Q-band electron paramagnetic resonance study of nitrosylprotoheme dimethyl ester complexes with N-,O-, and S-donor ligand as model systems for nitrosylhemoproteins

Electron paramagnetic resonance (epr) spectra (at X- and Q band frequency) of nitrosyl(proto-porphyrin IX dimethyl ester) iron( II) complexes with a trans axial ligand of nitrogen-, oxygen-, and sulfur-donor ligand, in the trozen glass state at 77°K, have been investigated in order to understand the epr spectra of nitrosylhemoproteins. The Q-band spectra resolved the spectral features more clearly than the X-band spectra and distinctly exhibited two groups of absorptions, which were attributable to two molecular species. Significant relations were found between two g values (e.g., g_x-g_z, g_x-g_y) and between the g value and the degree of the hyperfine splitting in central absorption. The epr parameters were not very sensitive to the π-bonding ability of the axial ligand, but registered the steric interaction of the axial ligand with porphynnato core. These findings can be utilized in the characterization of an axial ligand trans to the nitrosyl group in nitrosylhemoproteins.     

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.     

Multiple heme pocket subconformations of methemoglobin associated with distal histidine interactions

Electron paramagnetic resonance spectra of methemoglobin reveal that, in addition to the major tetragonal high-spin aqueous complex and the low-spin hydroxide complex, three other complexes associated with the interaction of the distal histidine are resolved. These are a rhombic high-spin and two classes of low-spin bis-histidine complexes. By freeze-quenching experiments it is shown that the rhombic high-spin and one of the low-spin bis-histidine complexes (B) are at equilibrium with the dominant species. Incubation in the 210-260 K temperature range shifts the total equilibrium toward a low-energy state with the distal histidine coordinated to the iron (complex C).     

Infrared and electron paramagnetic resonance study of nitrosyl(protoporphyrin IX dimethyl ester)iron(II) and its complexes with nitrogenous bases as model systems for nitrosylhemoproteins: effect of solvent polarity

Infrared and electron paramagnetic resonance spectra of nitrosyl(protoporphyrin IX dimethyl ester)iron(II)(Fe(PPDME)(NO)) and its complexes with nitrogenous bases (N bases) such as imidazoles, pyridines, aliphatic amines, and anilines have been measured in various solvents. At room temperature, giso, Aiso, and nu NO values of five-coordinate Fe(PPDME)(NO) decreased with an increase in solvent polarity parameter ET, indicating the interaction between the solvent and the vacant axial coordination position. It has been found that the nu NO value of six-coordinate species is very sensitive to the solvent polarity, while the giso value is less sensitive. The solvent effect on the equilibrium constants, which are evaluated from the intensity change of the NO stretching band for five- and six-coordinate species, is discussed.     

An infrared study of NO bonding to heme B and hemoglobin A. Evidence for inositol hexaphosphate induced cleavage of proximal histidine to iron bonds.

Five- and six-coordinate nitrosyl hemes have been prepared and their infrared, electron paramagnetic resonance (EPR), and visible-Soret spectra compared with the corresponding spectra for nitrosyl hemoglobin A (Hba-NO) determined both in the presence and the absence of inositol hexaphosphate (IHP). The five- and six-coordinate NO complexes prepared from either dipyridine or pyridine carbonyl protoheme dimethyl ester had N-O stretch bands (nuno) near 1675 and 1625 cm-1, respectively. These frequencies are sensitive to change in solvent (nuno decreased as the dipole moment of the solvent increased) and, with six-coordinate species, to changes in trans ligand. However, these solvent and trans ligand effects were small compared with the difference (ca. 50 cm-11) between five- and six -coordinate species. The nature of the trans ligand affected the relative proportions of the two...     

