ENDOR studies of VO2+: probing protein-metal ion interactions in nephrocalcin.

Nephrocalcin inhibits the growth of calcium oxalate monohydrate crystals in the mammalian kidney. Isoforms A and B contain three equivalents of gamma-carboxyglutamic acid (Gla) residues implicated in Ca2+-binding and exhibit strong inhibitor properties and high Ca2+-binding affinity (Kd approximately 10(-8) M). Isoforms C and D lack these properties and exhibit low Ca2+-binding affinity (Kd approximately 10(-6) M). With VO2+ as a structural probe, electron paramagnetic resonance (EPR) studies of the Ca2+-binding sites of isoforms B and D showed that VO2+ binds competitively with a metal ion:protein stoichiometry of 4:1. EPR spectral parameters of the VO2+ ion were consistent with only equatorial oxygen-donor ligands. EPR and angle-selected electron nuclear double resonance (ENDOR) spectra showed two equatorially positioned, metal coordinating waters in isoform D while in isoform B no ligands undergoing hydrogen exchange were found. Since isoform D showed no evidence for axially coordinated water, similarly to isoform B, it is likely that the protein residues occupying the axial sites are identical in both proteins. ENDOR spectra of VO2+-complexes of isoforms B and D were compared to spectra of the VO2+-complex with alpha-ethylmalonic acid (EMA), a molecular mimic of Gla. Spectra of the VO2+-complex of EMA showed axial water located trans to the V=O bond and outer shell water hydrogen-bonded to the vanadyl oxygen, consistent with the X-ray structure of Ca(EMA)2. We, therefore, conclude that the spatial disposition of carboxylate groups of Gla residues coordinating Ca2+ in isoforms A and B must differ from that observed in the crystal structure of Ca(EMA)2.     

Iron binding to horse spleen apoferritin: a vanadyl ENDOR spin probe study.

The role of the protein shell in the formation of the hydrous ferric oxide core of ferritin is poorly understood. A VO2+ spin probe study was undertaken to characterize the initial complex of Fe2+ with horse spleen apoferritin (96% L-subunits). A competitive binding study of VO2+ and Fe2+ showed that the two metals compete 1:1 for binding at the same site or region of the protein. Curve fitting of the binding data showed that the affinity of VO2+ for the protein was 15 times that of Fe2+. Electron nuclear double resonance (ENDOR) measurements on the VO(2+)-apoferritin complex showed couplings from two nitrogen nuclei, tentatively ascribed to the N1 and N3 nitrogens of the imidazole ligand of histidine. The possibility that the observed nitrogen couplings are from two different ligands is not precluded by the data, however. A pair of exchangeable proton lines with a coupling of approximately 1 MHz is tentatively assigned to the NH proton of the coordinated nitrogen. A 30-40% reduction in the intensity of the 1H matrix ENDOR line upon D2O-H2O exchange indicates that the metal-binding site is accessible to solvent and, therefore, to molecular oxygen as well. The ENDOR data provide the first evidence for a principle iron(II)-binding site with nitrogen coordination in an L-subunit ferritin. The site may be important in Fe2+ oxidation during the beginning stages of core formation.     

Oxo-vanadium as a spin probe for the investigation of the metal coordination environment of imidazole glycerol phosphate dehydratase

Imidazole glycerol phosphate dehydratase (IGPD) catalyses the dehydration of imidazole glycerol phosphate to imidazole acetol phosphate, an important late step in the biosynthesis of histidine. IGPD, isolated as a low molecular weight and inactive apo-form, assembles with specific divalent metal cations to form a catalytically active high molecular weight metalloenzyme. Oxo-vanadium ions also assemble the protein into, apparently, the same high molecular weight form but, uniquely, yield a protein without catalytic activity. The VO2+ derivative of IGPD has been investigated by electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopy. The spin Hamiltonian parameters indicate the presence of multiple 14N nuclei in the inner coordination sphere of VO2+ which is corroborated by ENDOR and ESEEM spectra showing resonances attributable to interactions with 14N nuclei. The isotropic superhyperfine coupling component of about 7 MHz determined by ENDOR is consistent with a nitrogen of coordinated histidine imidazole(s). The ESEEM Fourier-transform spectra further support the notion that the VO2+ substituted enzyme contains inner-sphere nitrogen ligands. The isotropic and anisotropic 14N superhyperfine coupling components are similar to those reported for other equatorially coordinated enzymatic histidine imidazole systems. ESEEM resonances from axial 14N ligands are discussed.     

