Ligation of the diiron site of the hydroxylase component of methane monooxygenase. An electron nuclear double resonance study.

Electron nuclear double resonance (ENDOR) spectroscopy is used to probe the coordination of the mixed valence (Fe(II).Fe(III)) diiron cluster of the methane monooxygenase hydroxylase component (MMOH-) isolated from Methylosinus trichosporium OB3b. ENDOR resonances are observed along the principal axis directions g1 = 1.94 and g3 = 1.76 from at least nine different protons and two different nitrogens. The nitrogens are strongly coupled and appear to be directly coordinated to the cluster irons. The ratio of their superhyperfine coupling constants is roughly 4:7, which equals the ratio of the spin expectation values of the Fe(II) and Fe(III) in the ground state and suggests that at least one nitrogen is coordinated to each iron of the mixed valence cluster. Moreover, the superhyperfine and quadrupole coupling constants assigned to the Fe(III) site (AN = 13.6 MHz, PN = 0.7 MHz) are comparable with those observed for semimethemerythrin sulfide (AN = 12.1 MHz, PN = 0.7 MHz), for which the nitrogen ligands are histidines. At least three of the coupled protons exchange slowly when MMOH- is incubated in D2O, and 2H ENDOR resonances are subsequently observed. These observations are also consistent with histidine ligation of the iron cluster. On addition of the inhibitor dimethyl sulfoxide (Me2SO) to MMOH- the EPR spectrum sharpens and shifts dramatically. Only one set of 14N ENDOR resonances is observed with frequencies equal to those assigned to the Fe(III)-histidine resonances of uncomplexed MMOH- suggesting that the nitrogen coordination to the Fe(II) site is altered or possibly lost in the presence of Me2SO. 2H ENDOR resonances are observed in the presence of d6-Me2SO indicating that the inhibitor Me2SO binds near or possibly to the diiron cluster. In contrast, no 2H ENDOR resonances are observed from d4-methanol upon addition to MMOH-. Thus, the changes observed in the EPR spectrum of MMOH- upon addition of methanol may result from binding to a site away from the diiron cluster or from bulk solvent effects on the protein structure.     

Electron spin echo envelope modulation spectroscopic study of iron-nitrogen interactions in myoglobin hydroxide and Fe(III) tetraphenylporphyrin models.

The electron-nuclear coupling in low-spin iron complexes including myoglobin hydroxide (MbOH) and two related model compounds, Fe(III) tetraphenylporphyrin(pyridine)(OR-) (R = H or CH3) and Fe(III) tetraphenylporphyrin(butylamine)(OR-) was investigated using electron spin echo envelope modulation (ESEEM) spectroscopy. The assignment of frequency components in ESEEM spectra was accomplished through the use of nitrogen isotopic substitution wherever necessary. For example, the proximal imidazole coupling in MbOH was investigated without interference from the contributions of porphyrin 14N nuclei after substitution of the heme in native Mb with 15N-labeled heme. Computer simulation of spectra using angle selected techniques enabled the assignment of parameters describing the hyperfine and quadrupole interactions for axially bound nitrogen of imidazole in MbOH, of axial pyridine and butylamine in the models, and for the porphyrin nitrogens of the heme in native MbOH. The isotropic component of axial nitrogen hyperfine interactions exhibits a trend from 5 to 4 MHz, with imidazole (MbOH) greater than pyridine greater than amine. The nuclear quadrupole interaction coupling constant e2Qq was near 2 MHz for all nitrogens in these complexes. The Qzz axis of the nuclear quadrupole interaction tensor for the proximal imidazole nitrogen in MbOH was found to be aligned near gz (gmax) in MbOH, suggesting that gz is near the heme normal. A crystal field analysis, that allows a calculation of rhombic and axial splittings for the d orbitals of the t2g set in a low-spin heme complex, based on the g tensor assignment gz greater than gy greater than gx, yielded results that are consistent with the poor pi-acceptor properties expected for the closed shell oxygen atom of the hydroxide ligand in MbOH. A discussion is presented of the unusual results reported in a linear electric field effect in EPR (LEFE) study of MbOH published previously [Mims, W. B., & Peisach, J. (1976) J. Chem. Phys. 64, 1074-1091].     