The acid-alkaline transition of a sea turtle myoglobin: coexistence at high pH of high- and low-spin forms

The microenvironment of the iron in a sea turtle Dermochelys coriacea myoglobin is studied using the spectroscopic techniques EPR and optical absorption. Optical absorption spectra in the visible region suggest a great homology between turtle Mb and other myoglobins, such as those from whale, human and elephant. The pK of the acid-alkaline transition is 8.4 slightly lower than the pK of whale and equal to that of elephant myoglobin. The EPR spectrum at pH 7.0 is characteristic of a high-spin configuration with axial symmetry (gx = gy = 5.95). At higher pH, this signal changes in a way different from that observed for whale myoglobin. We observe for turtle Mb both the formation of a low-spin configuration with rhombic symmetry (gx = 2.56, gy = 2.20, gz = 1.90) and of a high-spin species with rhombic distortion (gx = 6.79, gy = 5.18, gz = 2.12). This suggests a lowering of symmetry at the haem, so that now the x and y directions are no more equivalent. This can be explained by amino acid substitution at the distal positions of haem or to off-axial positioning of distal residues. The coexistence at high pH (pH 11.0) of these two spin forms could be explained by the existence of two protein conformations, in which the crystal field splitting factor, delta, and the electron exchange energy are of the same order, allowing the presence of different configurations simultaneously. The presence of different kinds of haem is ruled out by the experiments with nitrosyl turtle Mb and turtle Mb-F showing spectra very similar to those of whale myoglobin. The pk of the acid-alkaline transition, 8.5, obtained from EPR spectra, agrees very well with results from optical absorption.     

Electron paramagnetic resonance studies of heme c and its nitrosyl derivative in Vibrio (Achromobacter) fischeri nitrite reductase

Interactions of Vibrio (formerly Achromobacter) fischeri nitrite reductase were studied by electron paramagnetic resonance spectroscopy. The spectrum of the oxidized enzyme showed a number of features which were attributed to two low-spin ferric hemes. These comprised an unusual derivative peak at g = 3.7 and a spectrum at g = 2.88, 2.26, and 1.51. Neither heme was reactive in the oxidized state with the substrate nitrite and with cyanide and azide. When frozen under turnover conditions (i.e., reduction in the presence of excess nitrite), the enzyme showed the spectrum of a nitrosyl heme derivative. The g = 2.88, 2.26, and 1.51 signals reappeared partially on reoxidation by nitrite, indicating that the nitrosyl species which remained arose from the g = 3.7 heme. The nitrosyl derivative showed a 14N nuclear hyperfine splitting, Az = 1.65 mT. The nitrosyl derivative was produced by treatment of the oxidized nitrite reductase with nitric oxide or hydroxylamine. Exchange of nitric oxide between the nitrosyl derivative and NO gas in solution was observed by using the [15N]nitrosyl compound. A possible reaction cycle for the enzyme is discussed, which involves reduction of the enzyme followed by binding of nitrite to one heme and formation of the nitrosyl intermediate.     

Electron paramagnetic resonance study of ferrous cytochrome P-450scc-nitric oxide complexes: effects of cholesterol and its analogues

Electron paramagnetic resonance (EPR) spectra of nitric oxide (NO) complexes of ferrous cytochrome P-450scc were measured at 77 K for the first time without using the rapid-mixing and freeze-quenching technique. Without substrate the EPR spectra were very similar to those of cytochrome P-450cam (from Pseudomonas putida) and cytochrome P-450LM (from rat liver microsomes) with rhombic symmetry; gx = 2.071, gz = 2.001, gy = 1.962, and Az = 2.2 mT for 14NO complexes. Upon addition of substrates [such as cholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, 25-hydroxycholesterol, and 22-ketocholesterol], the EPR spectra exhibited many variations having rhombic symmetry in the major component and an additional minor component with less rhombic symmetry. Furthermore, addition of 20(S)-hydroxycholesterol caused a striking change in the EPR spectrum. The component with rhombic symmetry disappeared completely, and the component with less rhombic symmetry dominated (gx = 2.027, gz = 2.007, gy = 1.984, and Az = 1.76 mT for 14NO complexes). These observations suggest the existence of the following physiologically important natures: (1) the conformational flexibility of the active site of the enzyme due to the steric interaction between the substrate and the heme-bound ligand molecule and (2) the importance of the hydroxylation of the cholesterol side chain at the 20S position to proceed the side-chain cleavage reaction in cytochrome P-450scc.     

Resonance Raman and EPR of nitrosyl human hemoglobin and chains, carp hemoglobin, and model compounds. Implications for the nitrosyl heme coordination state.