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 configuration of the Cu2+ binding region in full-length human prion protein

The cellular prion protein (PrP^C) is a Cu²⁺ binding protein connected to the outer cell membrane. The molecular features of the Cu²⁺ binding sites have been investigated and characterized by spectroscopic experiments on PrP^C-derived peptides and the recombinant human full-length PrP^C (hPrP-[23-231]). The hPrP-[23-231] was loaded with ⁶³Cu under slightly acidic (pH 6.0) or neutral conditions. The PrP^C/Cu²⁺-complexes were investigated by extended X-ray absorption fine structure (EXAFS), electron paramagnetic resonance (EPR), and electron nuclear double resonance (ENDOR). For comparison, peptides from the copper-binding octarepeat domain were investigated in different environments. Molecular mechanics computations were used to select sterically possible peptide/Cu²⁺ structures. The simulated EPR, ENDOR, and EXAFS spectra of these structures were compared with our experimental data. For a stoichiometry of two octarepeats per copper the resulting model has a square planar four nitrogen Cu²⁺ coordination. Two nitrogens belong to imidazole rings of histidine residues. Further ligands are two deprotonated backbone amide nitrogens of the adjacent glycine residues and an axial oxygen of a water molecule. Our complex model differs significantly from those previously obtained for shorter peptides. Sequence context, buffer conditions and stoichiometry of copper show marked influence on the configuration of copper binding to PrP^C. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

17O electron nuclear double resonance characterization of substrate binding to the [4Fe-4S]1+ cluster of reduced active aconitase

To characterize the binding of substrate to aconitase, we have made 17O electron nuclear double resonance (ENDOR) measurements on reduced active ([4Fe-4S]1+) beef heart aconitase, both in H216O and H217O, in the presence of substrate and the inhibitors, tricarballylate, trans-aconitate, and 1-hydroxy-2-nitro-1, 3-propanedicarboxylate, referred to here as nitroisocitrate; the hydroxyl of the latter also was isotypically labeled with 17O. The hydroxyl oxygen of citrate and isocitrate is exchanged with solvent water by aconitase, but the hydroxyl of nitroisocitrate is not. Thus, the isotopic composition of nitroisocitrate can be chemically controlled, allowing direct identification of any 17O ENDOR signal associated with it. 17O ENDOR signals were observed from Hx17O (mean = 1 or 2) bound to the [4Fe-4S]1+ cluster in samples prepared with trans-aconitate and unlabeled nitroisocitrate. 17O-Labeled nitroisocitrate in H216O bound to the cluster showed a signal from the 17OH group; in H217O it showed 17O ENDOR resonances due to both Hx17O and 17OH of substrate. This result demonstrates that the cluster participates in substrate binding and can simultaneously coordinate the hydroxyl of a substrate (or analogue) and water (or hydroxyl). The sample with citrate in H217O showed only the Hx17O signal, although aconitase exchanges the hydroxyl of substrate with solvent water. The mechanism of action of aconitase is discussed in light of this observation. Comparison shows the ENDOR study to be in agreement with previous M?ssbauer and EPR spectroscopic results.     

EPR and 57Fe ENDOR investigation of 2Fe ferredoxins from Aquifex aeolicus

We have employed EPR and a set of recently developed electron nuclear double resonance (ENDOR) spectroscopies to characterize a suite of [2Fe?C2S] ferredoxin clusters from Aquifex aeolicus (Aae Fd1, Fd4, and Fd5). Antiferromagnetic coupling between the Fe^II, S?=?2, and Fe^III, S?=?5/2, sites of the [2Fe?C2S]⁺ cluster in these proteins creates an S?=?1/2 ground state. A complete discussion of the spin-Hamiltonian contributions to g includes new symmetry arguments along with references to related FeS model compounds and their symmetry and EPR properties. Complete ⁵⁷Fe hyperfine coupling (hfc) tensors for each iron, with respective orientations relative to g, have been determined by the use of ??stochastic?? continuous wave and/or ??random hopped?? pulsed ENDOR, with the relative utility of the two approaches being emphasized. The reported hyperfine tensors include absolute signs determined by a modified pulsed ENDOR saturation and recovery (PESTRE) technique, RD-PESTRE??a post-processing protocol of the ??raw data?? that comprises an ENDOR spectrum. The ⁵⁷Fe hyperfine tensor components found by ENDOR are nicely consistent with those previously found by M?ssbauer spectroscopy, while accurate tensor orientations are unique to the ENDOR approach. These measurements demonstrate the capabilities of the newly developed methods. The high-precision hfc tensors serve as a benchmark for this class of FeS proteins, while the variation in the ⁵⁷Fe hfc tensors as a function of symmetry in these small FeS clusters provides a reference for higher-nuclearity FeS clusters, such as those found in nitrogenase.     