Characterization of the [4Fe-4S]+ cluster at the active site of aconitase by 57Fe, 33S, and 14N electron nuclear double resonance spectroscopy

57Fe, 33S, and 14N electron nuclear double resonance (ENDOR) studies have been performed to characterize the [4Fe-4S]+ cluster at the active site of aconitase. Q-band 57Fe ENDOLR of isotopically enriched enzyme, both substrate free and in the enzyme-substrate complex, reveals four inequivalent iron sites. In agreement with M?ssbauer studies [Kent et al. (1985) J. Biol. Chem. 260, 6371-6881], one of the iron ions, Fea, which is easily removed by oxidation to yield the [3Fe-4S]+ cluster of inactive aconitase, shows a dramatic change in the presence of substrate. The remaining iron sites, Feb1,2,3, show minor changes when substrate is bound. Methods devised by us for analyzing and simulating ENDOR spectra of a randomly oriented paramagnet have been used to determine the principal values and orientation relative to the g tensor for the hyperfine tensors of three of the four inequivalent iron sites of the [4Fe-4S]+ cluster, Fea, Feb2, and Feb3, in the substrate-free enzyme and the enzyme-substrate complex. The full tensor for the fourth site, Feb1, could not be obtained because its signal is seen only over a limited range of the EPR envelope. 33S ENDOR data for the enzyme-substrate complex using enzyme reconstituted with 33S show that the four inorganic bridging sulfide ions of the [4Fe-4S]+ cube have isotropic hyperfine couplings of A(S) less than 12 MHz, and analysis indicates that they can be divided into two pairs, one with couplings of A(S1) approximately less than 1 MHz and the other with A(S2) approximately 6-12 MHz; the analysis further places these pairs within the cube relative to the iron sites. 33S data for substrate-free enzyme is qualitatively similar and can be completely simulated by two types of S2- ion, with A(S1) approximately 7.5 and A(S2) approximately 9 MHz; the full hyperfine tensors have been determined. The hyperfine values for the two enzyme forms correspond to surprisingly small unpaired spin density on S2-. 14N ENDOR at Q-band reveals a nitrogen signal that does not change upon substrate binding.     

Hydrogen bonding to the bound dioxygen in oxy cobaltous myoglobin reduces the superhyperfine coupling to the proximal histidine.

Electron spin echo envelope modulation (ESEEM) spectroscopy was used to study the electron-nuclear coupling in two oxygenated cobalt-substituted hemoproteins, myoglobin (oxyCoMb) and a monomeric hemoglobin from Glycera dibranchiata (oxyCoHbgly). The modulation frequency components in ESEEM spectra of both proteins arose from the coupling to the N epsilon of the proximal histidyl imidazole. The hyperfine and quadrupole coupling parameters for these two nitrogens, calculated by computer spectral simulation, are Aiso = 2.46 MHz, e2qQ = 2.15 MHz, and eta = 0.4 for oxyCoMb and Aiso = 3.70 MHz, e2qQ = 2.70 MHz, and eta = 0.5 for oxyCoHbgly. A hyperfine coupling of 0.6 MHz, found for oxyCoMb in D2O but not for oxyCoHbgly in D2O, was assigned to the coupling to a deuteron that is hydrogen-bonded to the O2 ligand in oxyCoMb. This hydrogen bonding is believed to be responsible for the reduction in hyperfine and nuclear quadrupole coupling to the proximal histidyl imidazole N epsilon in oxyCoMb. A molecular orbital model for O2 adducts of cobaltous compounds [Tovrog et al. (1976) J. Am. Chem. Soc. 98, 5144] was used to understand the hydrogen bond-induced reduction in 14N superhyperfine coupling in oxyCoMb.     

