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.     

The interaction of nitric oxide with soybean lipoxygenase-1.

The interaction of nitric oxide with the non-heme iron dioxygenase lipoxygenase is reported. This apparently resulted in a novel type of complex where an electron is donated to the NO molecule. In addition a new position for an EPR transition from iron was discovered which, it is suggested results from high spin ferric iron in a field of axial symmetry characterised by a very low value for D.     

The nitric oxide complex of ferrous soybean lipoxygenase-1. Substrate, pH, and ethanol effects on the active-site iron

Soybean lipoxygenase is a non-heme iron enzyme that catalyzes the hydroperoxidation of linoleic acid by dioxygen. Exposure of ferrous lipoxygenase to nitric oxide yields a species displaying an electron paramagnetic resonance spectrum characteristic of a nearly axial S = 3/2 electronic spin system arising from the ferrous-nitrosyl complex. That spectrum is pH-sensitive, reflecting changes in the environment of the metal ion between pH 7 and 11. Addition of ethanol abolishes the effects of pH in a saturable fashion, resulting in a spectrum similar to that seen at pH 7. Exchange of lipoxygenase into H2(17)O leads to no significant line broadening in the low field portion of the spectrum, suggesting no coordination of water. The ferrous enzyme displays greater affinity for NO at pH 9 (where the enzyme is most active) than at pH 7. The binding of linoleic acid is competitive with that of NO at pH 9, but not at pH 7. These results are interpreted in terms of a model including only one iron site for exogenous ligands and an otherwise relatively stable iron coordination environment.     

Direct spectroscopic evidence for the presence of a 6Fe cluster in an iron-sulfur protein isolated from Desulfovibrio desulfuricans (ATCC 27774)

A novel iron-sulfur protein was purified from the extract of Desulfovibrio desulfuricans (ATCC 27774) to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a monomer of 57 kDa molecular mass. It contains comparable amounts of iron and inorganic labile sulfur atoms and exhibits an optical spectrum typical of iron-sulfur proteins with maxima at 400, 305, and 280 nm. M?ssbauer data of the as-isolated protein show two spectral components, a paramagnetic and a diamagnetic, of equal intensity. Detailed analysis of the paramagnetic component reveals six distinct antiferromagnetically coupled iron sites, providing direct spectroscopic evidence for the presence of a 6Fe cluster in this newly purified protein. One of the iron sites exhibits parameters (delta EQ = 2.67 +/- 0.03 mm/s and delta = 1.09 +/- 0.02 mm/s at 140 K) typical for high spin ferrous ion; the observed large isomer shift indicates an iron environment that is distinct from the tetrahedral sulfur coordination commonly observed for the iron atoms in iron-sulfur clusters and is consistent with a penta- or hexacoordination containing N and/or O ligands. The other five iron sites are most probably high spin ferric. Three of them show parameters characteristic for tetrahedral sulfur coordination. In correlation with the EPR spectrum of the as-purified protein which shows a resonance signal at g = 15.3 and a group of signals between g = 9.8 and 5.4, this 6Fe cluster is assigned to an unusual spin state of 9/2 with zero field splitting parameters D = -1.3 cm-1 and E/D = 0.062. Other EPR signals attributable to minor impurities are also observed at the g = 4.3 and 2.0 regions. The diamagnetic M?ssbauer component represents a second iron cluster, which, upon reduction with dithionite, displays an intense S = 1/2 EPR signal with g values at 2.00, 1.83, and 1.31. In addition, an EPR signal of the S = 3/2 type is also observed for the dithionite-reduced protein.     