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     

An electron nuclear double resonance investigation of redox-induced electronic structural change at CuA2+ in cytochrome c oxidase

We measured an electronic change at cysteine ligand(s) of the CuA2+ center brought on by reduction of other metal centers within cytochrome c oxidase, notably cytochrome a. This change specifically manifested itself as a modification in magnetic hyperfine coupling to the beta-protons of the beta-carbons adjacent to the cysteine sulfur in the CuA2+ coordination sphere. The electron nuclear double resonance ENDOR signals of these beta-protons had previously been assigned through study of selectively deuterated yeast oxidase. In the present study the ENDOR signals of the CuA2+ center were compared from the following forms of oxidase: resting (a3+.CuA2+.a3+3.CuB2+); mixed valence, 2-electron-reduced CO-ligated oxidase (a3+.CuA2+.a2+3CO.CuB+), and a more completely reduced mixed-valence CO-ligated oxidase. In agreement with previous studies on 3-electron-reduced oxidase, the latter more completely reduced oxidase showed cytochrome a preferentially reduced with respect to CuA, implying that the majority of paramagnetic CuA2+ centers had reduced cytochrome a partners. The ENDOR-resolved splitting of the beta-proton hyperfine features substantially decreased in going from the first two more oxidized forms to the more fully reduced latter form. Thus, the electronic structure of the CuA2+ center specifically monitored by hyperfine couplings to cysteine protons changed in response to a reductive event elsewhere in the protein. This structural change may correlate with the anticooperative redox interaction recently reported between cytochrome a and CuA.     

Catalytic function and local proton structure at the type 2 copper of nitrite reductase: the correlation of enzymatic pH dependence,conserved residues,and proton hyperfine structure

Electron nuclear double resonance (ENDOR) of protons at Type 2 and Type 1 cupric active sites correlates with the enzymatic pH dependence, the mutation of nearby conserved, nonligating residues, and electron transfer in heterologously expressed Rhodobacter sphaeroides nitrite reductase. Wild-type enzyme showed a pH 6 activity maximum but no kinetic deuterium isotope effect, suggesting protons are not transferred in the rate-limiting step of nitrite reduction. However, protonatable Asp129 and His287, both located near the Type 2 center, modulated enzyme activity. ENDOR of the wild-type Type 2 center at pH 6.0 revealed an exchangeable proton with large hyperfine coupling. Dipolar distance estimates indicated that this proton was 2.50-2.75 or 2.25-2.45 A from Type 2 copper in the presence or absence of nitrite, respectively. This proton may provide a properly oriented hydrogen bond to enhance water formation upon nitrite reduction. This proton was eliminated at pH 5.0 and showed a diminished coupling at pH 7.5. Mutations of Asp129 and His287 reduced enzyme activity and altered the exchangeable proton hyperfine spectra. Mutation of Asp129 prevented a pH-dependent change at the Type 1 Cys167 ligand as observed by Cys C(beta) proton ENDOR, implying there is a Type 2 and pH-dependent alteration of the Type 1 center. Mutation of the Type 1 center ligand Met182 to Thr and mutation of Asp129 increased the activation energy for nitrite reduction. Involvement of both the Type 1 center and Asp129 in modulating activation energy shows that electron transfer from the Type 1 center to a nitrite-ligated Type 2 center is rate-limiting for nitrite reduction. Mutation of Ile289 to Ala and Val caused minor perturbation to enzyme activity, but as detected by ENDOR, allowed formate binding. Thus, bulky Ile289 may exclude non-nitrite ligands from the Type 2 active site.     

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.     