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

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

Geometries and electronic structures of cyanide adducts of the non-heme iron active site of superoxide reductases: vibrational and ENDOR studies

We have added cyanide to oxidized 1Fe and 2Fe superoxide reductase (SOR) as a surrogate for the putative ferric-(hydro)peroxo intermediate in the reaction of the enzymes with superoxide and have used vibrational and ENDOR spectroscopies to study the properties of the active site paramagnetic iron center. Addition of cyanide changes the active site iron center in oxidized SOR from rhombic high-spin ferric (S = 5/2) to axial-like low-spin ferric (S = 1/2). Low-temperature resonance Raman and ENDOR data show that the bound cyanide adopts three distinct conformations in Fe(III)-CN SOR. On the basis of 13CN, C15N, and 13C15N isotope shifts of the Fe-CN stretching/Fe-C-N bending modes, resonance Raman studies of 1Fe-SOR indicate one near-linear conformation (Fe-C-N angle approximately 175 degrees) and two distinct bent conformations (Fe-C-N angles <140 degrees). FTIR studies of 1Fe-SOR at ambient temperatures reveals three bound C-N stretching frequencies in the oxidized (ferric) state and one in the reduced (ferrous) state, indicating that the conformational heterogeneity in cyanide binding is a characteristic of the ferric state and is not caused by freezing-in of conformational substates at low temperature. 13C-ENDOR spectra for the 13CN-bound ferric active sites in both 1Fe- and 2Fe-SORs also show three well-resolved Fe-C-N conformations. Analysis of the 13C hyperfine tensors for the three substates of the 2Fe-SOR within a simple heuristic model for the Fe-C bonding gives values for the Fe-C-N angles in the three substates of ca. 123 degrees (C3) and 133 degrees (C2), taking a reference value from vibrational studies of 175 degrees (C1 species). Resonance Raman and ENDOR studies of SOR variants, in which the conserved glutamate and lysine residues in a flexible loop above the substrate binding pocket have been individually replaced by alanine, indicate that the side chains of these two residues are not involved in direct interaction with bound cyanide. The implications of these results for understanding the mechanism of SOR are discussed.     

Electron-nuclear double resonance studies of oxidized Escherichia coli sulfite reductase: 1H, 14N, and 57Fe measurements

We have employed electron-nuclear double resonance (ENDOR) spectroscopy to study the bridged siroheme--[Fe4S4] cluster that forms the catalytically active center of the oxidized hemoprotein subunit (SiRo) of Escherichia coli NADPH-sulfite reductase. The siroheme 57Fe hyperfine coupling (Az = 27.6 MHz, Ay = 26.8 MHz) is similar to that of other high-spin heme systems (A approximately equal to 27 MHz). Bonding parameters obtained from the 14N hyperfine coupling constants of the siroheme pyrrole nitrogens are consistent with a model of a nonplanar pi system of reduced aromaticity. The absence of hyperfine coupling to the 14N of an axial ligand, such as is observed for the histidine 14N of metmyoglobin (Az = 11.55 MHz), rules out the possibility that imidazolate acts as the bridge between the siroheme and the [Fe4S4] cluster. Proton ENDOR of the deuterium-exchanged protein indicates that H2O does not function as a sixth axial ligand and suggests that the ferrisiroheme is five-coordinate. 57Fe ENDOR measurements confirm the results of M?ssbauer spectroscopy for the [Fe4S4] cluster. They also disclose a slight anisotropy of the cluster 57Fe coupling that may be associated with the mechanism by which the siroheme and cluster spins are coupled.     

14,15N, 13C, 57Fe, and 1,2H Q-band ENDOR study of Fe-S proteins with clusters that have endogenous sulfur ligands.