EPR characterization of axial bond in metal center of native and cobalt-substituted guanylate cyclase

The nature of the metal-proximal base bond of soluble guanylate cyclase from bovine lung was examined by EPR spectroscopy. When the ferrous enzyme was mixed with NO, a new species was transiently produced and rapidly converted to a five-coordinate ferrous NO complex. The new species exhibited the EPR signal of six-coordinate ferrous NO complex with a feature of histidine-ligated heme. The histidine ligation was further examined by using the cobalt protoporphyrin IX-substituted enzyme. The Co2+-substituted enzyme exhibited EPR signals of a broad g perpendicular;1 component and a g;1 component with a poorly resolved triplet of 14N superhyperfine splittings, which was indicative of the histidine ligation. These EPR features were analogous to those of alpha-subunits of Co2+-hemoglobin in tense state, showing a tension on the iron-histidine bond of the enzyme. The binding of NO to the Co2+-enzyme markedly stimulated the cGMP production by forming the five-coordinate NO complex. We found that N3- elicited the activation of the ferric enzyme by yielding five-coordinate high spin N3- heme. These results indicated that the activation of the enzymes was initiated by NO binding to the metals and proceeded via breaking of the metal-histidine bonds, and suggested that the iron-histidine bond in the ferric enzyme heme was broken by N3- binding.     

Electron paramagnetic resonance spectroscopy of lactoperoxidase complexes: clarification of hyperfine splitting for the NO adduct of lactoperoxidase

Electron paramagnetic resonance (EPR) studies of the nitrosyl adduct of ferrous lactoperoxidase (LPO) confirm that the fifth axial ligand in LPO is bound to the iron via a nitrogen atom. Complete reduction of the ferric LPO sample is required in order to observe the nine-line hyperfine splitting in the ferrous LPO/NO EPR spectrum. The ferrous LPO/NO complex does not exhibit a pH or buffer system dependence when examined by EPR. Interconversion of the ferrous LPO/NO complex and the ferric LPO/NO2- complex is achieved by addition of the appropriate oxidizing or reducing agent. Characterization of the low-spin LPO/NO2- complex by EPR and visible spectroscopy is reported. The pH dependence of the EPR spectra of ferric LPO and ferric LPO/CN- suggests that a high-spin anisotropic LPO complex is formed at high pH and an acid-alkaline transition of the protein conformation near the heme site does occur in LPO/CN-. The effect of tris(hydroxymethyl)aminomethane buffer on the LPO EPR spectrum is also examined.     

Active site of bovine adrenocortical cytochrome P-450(11) beta studied by resonance Raman and electron paramagnetic resonance spectroscopies: distinction from cytochrome P-450scc

Cytochrome P-45011 beta was purified as the 11-deoxycorticosterone-bound form from bovine adrenocortical mitochondria and its active site was investigated by resonance Raman and EPR spectroscopies. Resonance Raman spectra of the purified sample revealed that the heme iron adopts the pure pentacoordinated ferric high-spin state on the basis of the nu 10 (1629cm-1) and nu 3 (1490 cm-1) mode frequencies, which are higher than those of the hexacoordinated ferric high-spin cytochrome P-450scc-substrate complexes. In the ferrous-CO state, a Fe2(+)-CO stretching mode was identified at 481.5 cm-1 on the basis of an isotopic substitution technique; this frequency is very close to that of cytochrome P-450scc in the cholesterol-complexed state (483 cm-1). The EPR spectra of the purified sample at 4.2 K showed ferric high-spin signals (at g = 7.98, 3.65, and 1.71) that were clearly distinct from the cytochrome P-450scc ferric high-spin signals (g = 8.06, 3.55, and 1.68) and confirmed previous assignments of ferric high-spin signals in adrenocortical mitochondria. The EPR spectra of the nitric oxide (NO) complex of ferrous cytochrome P-45011 beta showed EPR signals with rhombic symmetry (gx = 2.068, gz = 2.001, and gy = 1.961) very similar to those of the ferrous cytochrome P-450scc-NO complex in the presence of 22(S)-hydroxycholesterol and 20(R),22-(R)-dihydroxycholesterol at 77 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing subtle coordination changes in the iron-quinone complex of photosystem II during charge separation,by the use of NO