We report the joint resonance Raman (RR) and electron paramagnetic resonance (epr) study of five- and six-coordinate nitrosyl heme model compounds and of the titled nitrosyl hemoproteins. Both epr and RR spectra fall into two types which, in the models, correspond to five- and six-coordinate nitrosyl hemes. However, neither RR nor epr spectroscopy is highly sensitive to the nature of the bond between a nitrosyl heme and a coordinated nitrogenous base, nor do the results of one technique uniformly correlate with those of the other. It is not possible to use epr spectroscopy as a test for the coordination state of a nitrosyl heme. The position of the highest frequency (depolarized) RR band possibly provides such a test. Any breaking of the very weak bond between nitrosyl heme and proximal histidine in T state human HbNO is more a consequence of tertiary structural features unique to the human alphaNO chains than it is of properties of the T quaternary conformation.     

EPR spectroscopy studies on the structural transition of nitrosyl hemoglobin in the arterial-venous cycle of DEANO-treated rats as it relates to the proposed nitrosyl hemoglobin/nitrosothiol hemoglobin exchange

In vivo studies show a dynamic cycle in which alpha-nitrosylated hemoglobin is mainly in the relaxed state in arterial blood of rats treated with 2-(N,N-diethylamino)-diazenolate-2-oxide, but converts mainly to the tense state during the arterial-venous transit. A detailed analysis shows that different electron paramagnetic resonance spectra recorded for alpha-nitrosyl hemoglobin in arterial and venous blood at 77 K originate only from a different ratio between 5- and 6-coordinate heme without any change in the concentration of nitrosyl hemoglobin. In venous blood, the five- and six-coordination equilibrium of the alpha-nitrosyl heme is shifted in favor of the 5-coordinate state (58% venous vs. 20% arterial). These results are not consistent with the recently proposed exchange of nitrosyl heme with the beta-93 nitrosothiol group of hemoglobin during the arterial-venous cycle.     

Electron paramagnetic resonance study of nitrosylprotoheme dimethyl ester complexes with aliphatic nitrogenous bases: characterization of the axial ligand trans to the nitrosyl group in nitrosylhemoproteins

The interaction of nitrosyl(protoporphyrin IX dimethyl ester) iron (II) (Fe(PPDME)(NO)) with aliphatic amines, anilines, and cyclic imines has been studied by electron paramagnetic resonance (EPR) measurements at room temperature and at 77K. At room temperature, the bases studied here were divided into two groups according to the exchange rate between two EPR-positive species, Fe(PPDME)(NO) and Fe(PPDME)(NO)B, in equilibrium; Fe(PPDME)(NO) + B

Fe(PPDME)-(NO)B. The Fe(PPDME)(NO)-base system with a fast exchange rate on the EPR time scale had a smaller equilibrium constant (K) than that with a slow rate. The EPR spectra of the Fe(PPDME)(NO)-base system both at room temperature and at 77K were markedly influenced by the steric interaction of the base with the porphyrin core.     

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 study of water distribution in starch granules

An electron paramagnetic resonance (EPR) study was performed for potato and wheat starch containing Cu2+ ions as a paramagnetic probe. Distribution of water in the starch granules as well as the interactions between the copper and starch matrix of different crystalline structures were determined. EPR spectra of the native starches consisted of two different centers of Cu2+. One of them, giving at 293 and 77 K an EPR signal of axial symmetry with a well-resolved hyperfine structure (HFS), was assigned to the Cu2+ -starch complex in which Cu2+ ions strongly interacted with oxygen atoms of the starch matrix. Another Cu2+ species, exhibiting an isotropic signal at 293 K and an axial signal with resolved HFS at 77 K, was attributed to a [Cu(H2O)6]2+ complex freely rotating at room temperature and immobilized at low temperatures. Interaction of Cu2+ with the starch matrix and the relative number of the particular copper species depended on the crystallographic type of starch. Dehydration at 393 K resulted in elimination of the rotating complex signal and decrease of the total intensity of the EPR spectrum caused by clustering of the Cu2+ ions. Freezing at 77 K and thawing led to restoring of the spectrum intensity and reappearing of the signal of the [Cu(H2O)6]2+ complex. This effect, related to liberation of water molecules from the granule semicrystalline growth rings on freezing/thawing, was especially visible for wheat starch, indicating differences in the water retention ability of starch granules of different crystallographic structure.     