The benefits of performing ENDOR experiments at higher microwave frequency are demonstrated in a Q-band (35 GHz) ENDOR investigation of a number of proteins with [nFe-mS] clusters, n = 2, 3, 4. Each protein displays several resonances in the frequency range of 0-20 MHz. In all instances, features are seen near v approximately 13 and 8 MHz that can be assigned, respectively, to "distant ENDOR" from 13C in natural-abundance (1.1%) and from 14N (the delta m1 = +/- 2 transitions); the nuclei involved in this phenomenon are remote from and have negligible hyperfine couplings to the cluster. In addition, a number of proteins show local 13C ENDOR signals with resolved hyperfine interactions; these are assigned to the beta carbons of cysteines bound to the cluster [A(13C) approximately 1.0 MHz]. Five proteins show resolved, local delta m1 = +/- 2 ENDOR signals from 14N with an isotropic hyperfine coupling, 0.4 less than or equal to A(14N) less than or equal to 1.0, similar to those seen in ESEEM studies; these most likely are associated with N-H...S hydrogen bonds to the cluster. Anabaena ferredoxin further shows a signal corresponding to A(14N) approximately 4 MHz. Quadrupole coupling constants are derived for both local and distant 14N signals. The interpretation of the data is supported by studies on 15N- and 13C-enriched ferredoxin (Fd) from Anabaena 7120, where the 15N signals can be clearly correlated with the corresponding 14N signals and where the 13C signals are strongly enhanced. Thus, the observation of 14N delta m1 = +/- 2 signals at Q-band provides a new technique for examining weak interactions with a cluster.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of Mg2+ binding sites in isolated alpha and beta subunits of H(+)-ATPase from Bacillus PS3

The properties of the nucleotide binding sites in the isolated beta and alpha subunits of H(+)-ATPase from Bacillus PS3 (TF1) have been examined by studying the EPR properties of bound VO(2+), which is a paramagnetic probe for the native Mg2+ cation cofactor. The amino acid ligands of the VO2+ complexes with the isolated beta subunit, with the isolated alpha subunit, with different mixtures of both alpha and beta subunits, and with the catalytic alpha 3 beta 3 gamma subcomplex have been characterized by a combination of EPR, ESEEM, and HYSCORE spectroscopies. The EPR spectrum of the isolated beta subunit with bound VO2+ (1 VO2+/beta) is characterized by (51)V hyperfine coupling parameters (A( parallel) = 168 x 10(-)(4) cm(-)(1) and A( perpendicular) = 60 x 10(-)(4) cm(-)(1)) that suggest that VO2+ binds to the isolated beta subunit with at least one nitrogen ligand. Results obtained for the analogous VO2+ complex with the isolated alpha subunit are virtually identical. ESEEM and HYSCORE spectra are also reported and are similar for both complexes, indicating a very similar coordination scheme for VO2+ bound to isolated alpha and beta subunits. In the isolated beta (or alpha) subunit, the bound VO2+ cation is coordinated by one nitrogen ligand with hyperfine coupling parameters A( parallel)((14)N) = 4.44 MHz, and A( perpendicular)((14)N) = 4.3 MHz and quadrupole coupling parameters e(2)()qQ approximately 3.18 MHz and eta approximately 1. These are typical for amine-type nitrogen ligands equatorial to the VO2+ cation; amino acid residues in the TF1 beta and alpha subunits with nitrogen donors that may bind VO2+ are reviewed. VO2+ bound to a mixture of alpha and beta subunits in the presence of 200 mM Na2SO4 to promote the formation of the alpha 3 beta 3 hexamer has a second nitrogen ligand with magnetic properties similar to those of a histidine imidazole. This situation is analogous to that in the alpha 3 beta 3 gamma subcomplex and in the whole TF1 enzyme [Buy, C., Matsui, T., Andrianambinintsoa, S., Sigalat, C., Girault, G., and Zimmermann, J.-L. (1996) Biochemistry 35, 14281-14293]. These data are interpreted in terms of only partially structured nucleotide binding sites in the isolated beta and alpha subunits as compared to fully structured nucleotide binding sites in the alpha 3 beta 3 heterohexamer, the alpha 3 beta 3 gamma subcomplex, and the whole TF1 ATPase.     