The terminal electron acceptor of Photosystem II, PSII, is a linear complex consisting of a primary quinone, a non-heme iron(II), and a secondary quinone, Q(A)Fe(2+)Q(B). The complex is a sensitive site of PSII, where electron transfer is modulated by environmental factors and notably by bicarbonate. Earlier studies showed that NO and other small molecules (CN(-), F(-), carboxylate anions) bind reversibly on the non-heme iron in competition with bicarbonate. In the present study, we report on an unusual new mode of transient binding of NO, which is favored in the light-reduced state (Q(A)(-)Fe(2+)Q(B)) of the complex. The related observations are summarized as follows: (i) Incubation with NO at -30 degrees C, following light-induced charge separation, results in the evolution of a new EPR signal at g = 2.016. The signal correlates with the reduced state Q(A)(-)Fe(2+) of the iron-quinone complex. (ii) Cyanide, at low concentrations, converts the signal to a more rhombic form with g values at 2.027 (peak) and 1.976 (valley), while at high concentrations it inhibits formation of the signals. (iii) Electron spin-echo envelope modulation (ESEEM) experiments show the existence of two protein (14)N nuclei coupled to electron spin. These two nitrogens have been detected consistently in the environment of the semiquinone Q(A)(-) in a number of PSII preparations. (iv) NO does not directly contribute to the signals, as indicated by the absence of a detectable isotopic effect ((15)NO vs (14)NO) in cw EPR. (v) A third signal with g values (2.05, 2.03, 2.01) identical to those of an Fe(NO)(2)(imidazole) synthetic complex develops slowly in the dark, or faster following illumination. (vi) In comparison with the untreated Q(A)(-)Fe(2+) complex, the present signals not only are confined to a narrow spectral region but also saturate at low microwave power. At 11 K the g = 2.016 signal saturates with a P(1/2) of 110 microW and the g = 2.027/1.976 signal with a P(1/2) of 10 microW. (vii) The spectral shape and spin concentration of these signals is successfully reproduced, assuming a weak magnetic interaction (J values in the range 0.025-0.05 cm(-)(1)) between an iron-NO complex with total spin of (1)/(2) and the spin, (1)/(2), of the semiquinone, Q(A)(-). The different modes of binding of NO to the non-heme iron are examined in the context of a molecular model. An important aspect of the model is a trans influence of Q(A) reduction on the bicarbonate ligation to the iron, transmitted via H-bonding of Q(A) with an imidazole ligand to the iron.     

Lipoxygenase isoenzymes: a spectroscopic and structural characterization of soybean seed enzymes

Applying recent developments in protein purification techniques, a number of lipoxygenase isoenzymes have been isolated in satisfactory quantities for a detailed physical and structural characterization. Four seed isoenzymes from two soybean cultivars have been studied by peptide mapping, free thiol and iron content determinations, and C-terminal analysis as well as by uv-visible absorption and EPR spectroscopy. While differences between the type 1 enzyme and the other isoenzymes were readily detected using proteolytic peptide mapping, digestion with dilute hydrochloric acid and C-terminal analysis both revealed structural features which were similar in all of the isoenzymes. One clear difference between the lipoxygenases was in their free sulfhydryl group content. The uv-visible absorption spectrum of each native isoenzyme was consistent with expectations for the experimental aromatic amino acid content. All of the isoenzymes contained one non-heme iron atom per molecule of protein. The oxidation of each isoenzyme with product hydroperoxide resulted in the conversion of the iron from an EPR silent state into several forms with EPR signals characteristic of high spin iron(III). The EPR spectra of all isoenzymes were remarkably similar. A time course EPR and catalytic activity study revealed that the various EPR active states represent a complex equilibrium between iron atoms in different environments. The pH dependence for the EPR and absorption spectroscopy lends support to the hypothesis that acid/base chemistry represents an important aspect of lipoxygenase catalysis.     

Extensive studies of the heme coordination structure of indoleamine 2,3-dioxygenase and of tryptophan binding with magnetic and natural circular dichroism and electron paramagnetic resonance spectroscopy

