a β-band at 77 K which is identical in shape to that of native cytochrome c₁. In the EPR spectrum of a complex of ferrocytochrome c₁ and nitric oxide at pH 10.5, no hyperfine splitting derived from a second ligated nitrogen atom could be detected. The results indicate that methionine and cysteine are the axial ligands of heme in cytochrome c₁. The EPR spectrum of isolated ferricytochrome c₁ is that of a low-spin heme iron compound with a g_z value of 3.36 and a g_y value of 2.04.     

Spectral study of ascorbate oxidase

The electronic, CD and EPR spectra of ascorbate oxidase isolated from the green zucchini squash (Cucurbita pepo medullosa) in 0.1 M phosphate buffer (pH 6.8) have been investigated. The visible absorption bands are clearly resolved in the CD spectrum, where the extrema occur at 735, 610, 550, 475 and 330 nm, while weak additional CD activity possibly occurs near 420 nm. The near-UV spectrum is dominated by the absorption of the aromatic amino acid residues centered at 280 nm, while resolved CD bands occur at 296, 291, 283, 265 and 240 nm. In the far-UV region the protein CD spectrum reflects its secondary structure: a single negative maximum at 218 nm suggests a predominant anti-parallel β conformation for ascorbate oxidase. The frozen solution EPR spectrum of the protein has been fitted according to a new computer simulation procedure. The following parameters were obtained: for the type 1 copper g_z = 2.222, g_x = 2.032, g_y = 2.056, A_z = 59 G, A_x = 11 G, and A_y = 5 G; for the type 2 copper g ? = 2.240, g_⊥ = 2.057, A? = 179 G and A_⊥ = 1 G. Of the eight copper atoms present in the protein four are EPR-detectable: three of type 1 and one of type 2, as shown by computer simulation of the EPR spectrum. Ascorbate oxidase is a rather unstable protein when purified and it is sensitive to a number of environmental factors. Aging of the protein leads to a decrease in the ratio between the type 1 and type 2 coppers. A new species formed at the early stages of the aging process, that has been spectrally characterized, suggests that the loss of the type 1 copper is preceded by a change in the symmetry of the original type 1 site from pseudotetrahedral to pseudotetragonal.     

The EPR spectrum of isolated Complex III

It has been found that antimycin shifts the EPR signal of oxidized Complex III at g = 3.44 (ferricytochrome b-562) to g = 3.48, while the signal at g = 3.8 (ferricytochrome b-566) sharpens. Antimycin also affects the optical spectrum of ferricytochrome b by sharpening the α-band and splitting the γ-band. It is shown that nitric oxide reacts irreversibly with the non-heme iron components of Complex III. A reaction of NO with ferrocytochrome b-566 is suggested, resulting in lines at g = 2.10, 2.07 and 2.01.     

Thermodynamic and EPR characterization of iron-sulfur centers in the NADH-ubiquinone segment of the mitochondrial respiratory chain in pigeon heart

Several iron-sulfur centers in the NADH-ubiquinone segment of the respiratory chain in pigeon heart mitochondria and in submitochondrial particles were analyzed by the combined application of cryogenic EPR (between 30 and 4.2 °K) and potentiometric titration.Center N-1 (iron-sulfur centers associated with NADH dehydrogenase are designated with the prefix “N”) resolves into two single electron titrations with E_{m 7.2} values of ?380±20 mV and ?240±20 mV (Centers N-1a and N-1b, respectively). Center N-1a exhibits an EPR spectrum of nearly axial symmetry with g_// = 2.03, g_⊥ = 1.94, while that of Center N-1b shows more apparent rhombic symmetry with g_z = 2.03, g_y = 1.94 and g_x = 1.91. Center N-2 also reveals EPR signals of axial symmetry at g_// = 2.05 and g_⊥ = 1.93 and its principal signal overlaps with those of Centers N-1a and N-1b. Center N-2 can be easily resolved from N-1a and N-1b because of its high E_{m 7.2} value (?20±20 mV).Resolution of Centers N-3 and N-4 was achieved potentiometrically in submitochondrial particles. The component with E_{m 7.2} = ? 240±20 mV is defined as Center N-3 (g_z = 2.10, (g_y = 1.93?), g_x = 1.87); the ?405±20 mV component as Center N-4 (g_z = 2.11, (g_y = 1.93?), g_x = 1.88). At temperatures close to 4.2 °K, EPR signals at g = 2.11, 2.06, 2.03, 1.93, 1.90 and 1.88 titrate with E_{m 7.2} = ?260±20 mV. The multiplicity of peaks suggests the presence of at least two different ironsulfur centers having similar E_{m 7.2} values (?260±20 mV); hence, tentatively assigned as N-5 and N-6.Consistent with the individual E_{m 7.2} values obtained, addition of succinate results in the partial reduction of Center N-2, but does not reduce any other centers in the NADH-ubiquinone segment of the respiratory chain. Centers N-2, N-1b, N-3, N-5 and N-6 become almost completely reduced in the presence of NADH, while Centers N-1a and N-4 are only slightly reduced in pigeon heart submitochondrial particles. In pigeon heart mitochondria, the E_{m 7.2} of Center N-4 lies much closer to that of Center N-3, so that resolution of the Center N-3 and N-4 spectra is not feasible in mitochondrial preparations. E_{m 7.2} values and EPR lineshapes for the other ironsulfur centers of the NADH-ubiquinone segment in the respiratory chain of intact mitochondria are similar to those obtained in submitochondrial particle preparations. Thus, it can be concluded that, in intact pigeon heart mitochondria, at least five iron-sulfur centers show E_{m 7.2} values around -250 mV; Center N-2 exhibits a high E_{m 7.2} (?20±20 mV), while Center N-1a shows a very low E_{m 7.2} (?380±20 mV).     