Electron spin-echo envelope modulation study of multicrystalline Cu(2+)-insulin: effects of Cd(2+) on the nuclear quadrupole interaction of the Cu(2+)-coordinated imidazole remote nitrogen

A comparison of electron spin-echo envelope modulation (ESEEM) spectra from multi-crystalline Cu(2+)-insulin with and without additional Cd(2+) show a dramatic change in the quadrupole coupling parameters of the remote nitrogens of the two histidine imidazoles that ligate to copper. Without Cd(2+), the quadrupole parameters are like those observed in blue copper proteins and in copper substituted lactoferrin. With Cd(2+) soaked into the Cu(2+)-insulin crystals, the quadrupole parameters are similar to those found in galactose oxidase. Theoretical simulations of ESEEM spectra guided by structure modeling suggest that these changes originate from differences in the hydrogen bonding environments of the imidazole remote nitrogen. In addition, a compilation of results from previous ESEEM studies of copper proteins reveals that the asymmetry parameter, eta, may be an indicator of type of hydrogen bond the imidazole remote nitrogen makes. When eta > or = 0.9, the nitrogen hydrogen bonds to water, whereas when eta < 0.9, the nitrogen hydrogen bonds to the protein.     

Electron-nuclear double resonance spectroscopy of 15N-enriched phthalate dioxygenase from Pseudomonas cepacia proves that two histidines are coordinated to the [2Fe-2S] Rieske-type clusters

We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type [2Fe-2S] cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) [15N]histidine in a 14N background; (4) [14N]histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske [2Fe-2S] center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type [2Fe-2S] centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier M?ssbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled [Fe2+ (S = 2), Fe3+ (S = 5/2)] pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.     

An electron spin-echo envelope modulation (ESEEM) study of the QA binding pocket of PS II reaction centers from spinach and Synechocystis

We have used electron spin-echo envelope modulation spectroscopy (ESEEM) to characterize the protein-cofactor interactions present in the QA- binding pocket of PS II centers isolated from spinach and Synechocystis. We conclude that the ESEEM spectrum of QA- is the result of interactions of the S = 1/2 electron spin of QA- with the I = 1 nuclear spins of the peptide nitrogens of two different amino acids. One peptide nitrogen has ESEEM peaks near 0.7, 2.0, 2.85, and 5.0 MHz with isotropic and dipolar hyperfine couplings of Aiso = 2.0 MHz and Adip = 0.25 MHz, respectively. On the basis of these hyperfine couplings we predict the existence of a strong hydrogen bond between QA- and the peptide nitrogen with a hydrogen bond distance of about 2 A. We have not identified the amino acid origin of this peptide nitrogen. By using amino acid specific isotopic labeling in conjunction with site-directed mutagenesis, we demonstrate that the second peptide nitrogen is that of D2-Ala260, with ESEEM peaks near 0.6 and 1.5 MHz and an isotropic hyperfine coupling, Aiso, less than 0.2 MHz. This small isotropic coupling suggests that the D2-Ala260 peptide nitrogen at best forms a weak hydrogen bond with QA-.     

The cationic plastoquinone radical of the chloroplast water splitting complex: Hyperfine splitting from a single methyl group determines the EPR spectral shape of Signal II

