Q-band ENDOR Research Articles

X and Q-band endor study of the Fe+ (I) center in chlorinated SrCl2 single crystals

Previous Electron Paramagnetic Resonance (EPR) studies have shown that Fe2+ doped SrCl2 crystals grown in a chlorine atmosphere exhibit new trapped electron Fe+ centers after irradiation with X or γ-rays at 77 K. Two types of such centers, called Fe+ (I) and Fe+ (II), both exhibiting axial ⟨001⟩ symmetry, have been identified. We present here the results of an Electron Nuclear Double Resonance (ENDOR) study in X and Q-band on the Fe+ (I) center, which has been found to be thermally stable up to 700K. The superhyperfine and quadrupole interactions of the paramagnetic Fe+ ion with the nearest Cl− ions have been determined from the ENDOR angular dependence data. The results strongly suggest that the Fe+ (I) center consists of a Fe+ ion in the center of a cube of eight Cl− ions, compressed along an ⟨001⟩ direction.

Detailed Proton Q-Band ENDOR Study of the Electron Spin Population Distribution in the Reduced [4Fe-4S]1+ State

Proton ENDOR spectroscopy has been used in single crystals of the synthetic model compound [N(C2D5)4]2[Fe4S4(SCH2C6D5)4] in order to determine the electron spin population distribution within the [4Fe-4S]1+ paramagnetic state. This compound constitutes an excellent biomimetic model of the active sites of many [4Fe-4S] proteins, with the [4Fe-4S]1+ state corresponding to the reduced state of ferredoxin proteins and of numerous other related proteins. Because the information obtained by ENDOR and (or) by NMR from these protons constitutes the basis of the most detailed knowledge on these proteins from magnetic resonance methods, our purpose is to set a firm basis for their interpretation. Only by using Q-band ENDOR could we succeed in measuring the complete hyperfine tensors of a total of 11 protons, i.e., the eight protons of the four CH2 groups and three additional protons from nearest neighbors. By appealing to a new version of the multicentric point-dipole analysis of the dipolar parts of these tensors, we could deduce a detailed map of the electron spin population distribution. This new approach is based on the direct determination of the four spin projection coefficients centered on the four iron monomers constituting the cluster. Our findings confirm that two classes of reduced [4Fe-4S]1+ have to be distinguished, according to the magnitude of the spin coupling coefficients that best describe their fundamental magnetic spin states. Such a distinction has been observed already among oxidized [4Fe-4S]3+ centers. This opens the way for further research aiming at understanding the relations between structures, redox states, ligation, and magnetic spin states.

Q-Band ENDOR (Electron Nuclear Double Resonance) of the Heme o3 Liganding Environment at the Binuclear Center in Cytochrome bo3 from Escherichia coli

The liganding environment and electronic structure of high-spin ferric heme o3 in the binuclear center of bo3 ubiquinol oxidase were probed with Q-band (34.1 GHz) ENDOR. We studied forms of the enzyme where reduction eliminated antiferromagnetic coupling to the nearby CuB center. ENDOR comparisons were made to 14N heme and histidine nitrogen features, to exchangeable proton features, and to the 17O-water feature of aquometmyoglobin, a high-spin ferric heme protein with a known axial water ligand. Nitrogen features observed from heme and proximal histidine of cytochrome o3 occurred in the range of frequencies where they had previously been observed for aquometmyoglobin. However, the proximal histidine of cytochrome o3 was notable in revealing more disorder and a wider range in its hyperfine couplings than in aquometmyoglobin. Di-oxygen-induced turnover of the bo3 enzyme altered both the heme and histidine electronic structure so as to show after turnover a simpler, better resolved heme and histidine pattern with greater similarity to the pattern found in aquometmyoglobin. We saw no evidence from cytochrome o3 for the 6 MHz exchangeable water proton coupling and the 17.5 MHz 17O-water coupling exhibited by aquometmyoglobin. A plausible conclusion from such a negative result is that the high-spin ferric o3 heme which we studied has no covalently attached axial sixth OHX ligand when magnetically decoupled from CuB. Comparison of cytochrome o3 in protonated and deuterated solvents definitively indicated no exchangeable proton couplings greater than 3.5 MHz. An implication of our study is that in the magnetically decoupled high-spin ferric cytochrome o3 there is either no sixth OHX ligand or, if there is any “sixth” OHX ligand to cytochrome o3 that can exchange with 17O-water, it would have to be off-axis, disordered, and weakly liganded to the heme.