Structural basis for VO<Superscript>2+</Superscript> inhibition of nitrogenase activity (A): <Superscript>31</Superscript>P and <Superscript>23</Superscript>Na interactions with the metal at the nucleotide binding site of the nitrogenase Fe protein identified by ENDOR spectroscopy

We previously reported the vanadyl hyperfine couplings of VO(2+)-ATP and VO(2+)-ADP complexes in the presence of the nitrogenase Fe protein from Klebsiella pneumoniae (Petersen et al. in Biochemistry 41:13253-13263, 2002). It was demonstrated that different VO(2+)-nucleotide coordination environments coexist and are distinguishable by electron paramagnetic resonance (EPR) spectroscopy. Here orientation-selective continuous-wave electron-nuclear double resonance (ENDOR) spectra have been investigated especially in the low-radio-frequency range in order to identify superhyperfine interactions with nuclei other than protons. Some of these resonances have been attributed to the presence of a strong interaction with a 31P nucleus although no resolvable superhyperfine structure due to 31P or other nuclei was detected in the EPR spectra. The superhyperfine coupling component is determined to be about 25 MHz. Such a 31P coupling is consistent with an interaction of the metal with phosphorus from a directly, equatorially coordinated nucleotide phosphate group(s). Additionally, novel more prominent 31P ENDOR signals are detected in the low-frequency region. Some of these correspond to a relatively weak 31P coupling. This coupling is present with ATP for all pH forms but is absent with ADP. The ENDOR resonances of these weakly coupled 31P are likely to originate from an interaction of the metal with a nucleotide phosphate group of the nucleoside triphosphate and are attributed to a phosphorus with axial characteristics. Another set of resonances, split about the nuclear Zeeman frequency of 23Na, was detected, suggesting that a monovalent Na+ ion is closely associated with the divalent metal-nucleotide binding site. Na+ replacement by K+ unambiguously confirmed that ENDORs at radio frequencies between 3.0 and 4.5 MHz arise from an interaction with Na+ ions. In contrast to the low-frequency 31P signal, these resonances are present in spectra with both ADP and ATP, and for both low- and neutral-pH forms, although slight differences are detected, showing that these are sensitive to the nucleotide and pH.     

Electron-nuclear double resonance of copper complexes of human transferrin

Electron-nuclear double resonance (ENDOR) spectroscopy has been used to study ligand and copper hyperfine interactions in Cu(II) complexes of human transferrin. A nearly isotropic superhyperfine interaction of the Cu(II) spin with a single 14N nucleus was identified, and the principal values of its tensor were estimated. All principal values of the copper hyperfine tensor were also directly measured for the first time. Resonances from at least two exchangeable protons were observed, but their origin could not be ascertained. At physiological pH, and in the presence of bicarbonate, ENDOR spectra of the two metal-binding sites were virtually indistinguishable.     

Characterization of the Ni-Fe-C complex formed by reaction of carbon monoxide with the carbon monoxide dehydrogenase from Clostridium thermoaceticum by Q-band ENDOR.

Q-Band ENDOR studies on carbon monoxide dehydrogenase (CODH) from the acetogenic bacterium Clostridium thermoaceticum provided unambiguous evidence that the reaction of CO with CODH produces a novel metal center that includes at least one nickel, at least three iron sites, and the carbon of one CO. The 57Fe hyperfine couplings determined by ENDOR are similar to the values used in simulation of the M?ssbauer spectra [Lindahl et al. (1989) J. Biol. Chem. 265, 3880-3888]. EPR simulation using these AFe values is equally good for a 4Fe or a 3Fe center. The 13C ENDOR data are consistent with the binding of a carbon atom to either the Ni or the Fe component of the spin-coupled cluster. The 13C hyperfine couplings are similar to those determined earlier for the C0-bound form of the H cluster of the Clostridium pasteurianum hydrogenase, proposed to be the active site of hydrogen activation [Telser et al. (1987) J. Biol. Chem. 262, 6589-5694]. The 61 Ni ENDOR data are the first nickel ENDOR recorded for an enzyme. The EPR simulation using the ENDOR-derived hyperfine values for 61Ni is consistent with a single nickel site in the Ni-Fe-C complex. On the basis of our results and the M?ssbauer data [Lindahl et al. (1989) J. Biol. Chem. 265, 3880-3888], we propose the stoichiometry of the components of the Ni-Fe-C complex to be Ni1Fe3-4S greater than or equal to 4C1, with four acid-labile sulfides.     