Isolation from bovine liver mitochondria of a soluble ferredoxin active in a reconstituted steroid hydroxylation reaction.

An iron-sulfur protein has been isolated from bovine liver mitochondria and purified 140-fold on DEAE-cellulose and Sephadex G-100. During the isolation the protein was detected by its NADPH-cytochrome $\text{c}$ reductase activity in the presence of adrenal NADPH-ferredoxin reductase. The molecular weight of the protein (12,400), the optical spectrum (peaks at 414 nm and 455 nm which disappear upon reduction), and the EPR spectrum (g_x = g_y = 1.935 and g_z = 2.02) were typical for a ferredoxin. In the presence of soluble adrenal cytochrome P₄₅₀, ferredoxin reductase and NADPH, this protein could support the formation of pregnenolone from cholesterol. Under similar conditions, but in the presence of a cytochrome P₄₅₀ solubilized from rat liver mitochondria, cholesterol was transformed into a more polar compound tentatively identified as 26-hydroxycholesterol.     

Effect of inositol hexakisphosphate on the EPR properties of the nitric oxide derivative of ferrous dromedary (Camelus dromedarius) hemoglobin. Evidence for two polyanion binding sites

The effect of inositol hexakisphosphate on the EPR properties of the nitric oxide derivative of ferrous dromedary (Camelus dromedarius) hemoglobin has been investigated at 110 K. In the absence of inositol hexakisphosphate, the nitrosyl derivative of dromedary hemoglobin shows an EPR spectrum with a rhombic shape and a weak hyperfine splitting in the gz region, a feature that is generally taken as characteristic of the high-affinity state of tetrameric hemoproteins. On addition of 1 mole of inositol hexakisphosphate/tetramer, three new hyperfine lines (Az = 1.7 mT), centered at gz = 2.01, appear; this type of spectrum is indicative of the low-affinity state of hemoglobins. A further addition of inositol hexakisphosphate, corresponding to a 20-fold molar excess, completely reverses the polyphosphate-dependent transition, giving an EPR spectrum that is exactly superimposable to that observed in the absence of the allosteric effector, i.e., is typical of the high-affinity state of the macromolecule. Both in the absence and presence of inositol hexakisphosphate, the EPR spectra are virtually independent of pH in the range explored (from 5.5 to 7.5). These results, taken together with the behavior of the nitric oxide derivative of human hemoglobin, provide further evidence for the existance in dromedary hemoglobin of two polyanion binding sites that affect in an opposite way the conformational equilibrium of the macromolecule.     

Metallobleomycins: electron spin resonance study on nitrosyl complex of iron(II)-Bleomycin

The ESR spectrum of the bleomycin-Fe(II)NO complex shows rhombic symmetry with a triplet hyperfine interaction in the g_z signal, and its ESR parameters have been compared with those of the ferrous-NO complexes of hemoproteins. The substitution of ₁₄NO by ₁₅NO gives the transition from a triplet to a doublet in the g_z absorption with a concomitant change in the nitrogen hyperfine constant. The addition of DNA to the ferrous-NO complex of bleomycin induces the greater separation of the g_x and g_y absorptions in comparison with the original ESR spectrum. The present three-line g_z signal for the bleomycin-Fe(II)-NO complex is indicative of weakened fifth axial nitrogen ligand-to-iron bonding with concomitantly stronger NO-to-iron bonding. On the other hand, the ESR feature of the bleomycin-Fe(III) complex is typical of the rhombic low-spin type, and no stable ferric-NO complex of bleomycin is formed.     