The Core Structure of X Generated in the Assembly of the Diiron Cluster of Ribonucleotide Reductase: 17O2 and H217O ENDOR

The intermediate, designated X, formed during the self-assembly reaction of the tyrosyl radical/μ-oxo-bridged diferric cofactor in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) is directly involved in the oxidation of Y122 to the catalytically essential ·Y122. Earlier rapid freeze-quench (RFQ) Q-band ENDOR studies led to the formulation of X as a spin-coupled FeIII/FeIV center, with an S = 1/2 ground state, and showed that X contains a single terminal aqua ligand (water molecule or 2-fold disordered hydroxyl) bound to FeIII but does not contain an hydroxyl bridge. That ENDOR data, coupled with RFQ-EXAFS data, plus the strong spin coupling between the iron ions constrain the structure of X to a di- or tribridged species whose inorganic core (defined as iron and exogenous ligands) contains the [(HxO)FeIIIOFeIV] fragment. To determine whether the core contains a second oxo bridge and to establish the fate of the atoms derived from O2, we have now performed CW and pulsed Q-band 17O ENDOR e...

High-Frequency EPR and Pulsed Q-Band ENDOR Studies on the Origin of the Hydrogen Bond in Tyrosyl Radicals of Ribonucleotide Reductase R2 Proteins from Mouse and Herpes Simplex Virus Type 1

The g tensor of the tyrosyl radical present in the active R2 protein of ribonucleotide reductase is anisotropic, and the g1 component is influenced by hydrogen bonding to the oxygen of the tyrosyl ...

Electronic structural information from Q-band ENDOR on the type 1 and type 2 copper liganding environment in wild-type and mutant forms of copper-containing nitrite reductase.

Q-band ENDOR elucidated proton and nitrogen hyperfine features to provide spin density information at ligands of blue-green Type 1 and catalytic Type 2 copper centers in nitrite reductase. The blue-green Type 1 center of nitrite reductase has a redox, electron-transfer role, and compared to the blue center of plastocyanin, it has the following structural differences: a shortened Cu-Smet bond length, a longer Cu-Scys bond length, and altered ligand-copper-ligand bond angles (Adman, E. T., Godden, J. W., and Turley, S. (1995) J. Biol. Chem. 270, 27458-27474). The hyperfine couplings of the two Type 1 histidine (N delta) ligands showed a larger percentage difference from each other in electron spin density than previously reported for other blue Type 1 proteins, while the cysteine beta-proton hyperfine couplings, a measure of unpaired p pi spin density on the liganding cysteine sulfur, showed a smaller electron spin density. A mutation of the Type 1 center, M182T, having the copper-liganding Met182 transformed to Thr182, caused the center to revert to an optically "blue" center, raised its redox potential by approximately 100 mV, and led to the loss of activity (prior paper). Surprisingly, in M182T there was no change from native Type 1 copper either in the histidine or cysteine hyperfine couplings or in g values and Cu nuclear hyperfine couplings. The conclusion is that the optical and redox alterations due to changed Type 1 methionine ligation need not be concurrent with electron spin delocalization changes in the HOMO as reported from its essential cysteine and histidines. A detailed picture of the nitrogen couplings from the three histidine (N epsilon) ligands of the Type 2 center indicated a substantial ( approximately 200%) electronic hyperfine inequivalence of one of the histidine nitrogens from the other two within the Type 2 HOMO and thus provided evidence for electronic distortion of the Type 2 site. In the presence of the nitrite substrate, hyperfine couplings of all histidines diminished. We suggest that this nitrite-induced decreased covalency would correlate with an increased Type 2 redox potential to assist electron transfer to the Type 2 center. Dipole-coupled, angle-selected exchangeable proton features, observed over a range of g values, predicted a ligand-water proton distance of 2.80 A from copper, and these water protons were eliminated by nitrite. His287 is not a Type 2 ligand but is positioned to perturb an axial water or a nitrite of Type 2 copper. In the presence of nitrite the mutant H287E showed no evidence for the loss of water protons and no diminished ligand histidine covalency. H287E has vastly diminished activity (prior paper), and the ENDOR information is that NO2- does not bind to Type 2 copper of H287E. In summary, the electronic information from this study of native and suitably chosen mutants provided a test of the highest occupied molecular orbital (HOMO) wave function at Type 1 and Type 2 coppers and an intimate electronic insight into functional enzymatic properties.