The proposal that EPR Signal II in spinach chloroplasts is due to a plastoquinone cation radical (O'Malley, P.J. and Babcock, G.T. (1983) Biophys. J. 41, 315a) has been investigated in further detail. The similarity in spectral shape between Signal II and the 2-methyl-5-isopropylhydroquinone cation radical is shown to arise from hyperfine coupling to one methyl group for both radicals. A well-resolved four line EPR spectrum of approximate relative intensity 1:3:3:1 for membrane orientation parallel and perpendicular to the applied magnetic field direction also indicates that the partially resolved structure of Signal II is due to hyperfine interaction with one methyl group, i.e., the 2-CH₃ group of the plastoquinone cation radical. The ENDOR band observed for this coupling is similar to that observed for methyl group bands of model quinone radicals. The principal hyperfine tensor values obtained for the methyl group interactions are A_⊥ = 27.2 MHz and $\text{A}{}_{\mathrm{}}\text{= 31.4}\text{MHz}$. The large isotropic coupling value (28.6 MHz) of the plastoquinone cation radical's 2-methyl group in vivo indicates that the antisymmetric orbital is the sole contributor to the spin-density distribution of Signal II. The orientation data also suggest that the plastoquinone cation radical is oriented such that the C-CH₃ bond direction, and hence the aromatic ring plane, lies perpendicular to the membrane plane.     

The identification of histidine ligands to cytochrome a in cytochrome c oxidase

A histidine auxotroph of Saccharomyces cerevisiae has been used to metabolically incorporate [1,3-15N2] histidine into yeast cytochrome c oxidase. Electron nuclear double resonance (ENDOR) spectroscopy of cytochrome a in the [15N]histidine-substituted enzyme reveals an ENDOR signal which can be assigned to hyperfine coupling of a histidine 15N with the low-spin heme, thereby unambiguously identifying histidine as an axial ligand to this cytochrome. Comparison of this result with similar ENDOR data obtained on two 15N-substituted bisimidazole model compounds, metmyoglobin-[15N]imidazole and bis[15N]imidazole tetraphenyl porphyrin, provides strong evidence for bisimidazole coordination in cytochrome a.     

Characterization of nonsymbiotic tomato hemoglobin

The nonsymbiotic tomato hemoglobin SOLly GLB1 (Solanum lycopersicon) is shown to form a homodimer of approximately 36 kDa with a high affinity for oxygen. Furthermore, our combined ultraviolet/visible, resonance Raman, and continuous wave electron paramagnetic resonance (EPR) measurements reveal that a mixture of penta- and hexacoordination of the heme iron is found in the deoxy ferrous form, whereas the ferric form shows predominantly a bis-histidine ligation (F8His-Fe(2+/3+)-E7His). This differs from the known forms of vertebrate hemoglobins and myoglobins. We have successfully applied our recently designed pulsed-EPR strategy to study the low-spin ferric form of tomato hemoglobin. These experiments reveal that, in ferric SOLly GLB1, one of the histidine planes is rotated 20 degrees (+/-10 degrees ) away from a N(heme)-Fe-N(heme) axis. Additionally, the observed g-values indicate a quasicoplanarity of the histidine ligands. From the HYSCORE (hyperfine sublevel correlation) measurements, the hyperfine and nuclear quadrupole couplings of the heme and histidine nitrogens are identified and compared with known EPR/ENDOR data of vertebrate Hbs and cytochromes. Finally, the ligand binding kinetics, which also indicate that the ferrous tomato Hb is only partially hexacoordinated, will be discussed in relation with the heme-pocket structure. The similarities and differences with other known nonsymbiotic plant hemoglobins will be highlighted.     

An EPR and electron nuclear double resonance investigation of carbon monoxide binding to hydrogenase I (bidirectional) from Clostridium pasteurianum W5