Dinitrosyl iron complexes with glutathione largely relieve rats of experimental endometriosis

A study was made of the effect of binuclear dinitrosyl iron complexes (DNIC) with glutathione in rats with experimental endometriosis. The latter was induced in an autotransplantation model, where two fragments of endometrium with myometrium (2 × 2 mm) from the left uterine horn were grafted to the inner surface of the anterior abdominal wall. After 4 weeks, the test animals received i.p. injections of 0.5 mL DNIC-glutathione at a dose of 12.5 μmol/kg daily for 12 days. This treatment more than halved the total volume of endometrioid tumors. Remarkably, tumor growths from grafts in control rats were often attended by tumors spontaneously arising nearby or in other locations; no such secondary tumors were observed in DNIC-treated animals. The EPR signal with g _av = 2.03 characteristic of protein-bound DNIC with thiol ligands was recorded in liver and endometrioid implants of control as well as treated animals. Activation of ribonucleotide reductase, detected by a doublet EPR signal at g = 2.0 with 2.3-mT hyperfine splitting, was found in small tumors. The beneficial effect of DNIC-glutathione was suggested to be due to DNIC breakdown near the tumors, with release of a large amount of molecular nitric oxide and nitrosonium ions that resulted in selective local cytotoxicity.     

EPR spectral changes of nitrosyl hemes and their relation to the hemoglobin T-R transition

EPR spectra of nitrosyl hemes were used to study the quaternary structure of hemoglobin. Human adult hemoglobin has been titrated with nitric oxide at pH 7.0 and 25 degrees C. After the equilibration of NO among the alpha and beta subunits the samples were frozen for EPR measurements. The spectra were fitted by linear combinations of three standard signals: the first arising from NO-beta-hemes and the other two arising for NO-alpha-hemes of molecules in the high- and low-affinity conformations. The fractional amounts of alpha subunits exhibiting the high-affinity spectrum fitted the two-state model (Edelstein, S.J. (1974) Biochemistry 13, 4998-5002) with the allosteric constant L = 7.10(6) and relative affinities cNO alpha and cNO beta approx. 0.01. Hemoglobin has been marked with nitric oxide one chain using low-saturation amounts of nitric oxide. The EPR spectra was studied as a function of oxygen saturation. Linear combinations of the three standard signals above fitted these spectra. The fractions of molecules exhibiting the high-affinity spectrum fitted the two-state model with L = 7 . 10(6), c)2 = 0.0033 and cNO alpha = 0.08, instead of cNO alpha = 0.01. Thus, the two-state model is not adequate to describe the conformational transition of these hybrids. The results present evidence of the non-equivalence between oxygen and nitric oxide as ligands.     

Sources of divalent sulfur allow recovery of the Fnr [4Fe-4S]<Superscript>2+</Superscript> center in <Emphasis Type="Italic">Escherichia coli</Emphasis> incubated with nitric oxide donors

Dinitrosyl iron complexes (DNICs) with various thiol ligands, the known donors of nitric oxide, markedly inhibited aidB gene expression in E. coli cells by destroying the [4Fe-4S]²⁺ center of its regulator protein Fnr. Therewith, the cells accumulated DNICs in the protein-bound form, identified by the EPR signal with g _⊥ = 2.04 and g _‖ = 2.014. Subsequent addition of sulfur sources L-cysteine or N-acetylcysteine, DTT as well as Na₂S to the DNIC-treated cells significantly restored the reporter gene expression. Simultaneously, the above-specified EPR signal was partly or completely replaced with a narrower signal (g _⊥ = 2.032, g _‖ = 2.02) identical to that of DNICs with persulfide (R-S-S⁻) ligands, which result from interaction of S²⁻ with thiols; inorganic sulfide proved to be the most efficient agent. These data corroborate the central role of S²⁻ in recovery of the protein [4Fe-4S] center disrupted by the NO donors.     