In order to probe the active site of the heme protein indoleamine 2,3-dioxygenase, magnetic and natural circular dichroism (MCD and CD) and electron paramagnetic resonance (EPR) studies of the substrate (L-tryptophan)-free and substrate-bound enzyme with and without various exogenous ligands have been carried out. The MCD spectra of the ferric and ferrous derivatives are similar to those of the analogous myoglobin and horseradish peroxidase species. This provides strong support for histidine imidazole as the fifth ligand to the heme iron of indoleamine 2,3-dioxygenase. The substrate-free native ferric enzyme exhibits predominantly high-spin EPR signals (g perpendicular = 6, g parallel = 2) along with weak low-spin signals (g perpendicular = 2.86, 2.28, 1.60); similar EPR, spin-state and MCD features are found for the benzimidazole adduct of ferric myoglobin. This suggests that the substrate-free ferric enzyme has a sterically hindered histidine imidazole nitrogen donor sixth ligand. Upon substrate binding, noticeable MCD and EPR spectral changes are detected that are indicative of an increased low spin content (from 30 to over 70% at ambient temperature). Concomitantly, new low spin EPR signals (g = 2.53, 2.18, 1.86) and MCD features characteristic of hydroxide complexes of histidine-ligated heme proteins appear. For almost all of the other ferric and ferrous derivatives, only small substrate effects are observed with MCD spectroscopy, while substantial substrate effects are seen with CD spectroscopy. Thus, changes in the heme coordination structure of the ferric enzyme and in the protein conformation at the active site of the ferric and ferrous enzyme are induced by substrate binding. The observed substrate effects on the ferric enzyme may correlate with the previously observed kinetic substrate inhibition of indoleamine 2,3-dioxygenase activity, while such effects on the ferrous enzyme suggest the possibility that the substrate is activated during turnover.     

Evidence for participation of iron in lipoxygenase reaction from optical and electron spin resonance studies.

Optical and EPR studies indicate that the iron present in lipoxygenase participates in catalysis. Addition of linoleic acid hydroperoxide to lipoxygenase 1 causes an increase in abosrbance over the range of 350 to 650 nm which is reversed when linoleic acid hydroperoxide is destroyed upon the addition of linoleic acid under anaerobic conditions. Lipoxygenase 1 alone exhibits no EPR signal but upon addition of linoleic acid hydroperoxide or linoleic acid several signals appear. Addition of linoleic acid hydroperoxide results in an EPR signal at g approximately equal to 6 accompanied by a small but relatively sharp signal at g approximately equal to 2. Under anaerobic conditions the latter is replaced by a broad anisotropic signal around g approximately equal to 2. The appearance of the EPR signal at g approximately equal to 6 coincides with the change in the optical spectrum of the enzyme. When linoleic acid is added under anaerobic conditions a broad anisotropic EPR signal around g approximately equal to 2 is observed. Thus it appears that lipoxygenase can exist in two forms: (a) a resting form with a very weak absorbance in the visible range of the light spectrum and no EPR signal and (b) an active form (after addition of linoleic acid hydroperoxide) with an increased optical absorbance and EPR signal at g approximately equal to 6. This observation may be related to the earlier discovery that the lipoxygenase reaction occurs with a lag which can be overcome by addition of product hydroperoxide. The EPR experiments indicate that lipoxygenase in the active form contains high spin ferric ion. Although EPR signals in the g approximately equal to 6 region are frequently observed with heme proteins, the only nonheme protein, other than lipoxygenase, reported to show an EPR signal in this region is the phenolytic dioxygenase, protocatechuate 3,4-dioxygenase (Peisach, J., Fujisawa, H., Blumberg, W. E., and Hayaishi, O. (1972) Fed. Proc. 31, 448).     

Spectroscopic characterization and ligand-binding properties of chlorite dismutase from the chlorate respiring bacterial strain GR-1.