Temperature dependence of NO binding modes in human neuroglobin

Both the ferrous and ferric forms of wild-type neuroglobin are found to be hexacoordinated with axial ligation of the F8-His and E7-His. Rapidly growing Escherichia coli cell cultures with low O2 concentration generate nitric oxide (NO). Combined electron paramagnetic resonance (EPR) and optical measurements show that wild-type human recombinant neuroglobin, overexpressed in such E. coli cells, still favors the F8His-Fe2+ -E7His conformation, whereby only a small fraction of the protein binds NO. Upon mutation of the E7-His to Leu and Gln, the competition with the distal histidine disappears and the nitrosyl ferrous form is readily observed. At low temperature, the EPR spectra of the NO-ligated Ngb proteins consist of contributions from two geometrically different NO-heme conformations. In combination with EPR data of vertebrate hemoglobins and myoglobins, the temperature dependence of the EPR spectra of the NO adducts of ferrous hNgb and its E7-mutants proves a strong stabilization of one isomer by the E7-histidine in wt hNgb. It is shown that this is not related to the polarity of histidine, but to its specific binding characteristics.     

Electron-conformational interactions at the active site of reduced bacterial cytochrome P450cam induced by a substrate and analysis of the electron structure of heme]

Magnetic circular dichroism (MCD) spectra in the Soret region (360-480 nm) of camphor-free and camphor-bound reduced bacterial cytochrome P450cam from Pseudomonas putida were recorded and analysed in the temperature range from 2 K to 290 K. The temperature dependences of the MCD intensity are qualitatively changed by binding of substrate to the enzyme. In the absence of camphor the linear increase of the MCD intensity with 1/T at T < 4.2 K gives evidence for degeneracy or near degeneracy of the ground electronic state. In the presence of substrate the degeneracy is removed and temperature profiles show saturation behaviour at T < 4.2 K and wavelength dependence of their high-temperature parts. The temperature profiles for the long-wavelength region of the Soret band have a maximum approximately at 15 K, whereas the MCD intensity increases in a monotonous manner up to saturation in the short-wavelength region. The wavelength dependence of temperature profiles gives evidence for the co-existence of two different forms of substrate-bound reduced P450cam. The following conclusions were obtained from a theoretical analysis of the temperature profiles. In the absence of substrate there are very small if any rhombic distortions at the heme iron, and a parameter D of axial zero-field splitting is negative (D = -8.3 cm-1 and -6.2 cm-1 for P450cam and P450LM2, respectively). In the presence of substrate the two forms of reduced P450cam have positive parameters D but of different values (D1 = 12 cm-1 and D2 = 28 cm-1), and there are large rhombic distortions at the heme iron. More than two-fold difference between the D values made it possible to isolate temperature-dependent contributions of the two enzyme forms from the total MCD spectra and to simulate the alterations of the MCD spectra with temperature for reduced P450cam in the presence of substrate. Taking into account the drastic effect of substrate binding on the ground electronic state of reduced P450cam one can suggest that substrate binding induces the transition of enzyme from an inactive to an active state.     

An Analysis of the Electron Spin Resonance of Low Spin Ferric Heme Compounds

The electron spin resonance spectra of low spin ferric heme proteins are calculated and compared with experimental spectra recently obtained for a large number of heme proteins. On the basis of observed g values, these heme proteins have been categorized into five prototypes. Since electron spin resonance spectra are quite sensitive to small changes in the local environment about the paramagnetic ion, we have explored in some detail, the possibility that the g value variation in these different types of proteins is due to systematic environmental changes. To this end, we have calculated the g values as a function of tetragonal and rhombic distortions and compared our results with observed behavior. In addition we have also calculated the energy intervals between the low spin states in zero magnetic field and the temperature dependent magnetic moments and studied the effect on these properties also of small symmetry changes.     

New pulsed EPR methods and their application to characterize mitochondrial complex I

Electron Paramagnetic Resonance (EPR) spectroscopy is the method of choice to study paramagnetic cofactors that often play an important role as active centers in electron transfer processes in biological systems. However, in many cases more than one paramagnetic species is contributing to the observed EPR spectrum, making the analysis of individual contributions difficult and in some cases impossible. With time-domain techniques it is possible to exploit differences in the relaxation behavior of different paramagnetic species to distinguish between them and separate their individual spectral contribution. Here we give an overview of the use of pulsed EPR spectroscopy to study the iron-sulfur clusters of NADH:ubiquinone oxidoreductase (complex I). While FeS cluster N1 can be studied individually at a temperature of 30 K, this is not possible for FeS cluster N2 due to its severe spectral overlap with cluster N1. In this case Relaxation Filtered Hyperfine (REFINE) spectroscopy can be used to separate the overlapping spectra based on differences in their relaxation behavior.     