Electron paramagnetic studies of the copper and iron containing soluble ammonia monooxygenase from <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis>

Soluble ammonia monooxygenase (AMO) from Nitrosomonas europaea was purified to homogeneity and metals in the active sites of the enzyme (Cu, Fe) were analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR spectra were obtained for a type 2 Cu(II) site with g_|| = 2.24, A_|| = 18.4 mT and g_⊥ = 2.057 as well as for heme and non heme iron present in purified soluble AMO from N. europaea. A second type 2 Cu(II) EPR signal with g_|| = 2.29, A_|| = 16.1 mT and g_⊥ = 2.03 appeared in the spectrum of the ferricyanide oxidized enzyme and was attributed to oxidation of cuprous sites. Comparison of EPR-detectable Cu²⁺ with total copper determined by inductively coupled plasma-mass spectrometry (ICP-MS) suggests that there are six paramagnetic Cu²⁺ and three diamagnetic Cu¹⁺ per heterotrimeric soluble AMO (two paramagnetic and one diamagnetic Cu per αβγ-protomer). A trigonal EPR signal at g = 6.01, caused by a high-spin iron, indicative for cytochrome bound iron, and a rhombic signal at g = 4.31, characteristic of specifically bound Fe³⁺ was detectable. The binding of nitric oxide in the presence of reductant resulted in a ferrous S = 3/2 signal, characteristic of a ferrous nitrosyl complex. Inactivation of soluble AMO with acetylene did neither diminish the ferrous signal nor the intensity of the Cu²⁺-EPR signal.     

EPR studies of the photodissociation reactions of cytochrome c oxidase-nitric oxide complexes

Three complexes of NO with cytochrome c oxidase are described which are all photodissociable at low temperatures as measured by EPR. The EPR parameters of the cytochrome a²⁺₃-NO complex are the same both in the fully reduced enzyme and in the mixed-valence enzyme. The kinetics of photodissociation of cytochrome a²⁺₃-NO and recombination of NO with cytochrome a²⁺₃ (in the 30–70 K region) revealed no differences in structure between cytochrome a²⁺₃ in the fully reduced and the mixed-valence states. The action spectrum of the photodissociation of cytochrome a²⁺₃-NO as measured by EPR has maxima at 595, 560 and 430 nm, and corresponds to the absorbance spectrum of cytochrome a²⁺₃-NO. Photodissociation of cytochrome a²⁺₃-NO in the mixed-valence enzyme changes the EPR intensity at g 3.03, due to electron transfer from cytochrome a²⁺₃ to cytochrome a³⁺. The extent of electron transfer was found to be temperature dependent. This suggests that a conformational change is coupled to this electron transfer. The complex of NO with oxidized cytochrome c oxidase shows a photodissociation reaction and recombination of NO (in the 20–40 K region) which differ completely from those observed in cytochrome a²⁺₃-NO. The observed recombination occurs at a temperature 15 K lower than that found for the cytochrome a²⁺₃-NO complex. The action spectrum of the oxidized complex shows a novel spectrum with maxima at 640 and below 400 nm; it is assigned to a Cu²⁺_B-NO compound. The triplet species with Δm_s = 2 EPR signals at g 4 and Δm_s = 1 signals at g 2.69 and 1.67, that is observed in partially reduced cytochrome c oxidase treated with azide and NO, can also be photodissociated.     

The orientation of iron-sulfur clusters and a spin-coupled ubiquinone pair in the mitochondrial membrane

Oriented multilayers made from beef heart and yeast mitochondria and submitochondrial particles were studied using electron paramagnetic resonance. EPR signals from membrane-bound iron-sulfur clusters and from a spin-coupled ubiquinone pair are highly orientation dependent, implying that these redox centers are fixed in the membrane at definite angles relative to the membrane plane. Typically the iron-iron axis (g_z) of the binuclear iron-sulfur clusters is in the membrane plane. This finding is discussed in terms of the protein structure. the tetranuclear iron-sulfur clusters can have their g_z axis either perpendicular or parallel to the membrane plane, but intermediate orientation was not observed.     

Spectroscopic and electrochemical properties of a new nitrosyl-complex of Ru(II): trans-[Ru(NO){(CH3CH2)2PCH2CH2P(CH2CH3)2}2Cl](PF6)2

A new ruthenium nitric oxide complex with the bidentate phosphine, 1,2-bis(diethylphosphino)ethane (depe), has been synthesized and characterized by UV-Vis, infrared, EPR, NMR, electrochemical techniques and X-ray structure determination. The electronic spectrum showed a typical band of dπ→pπ* charge-transfer (CT) transition, assigned to Ru(II)NO transition, and the vibrational spectrum exhibited a peak of nitrosyl ligand at (ν_NO=1851 cm⁻¹). A model structure for this complex has been proposed based on ¹H, ¹H{³¹P}, ³¹P{¹H}, ¹³C{¹H}, COSY ¹H¹H{³¹P}, J-Resolved, HSQC, HMBC, HSQC ¹H¹³C{³¹P} and ¹H¹³C HSQC/¹H¹H TOCSY spectral data, and confirmed by X-ray diffraction. The nitrosonium character for the NO ligand become evident through both electron paramagnetic resonance and X-ray data (angle RuNO=177.4(3)°). The reversible monoeletronic process at E_1/2=0.040 V versus SHE was assigned to the ligand NO⁺/NO redox couple. Under treatment with Cd(Hg) solutions containing the [Ru(NO)(depe)₂Cl](PF₆)₂ yields a signal in the EPR spectrum (g_⊥=1.99 and g_//=1.88) which fitted quite well with the simulated spectra of coordinated NO species.     