Effects of substrates (methyl isocyanide,C2H2) and inhibitor (CO) on resting-state wild-type and NifV(-)Klebsiella pneumoniae MoFe proteins

We report the use of electron nuclear double resonance (ENDOR) spectroscopy to examine how the metal sites in the FeMo-cofactor cluster of the resting nitrogenase MoFe protein respond to addition of the substrates acetylene and methyl isocyanide and the inhibitor carbon monoxide. 1H, 57Fe and 95Mo ENDOR measurements were performed on the wild-type and the NifV(-)proteins from Klebsiella pneumoniae. Among the molecules tested, only the addition of acetylene to either protein induced widespread changes in the 57Fe ENDOR spectra. Acetylene also induced increases in intensity from unresolved protons in the proton ENDOR spectra. Thus we conclude that acetylene may bind to the resting-state MoFe protein to perturb the FeMo-cofactor environment. On the other hand, the present results show that methyl isocyanide and carbon monoxide do not substantially alter the FeMo cofactor's geometric and electronic structures. We interpret this as lack of interaction between those two molecules and the FeMo cofactor in the resting state MoFe protein. Thus, although it is generally accepted that substrates or inhibitors bind to the FeMo-cofactor only under turnover condition, this work provides evidence that at least one substrate can perturb the active site of nitrogenase under non-catalytic conditions.     

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.     

Model for the porphyrin-DNA binding site: ENDOR investigations of Cu-porphyrins binding to DNA.

Proton ENDOR has been observed from frozen solutions (ca. 38K degrees) of copper meso-(4-N-tetra-methylpyridyl)porphyrin (CuTMpyP(4)) complexed with Salmon sperm DNA in water and D2O. Lines from exchangeable protons of the DNA bases have been observed in these ENDOR spectra. Analyses of these ENDOR data show that the separations of these DNA protons from the copper atom are between 3.76 and 3.84 A with angles of 19.5 to 22.5 degrees between the Cu-H vectors and the gz axis. A distant ENDOR response has also been observed from phosphorous nuclei in the DNA backbone. We estimate that the phosphorous atoms producing this ENDOR signal are 7.5-10 A from the copper center of the porphyrin. These ENDOR data combined with results from an earlier NMR investigation have been used to construct a computer simulated model of the binding site in which the porphyrin is partially intercalated and extends into the major groove of DNA. The two GC base pairs at this site are slightly inequivalent. For each, the G imino proton and one of the C amino protons are at appropriate positions to account for the ENDOR signals arising from exchangeable protons. It is unlikely that this inequivalence would persist at room temperature where dynamic processes would give an apparently symmetric interaction. Although the model accounts for all reported experimental data involving tetracationic porphyrin species which have been suggested to be intercalators, it is not a unique solution.     

57Fe and 1H electron-nuclear double resonance of three doubly reduced states Escherichia coli sulfite reductase

We have employed electron-nuclear double resonance (ENDOR) spectroscopy to study the 57Fe hyperfine interactions in the bridged-siroheme [4Fe-4S] cluster that forms the catalytically active center of the two-electron-reduced hemoprotein subunit of Escherichia coli NADPH-sulfite reductase (SiR2-). Previous electron paramagnetic resonance (EPR) and M?ssbauer studies have shown that this enzyme oxidation state can exist in three distinct spectroscopic forms: (1) a "g = 2.29" EPR species that predominates in unligated SiR2-, in which the siroheme Fe2+ is believed to be in an S = 1 state; (2) a "g = 4.88" type of EPR species that predominates in SiR2- in the presence of small amounts of guanidinium sulfate, in which the siroheme Fe2+ is in an S = 2 state; and (3) a classical "g = 1.94" type of EPR species that is seen in SiR2- ligated with CO, in which the siroheme Fe2+ is in an S = 0 state. In all three species, the cluster is in the [4Fe-4S]1+ state, and two distinct types of Fe site are seen in M?ssbauer spectroscopy. ENDOR studies confirm the M?ssbauer assignments for the cluster 57Fe in the g = 1.94 state, with A values of 37, 37, and 32 MHz for site I and ca. 19 MHz for site II. The hyperfine interactions are not too different on the g = 2.29 state, with site I Fe showing more anisotropic A values of 32, 24, and 20 MHz (site II was not detected).(ABSTRACT TRUNCATED AT 250 WORDS)