Suppression of axial methionine fluxion in Hydrogenobacter thermophilus Gln64Asn cytochrome c552

Proteins in the cytochrome c (cyt c) family with His-Met heme axial ligation display diverse heme electronic structures as revealed by the NMR spectra of their oxidized (paramagnetic) forms. These variations in electronic structure are thought to result primarily from differences in heme axial Met orientation among cyt c species. The factors determining Met orientation in cyts c, however, remain poorly understood. An additional layer of complexity was revealed with the recent finding that the axial Met in Hydrogenobacter thermophilus cytochrome c(552) (Ht cyt c(552)) is fluxional, sampling two conformations rapidly on the NMR time scale, resulting in an unusual compressed range of heme substituent hyperfine shifts [Zhong, L., Wen, X., Rabinowitz, T. M., Russell, B. S., Karan, E. F., and Bren, K. L. (2004) Proc.Natl. Acad. Sci. U.S.A. 101, 8637-8642]. In this work, the (1)H NMR hyperfine shift pattern of Ht cyt c(552) is drastically altered by making the conservative heme pocket mutation Gln64Asn. The mutant (Ht Q64N) displays a pattern of heme hyperfine shifts with a remarkable resemblance to that of structurally homologous Pseudomonas aeruginosa cyt c(551), which has Asn at position 64 and a single heme axial Met conformation. NMR analysis reveals that Asn64 in Ht Q64N is positioned to interact with the axial Met61, whereas the Gln64 in wild-type Ht cyt c(552) is not. It also is found that the heme axial Met is not fluxional in Ht Q64N and has an orientation similar to that in P. aeruginosa cyt c(551). These results indicate that peripheral interactions with the axial Met play an important role in determining axial Met orientation and heme electronic structure in cyts c.     

Electron paramagnetic resonance studies on Pseudomonas nitrosyl nitrite reductase. Evidence for multiple species in the electron paramagnetic resonance spectra of nitrosyl haemoproteins.

The e.p.r. spectra of reduced 14NO- and 15NO-bound Pseudomonas nitrite reductase have been investigated at pH 5.8 and 8.0 in four buffer systems. At pH 8.0, absorption spectra indicated that only the haem d1 was NO-bound, but, although quantification of the e.p.r. signals in all cases accounted for NO bound the the haem d1 in both subunits of the enzyme, the precise form of the signals varied with buffer and temperature. A rhombic species, with gx = 2.07, gz = 2.01 and gy = 1.96, represented in the low-temperature spectra seen in all the buffers was converted at high temperatures (approx. 200K) into a form showing a reduced anisotropy. Hyperfine splitting on the gz component of this rhombic signal indicated a nitrogen atom trans to NO and it is proposed that histidine provides the endogenous axial ligand for haem d1. At pH 5.8, absorption spectra indicated NO binding to both haems c and d1 and e.p.r. quantifications accounted for NO-bound haems c and d1 in both enzyme subunits. The e.p.r. spectra at pH 5.8 were generally similar to those at pH 8.0 with respect to g-values and hyperfine coupling constants, but were broader with less well defined hyperfine splittings. As at pH 8, rhombic signals present in spectra at low temperatures were converted to less anisotropic forms at high temperatures. The results are discussed in relation to work on model nitrosyl-protohaem complexes [Yoshimura, Ozaki, Shintani & Watanabe (1979) Arch. Biochem, Biophys. 193, 301-313]. No. e.p.r. signal was observed from oxidized NO-bound Pseudomonas nitrite reductase at pH 6.0, over the temperature range 6-100K.     

The influence of amino acid side chains on water binding to the copper(II) in bis(N,N-dimethyl-L-alpha-isoleucinato)-copper(II): an EPR and molecular mechanics study

Simulations were done of the electron paramagnetic resonance (EPR) spectra for bis(N,N-dimethyl-L-alpha-isoleucinato)copper(II) dissolved in deuterated methanol as a function of temperature. They indicated different behaviour of the complex below and above 300 degrees K. The effect was examined by the conformational analysis of the copper(II) complex with a new molecular mechanics force field.