Analysis of the electron paramagnetic resonance spectrum of the cobalamin intermediate in ribonucleotide reduction

EPR absorption-derivative lineshapes have been computed and least-squares fitted to the spectrum of the intermediate derived from 5'-deoxy-5'-adenosyl-cobalamin in the ribonucleotide reductase reaction. A Gausian-type intrinsic lineshape was assumed and the effects of inhomogenous broadening, rotation of coordinate axes of the A-tensor relative to the g-tensor, angular dependence of transition probability and ligand hyperfine splitting have also been investigated.When the overall spectrum was computed as the sum of the linshapes corresponding to two distinct Co(II) species, A and B, each having rhombic asymmetry, the least squares procedure converged to a much better fit than with a single species, and matched almost all of the features of the experimental spectrum.The magnetic properties of A and B were compared with those of a series of other Co(II) complexes by a plot of g_|?g₆ versus ∥A₆∥?∥A_|∥. The results eliminate cobalt with 5-coordination to nitrogen for A and B, suggest low-spin cobalt complexes having strongly distorted 6-fold coordination. The possibility that the sixth, symmetry-decreasing ligand is the oxygen molecule is excluded by the chemistry of the system and by the EPR properties of previously reported cob(II)alamins. It is suggested that the sixth ligand is carbonyl, amide or sulfhydryl group of an enzyme sidechain which is inserted off-axis into the coordination position so as to exert the observed symmetry-lowering effect.     

The metal site of Pseudomonas aeruginosa azurin,revealed by a crystal structure determination of the co(II) derivative and co-EPR spectroscopy

The crystal structure of cobalt-substituted azurin from Pseudomonas aeruginosa has been determined to final crystallographic R value of 0.175 at 1.9 Å resolution. There are four molecules in the asymmetric unit in the structure, and these four molecules are packed as a dimer of dimers. The dimer packing is very similar to that of the wild-type Pseudomonas aeruginosa azurin dimer. Replacement of the native copper by the cobalt ion has only small effects on the metal binding site presumably because of the existence of an extensive network of hydrogen bonds in its immediate neighborhood. Some differences are obvious, however. In wild-type azurin the copper atom occupies a distorted trigonal bipyramidal site, while cobalt similar to zinc and nickel occupy a distorted tetrahedral site, in which the distance to the Met121,S^δ atom is increased to 3.3–3.5 Å and the distance to the carbonyl oxygen of Gly45 has decreased to 2.1–2.4 Å. The X-band EPR spectrum of the high-spin Co(II) in azurin is well resolved (apparent g values g_x′ = 5.23; g_y′ = 3.83; g_z′ = 1.995, and hyperfine splittings A_x′ = 31; A_y′ = 20–30; A_z′ = 53 G) and indicates that the ligand field is close to axial. Proteins 27:385–394, 1997. © 1997 Wiley-Liss, Inc.     

The Origin of the Multiline and g = 4.1 Electron Paramagnetic Resonance Signals from the Oxygen-Evolving System of Photosystem II

Continuous illumination at 200 K of photosystem (PS) II-enriched membranes generates two electron paramagnetic resonance (EPR) signals that both are connected with the S₂ state: a multiline signal at g 2 and a single line at g = 4.1. From measurements at three different X-band frequencies and at 34 GHz, the g tensor of the multiline species was found to be isotropic with g = 1.982. It has an excited spin multiplet at ~30 cm^-1, inferred from the temperature-dependence of the linewidth. The intensity ratio of the g = 4.1 signal to the multiline signal was found to be almost constant from 5 to 23 K. Based on these findings and on spin quantitation of the two signals in samples with and without 4% ethanol, it is concluded that they arise from the ground doublets of paramagnetic species in different PS II centers. It is suggested that the two signals originate from separate PS II electron donors that are in a redox equilibrium with each other in the S₂ state and that the g = 4.1 signal arises from monomeric Mn(IV).