Chlorite dismutase (EC 1.13.11.49), an enzyme capable of reducing chlorite to chloride while producing molecular oxygen, has been characterized using EPR and optical spectroscopy. The EPR spectrum of GR-1 chlorite dismutase shows two different high-spin ferric heme species, which we have designated 'narrow' (gx,y,z = 6.24, 5.42, 2.00) and 'broad' (gz,y,x = 6.70, 5.02, 2.00). Spectroscopic evidence is presented for a proximal histidine co-ordinating the heme iron center of the enzyme. The UV/visible spectrum of the ferrous enzyme and EPR spectra of the ferric hydroxide and imidazole adducts are characteristic of a heme protein with an axial histidine co-ordinating the iron. Furthermore, the substrate analogs nitrite and hydrogen peroxide have been found to bind to ferric chlorite dismutase. EPR spectroscopy of the hydrogen peroxide adduct shows the loss of both high-spin and low-spin ferric signals and the appearance of a sharp radical signal. The NO adduct of the ferrous enzyme exhibits a low-spin EPR signal typical of a five-co-ordinate heme iron nitrosyl adduct. It seems that the bond between the proximal histidine and the iron is weak and can be broken upon binding of NO. The midpoint potential, Em(Fe3+/2+) = -23 mV, of chlorite dismutase is higher than for most heme enzymes. The spectroscopic features and redox properties of chlorite dismutase are more similar to the gas-sensing hemoproteins, such as guanylate cyclase and the globins, than to the heme enzymes.     

Access of ligands to the ferric center in lipoxygenase-1.

A form of ferric lipoxygenase-1 has been isolated that gives an EPR spectrum that is dominated by a species of intermediate rhombicity (E/D = 0.065). This species is obtained in the presence of a number of buffers of high concentration and in the absence of fatty acid byproducts of the iron oxidation. The species is unstable over a period of one day with respect to symmetry of the iron. The EPR lineshapes of the unstable species are highly sensitive to the anionic composition of the buffer and to the addition of neutral ligands. These results suggest that newly formed ferric lipoxygenase has weak affinity for a number of ligands. Affinity of charged ligands for the iron center may provide a mechanism for charge compensation as the iron center alternates between ferric and ferrous in the catalytic cycle. We use spectral simulation to evaluate quantitatively the interaction of the ferric center with ligands and also show that a transition in the middle Kramers doublet makes a significant contribution to the EPR spectrum of the more rhombic species.     

Multiple frequency EPR studies on three forms of oxidized cytochrome c oxidase

Bovine heart mitochondrial cytochrome c oxidase (cytochrome aa3) (EC 1.9.3.1) has been demonstrated to occur in several forms when the redox centers in the protein are thought to be fully oxidized. We report here the results of extensive EPR studies at 3, 8.9, 9.2, 9.4, 15 and 34 GHz on the resting state, the alternative resting state (with g = 12 at 9 GHz) and pulsed state (with g = 5 signal at 9 GHz). Theoretical consideration is given to all binary spin-coupling possibilities under the constraint that the iron atoms are either ferric or ferrous and the copper atoms are either cupric or cuprous. We conclude that the g = 12 signal can arise from any spin system with S greater than 1 and D = 0.15 cm-1. The g = 5 signals originate from an excited, integer-spin system with D = 0.035 cm-1, which is approximately 7 cm-1 above the ground state (not observed in EPR). It is pointed out that in interpretations of data and elaboration of suitable models in this field, the implications of spin-coupling should be considered in a comprehensive and not in a selective way. At 3 GHz, EPR spectra of CuA in the resting, pulsed and anaerobically oxidized states show that this center is identical in its EPR for all three states.     

Chloroperoxidase compound I: Electron paramagnetic resonance and M?ssbauer studies

The green primary compound of chloroperoxidase was prepared by freeze-quenching the enzyme after rapid mixing with a 5-fold excess of peracetic acid. The electron paramagnetic resonance (EPR) spectra of these preparations consisted of at least three distinct signals that could be assigned to native enzyme, a free radical, and the green compound I as reported earlier. The absorption spectrum of compound I was obtained through subtraction of EPR signals measured under passage conditions. The signal is well approximated by an effective spin Seff = 1/2 model with g = 1.64, 1.73, 2.00 and a highly anisotropic line width. M?ssbauer difference spectra of compound I samples minus native enzyme showed well-resolved magnetic splitting at 4.2 K, an isomer shift delta Fe = 0.15 mm/s, and quadrupole splitting delta EQ = 1.02 mm/s. All data are consistent with the model of an exchange-coupled spin S = 1 ferryl iron and a spin S' = 1/2 porphyrin radical. As a result of the large zero field splitting, D, of the ferryl iron and of intermediate antiferromagnetic exchange, S.J.S'.J approximately 1.02 D, the system consists of three Kramers doublets that are widely separated in energy. The model relates the EPR and M?ssbauer spectra of the ground doublet to the intrinsic parameters of the ferryl iron, D/k = 52 K, E/D congruent to 0.035, and A perpendicular (gn beta n) = 20 T, and the porphyrin radical.(ABSTRACT TRUNCATED AT 250 WORDS)     