Reconsideration of X, the Diiron Intermediate Formed during Cofactor Assembly in E. coli Ribonucleotide Reductase

The R2 subunit of Escherichia coli ribonucleotide reductase (RNR) contains a stable tyrosyl radical (•Y122) diferric cluster cofactor. Earlier studies on the cofactor assembly reaction detected a paramagnetic intermediate, X, that was found to be kinetically competent to oxidize Y122. Studies using rapid freeze-quench (RFQ) Mossbauer and EPR spectroscopies led to the proposal that X is comprised of two high spin ferric ions and a S = 1/2 ligand radical, mutually spin coupled to give a S = 1/2 ground state (Ravi, N.; Bollinger, J. M., Jr.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J. J. Am. Chem. Soc. 1994, 116, 8007−8014). An extension of RFQ methodology to Q-band ENDOR spectroscopy using 57Fe has shown that one of the irons has a very nearly isotropic hyperfine tensor (A(FeA) = −[74.2(2), 72.2(2), 73.2(2)] MHz) as expected for FeIII, but that the other iron site displays considerable anisotropy (A(FeB) = +[27.5(2), 36.8(2), 36.8(2)] MHz), indicative of substantial FeIV character. Reanalysis of the Mossbaue...

ENDOR Spectroscopic Study of Macroradicals in Irradiated Oriented Nylon-6

Abstract Macroradicals formed by hydrogen atom elimination in uniaxially and biaxially oriented nylon-6 have been investigated at low temperature using a Q-band ENDOR technique. Hyperfine coupling constants were obtained from the observed 1H ENDOR spectra for the protons located at α-, β-, and γ-positions relative to the unpaired electron in the radical -CO-NH-CH-CH2-CH2-. Principal values for α-proton anisotropic coupling tensor have been determined. A degree of angular disorientation of the radical centers -CH- along the axes of biaxially oriented polymer was evaluated. The protons of the β-methylene group were found to be magnetically nonequivalent and therefore the plane of the free carbon was twisted. The deviation from the ideally planar transconfiguration corresponds to the rotation around the Cα-Cβ bond on average angle 11°. A change in β-proton ENDOR spectrum has been revealed under mechanical loading of oriented specimens. The results obtained evidenced for be appearance of reversible torsional ...

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)

Characterization of the nickel-iron-carbon 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 Mössbauer 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 Mössbauer 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.

Detection and characterization of exchangeable protons bound to the hydrogen-activation nickel site of Desulfovibrio gigas hydrogenase: a proton and deuterium Q-band ENDOR study

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDetection and characterization of exchangeable protons bound to the hydrogen-activation nickel site of Desulfovibrio gigas hydrogenase: a proton and deuterium Q-band ENDOR studyChaoliang Fan, Miguel Teixeira, Jose Moura, Isabel Moura, Huynh Boi Hanh, Jean Le Gall, Harry D. Peck Jr., and Brian M. HoffmanCite this: J. Am. Chem. Soc. 1991, 113, 1, 20–24Publication Date (Print):January 1, 1991Publication History Published online1 May 2002Published inissue 1 January 1991https://doi.org/10.1021/ja00001a003RIGHTS & PERMISSIONSArticle Views243Altmetric-Citations103LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (702 KB) Get e-Alerts Get e-Alerts