Previous M?ssbauer and electron nuclear double resonance (ENDOR) studies of oxidized hydrogenase I (bidirectional) from Clostridium pasteurianum W5 demonstrated that this enzyme contains two diamagnetic [4Fe-4S]2+ clusters and an iron-sulfur center of unknown structure and composition that is characterized by its novel M?ssbauer and ENDOR properties. In the present study we combine ENDOR and EPR measurements to show that the novel cluster contains 3-4 iron atoms. In addition, we have used EPR and ENDOR spectroscopies to investigate the effect of binding the competitive inhibitor carbon monoxide to oxidized hydrogenase I, using 13C-labeled CO and enzyme isotopically enriched in 57Fe. Treatment of oxidized enzyme with CO causes the g-tensor of the paramagnetic center to change from rhombic to axial symmetry. The observation of a 13C signal by ENDOR spectroscopy and analysis of the EPR broadening show that a single CO covalently binds to the paramagnetic center. The 13C hyperfine coupling constant (Ac approximately equal to 21 MHz) is within the range observed for inorganic iron-carbonyl clusters. The observation of 57Fe ENDOR signals from two types of iron site ([A1c] approximately 30-34 MHz; [A2c] approximately 6 MHz) and resolved 57Fe hyperfine interactions in the EPR spectrum from two nuclei characterized by [A1c] confirm that the iron-sulfur cluster remains intact upon CO coordination, but show that CO binding greatly changes the 57Fe hyperfine coupling constants.     

The metal-binding site of imidazole glycerol phosphate dehydratase; EPR and ENDOR studies of the oxo-vanadyl enzyme

 The apo protein of imidazole glycerol phosphate dehydratase (IGPD) from Saccharomyces cerevisiae combines stoichiometrically with certain specific divalent metal cations to assemble the catalytically active form comprising 24 protein subunits and tightly bound metal. VO²⁺ ions react similarly but, uniquely, result in a metallo-protein (VO-IGPD) with neither catalytic activity nor the ability to bind to the reaction intermediate analogue, 2-hydroxy-3-(1,2,4-triazol-1-yl) propylphosphonate. Since VO²⁺ apparently assembles the quaternary structure correctly, it is used in the present study as a spin probe to investigate the metal centre coordination environment by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopy. At neutral pH, the EPR spectrum of VO-IGPD reveals at least three distinct VO²⁺ sub-spectra with one predominant at low pH. The spin Hamiltonian parameters for some of the sub-spectra are consistent with ⁵¹V having nitrogen in the inner-sphere equatorial coordination environment from, most probably, multiple coordinating histidines. Further evidence for inner-sphere nitrogen ligands is obtained from ENDOR spectroscopy. The spectra of the low rf region show signals from interactions with ¹⁴N which are consistent with couplings to the imino nitrogen of coordinated histidine residues. In addition a number of proton ENDOR line pairs are resolved. Of the few that disappear upon exchange of the protein into D₂O, one most likely originates from the exchangeable proton of the N-H group of a coordinated histidine imidazole. ¹H-ENDOR line pairs from non-exchangeable protons with splittings of approximately 3 MHz can be attributed to imidazole carbon protons. Thus, most of the couplings observed by ENDOR are consistent with being from the imidazole heterocycle of one or more histidine ligands. Received: 27 June 1996 / Accepted: 14 March 1997     

Characterization of the MCRred2 form of methyl-coenzyme M reductase: a pulse EPR and ENDOR study

Methyl-coenzyme M reductase (MCR), which catalyses the reduction of methyl-coenzyme M (CH(3)-S-CoM) with coenzyme B (H-S-CoB) to CH(4) and CoM-S-S-CoB, contains the nickel porphinoid F430 as prosthetic group. The active enzyme exhibits the Ni(I)-derived axial EPR signal MCR(red1) both in the absence and presence of the substrates. When the enzyme is competitively inhibited by coenzyme M (HS-CoM) the MCR(red1) signal is partially converted into the rhombic EPR signal MCR(red2). To obtain deeper insight into the geometric and electronic structure of the red2 form, pulse EPR and ENDOR spectroscopy at X- and Q-band microwave frequencies was used. Hyperfine interactions of the four pyrrole nitrogens were determined from ENDOR and HYSCORE data, which revealed two sets of nitrogens with hyperfine couplings differing by about a factor of two. In addition, ENDOR data enabled observation of two nearly isotropic (1)H hyperfine interactions. Both the nitrogen and proton data indicate that the substrate analogue coenzyme M is axially coordinated to Ni(I) in the MCR(red2) state.