M?ssbauer and EPR spectroscopy of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) from Pseudomonas aeruginosa has been investigated by EPR and M?ssbauer spectroscopy. Low temperature M?ssbauer data on the native enzyme (Fe3+, S = 5/2) yields a hyperfine field Hsat=-525 kG at the nucleus. This observation is inconsistent with earlier suggestions, based on EPR data of a rubredoxin-like ligand environment around the iron, i.e. a tetrahedral sulfur coordination. Likewise, the dithionite-reduced enzyme has M?ssbauer parameters unlike those of reduced rubredoxin. We conclude that the iron atoms are in a previously unrecognized environment. The ternary complex of the enzyme with 3,4-dihydroxyphenylpropionate and O2 yields EPR signals at g = 6.7 and g = 5.3; these signals result from an excited state Kramers doublet. The kinetics of the disappearance of these signals parallels product formation and the decay of the ternary complex as observed in the optical spectrum. The M?ssbauer and EPR data on the ternary complex establish the iron atoms to be a high-spin ferric state characterized by a large and negative zero-field splitting, D = approximately -2 cm-1.     

Purification and characterization of the MQH2:NO oxidoreductase from the hyperthermophilic archaeon Pyrobaculum aerophilum

The membrane-bound NO reductase from the hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum was purified to homogeneity. The enzyme displays MQH2:NO oxidoreductase (qNOR) activity, consists of a single subunit, and contains heme and nonheme iron in a 2:1 ratio. The combined results of EPR, resonance Raman, and UV-visible spectroscopy show that one of the hemes is bis-His-coordinated low spin (gz = 3.015; gy = 2.226; gx = 1.45), whereas the other heme adopts a high spin configuration. The enzyme also contains one nonheme iron center, which in the oxidized enzyme is antiferromagnetically coupled to the high spin heme. This binuclear high spin heme/nonheme iron center is EPR-silent and the site of NO reduction. The reduced high spin heme is bound to a neutral histidine and can bind CO to form of a low spin complex. The oxidized high spin heme binds NO, yielding a ferric nitrosyl complex, the intermediate causing the commonly found substrate inhibition in NO reductases (Ki(NO) = 7 microm). The qNOR as present in the membrane is, in contrast to the purified enzyme, quite thermostable, incubation at 100 degrees C for 86 min leading to 50% inhibition. The pure enzyme lacks heme b and instead contains stoichiometric amounts of hemes Op1 and Op2, ethenylgeranylgeranyl and hydroxyethylgeranylgeranyl derivatives of heme b, respectively. The archaeal qNOR is the first example of a NO reductase, which contains modified hemes reminiscent of cytochrome bo3 and aa3 oxidases. This report is the first describing the purification and structural and spectroscopic properties of a thermostable NO reductase.     

Nitrogenase. VIII. M?ssbauer and EPR spectroscopy. The MoFe protein component from Azotobacter vinelandii OP.

We have studied the molybdenum-iron protein (MoFe protein, also known as component I) from Azobacter vinelandi using M?ssbauer spectroscopy and electron paramagnetic resonance on samples enriched with 57Fe. These spectra can be interpreted in terms of two EPR active centers, each of which is reducible by one electron. A total of four different chemical environments of Fe can be discerned. One of them is a cluster of Fe atoms with a net electronic spin of 3/2, one of them is high-spin ferrous iron and the remaining two are iron in a reduced state (probably in clusters). The results are as follows: Chemical analysis yields 11.5 Fe atoms and 12.5 labile sulfur atoms per molybdenum atom; the molecule contains two Mo atoms per 300 000 daltons. The EPR spectrum of the MoFe protein exhibits g values at 4.32, 3.65 and 2.01, associated with the ground state doublet of a S = 3/2 spin system. The spin Hamiltonian H = D(S2/z minus 5/4 + lambda(S2/x minus S2/y)) + gbeta/o S-H fits the experimental data for go = 2.00 and lambda = 0.055. Quantitative analysis of the temperature dependence of the EPR spectrum yields D/k = 7.5 degrees K and 0.91 spins/molybdenum atom, which suggests that the MoFe protein has two EPR active centers. Quantitative evaluation of M?ssbauer spectra shows that approximately 8 iron atoms give rise to one quadrupole doublet; at lower temperatures magnetic spectra, associated with the groud electronic doublet, are observed; at least two magnetically inequivalent sites can be distinguished. Taken together the data suggest that each EPR center contains 4 iron atoms. The EPR and M?ssbauer data can only be reconciled if these iron atoms reside in a spin-coupled (S = 3/2) cluster. Under nitrogen fixing conditions the magnetic M?ssbauer spectra disappeared concurrently with the EPR signal and quadrupole doublets are obserced at all temperatures. The data suggest that each EPR active center is reduced by one electron. The M?ssbauer investigation reveals three other spectral components characteristic of iron nuclei in an environment of integer or zero electronic spin, i.e. they reside in complexes which are "EPR-silent". One of the components (3-4 iron atoms) has M?ssbauer parameters characteristic of the high-spin ferrous iron as in reduced ruberdoxin. However, measurements in strong fields indicate a diamagnetic environment. Another component, representing 9-11 iron atoms, seems to be diamagnetic also. It is suggested that these atoms are incorporated in spin-coupled clusters.     

EPR studies on the photo-induced intermediates of ferric NO complexes of rat neuronal nitric oxide synthase trapped at low temperature.

Rat neuronal nitric oxide synthase (nNOS) was expressed in Escherichia coli and purified. Although the nitric oxide (NO) complex of the ferric heme was EPR-silent, photo-illumination at 5 K to the NO complex of the ferric nNOS in the substrate-free form produced a new high spin EPR signal similar to that of the ferric heme of N(omega)-nitro-L-arginine-bound nNOS, suggesting that the photo-dissociated NO might move away from the heme. Low photo-dissociability of NO in this complex indicated less restricted movement of the dissociated NO in the distal region of the heme, which might result in the rapid rebinding of the NO to the ferric heme at 5 K. In the presence of substrate L-arginine, derivatives, or product L-citrulline, the photo-products from the ferric NO complexes exhibited large novel EPR signals with a spin-coupled interaction between the ferric heme (S = 5/2) and the photolyzed NO (S = 1/2), suggesting a stereochemically restricted interaction between the photo-dissociated NO and the guanidino- or the ureido-group of the substrate analogues at the distal heme region of nNOS. The photo-product from the NO complex produced from citrulline-bound nNOS might be the same intermediate species as that formed in the last step of the catalytic cycle.     

Semi-met oxidation level of chalcogenide derivatives of methemerythrin. M?ssbauer and EPR studies

Conclusive evidence is presented for an S = 1/2 spincoupled pair of high spin ferric and ferrous ions in the major reaction product of sulfide with the met form of the non-heme iron oxygen-carrying protein hemerythrin. Evidence for an analogous selenide derivative is also reported. M?ssbauer and EPR spectroscopy establish (a) the charge and spin states of the individual iron atoms in sulfidehemerythrin as Fe(III), S = 5/2, and Fe(II), S = 2, and (b) the existence of an antiferromagnetic exchange interaction that couples the two spins to a resultant spin S = 1/2. The combined M?ssbauer and EPR data confirm the correctness of the formulation first proposed for semi-methemerythrin by Harrington, P.C., de Waal, D.J.A., and Wilkins, R.G. ((1978) Arch. Biochem. Biophys. 191, 444-451) and furthermore show that a majority of the iron centers in the protein can be stabilized at this oxidation level. The results also demonstrate a new route to semi-methemerythrin. A titration of methemerythrin with selenide indicates that this derivative forms by a two step process consisting of first, reduction to the semi-met oxidation level by selenide and second, binding of selenide to either one or both irons.