he iron-molybdenum cofactor of nitrogenase (FeMoco) catalyzes fixation of N-2 via Fe hydride intermediates. Our understanding of these species has relied heavily on the characterization of well-defined 3d metal hydride complexes, which serve as putative spectroscopic models. Although the Fe ions in FeMoco, a weak-field cluster, are expected to adopt locally high-spin Fe2+/3+ configurations, synthetically accessible hydride complexes featuring d(5) or d(6) electron counts are almost exclusively low-spin. We report herein the isolation of a terminal hydride complex of four-coordinate, high-spin (d(5); S = 5/2) Mn2+. Electron paramagnetic resonance and electron-nuclear double resonance studies reveal an unusually large degree of spin density on the hydrido ligand. In light of the isoelectronic relationship between Mn2+ and Fe3+, our results are expected to inform our understanding of the valence electronic structures of reactive hydride intermediates derived from FeMoco.

We examine the electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopy of three quite distinct high-spin Mn(II) systems and describe experimental techniques and methods of analysis that are useful in their study. We demonstrate that this S = 5/2 metal center provides useful orientation-selection through the Zero-Field Splitting (ZFS) tensor that enables determination of a 13C hyperfine-coupling tensor with extremely small hyperfine interaction. We also demonstrate that Mims suppression effects can be used in concert with orientation-selection to edit complex [1,2]H ENDOR patterns that can be produced by even a ‘simple’ center with a single Mn(II). We develop a perturbation-based approach to understanding second-order shifts in Mn(II) ENDOR responses that occur in systems with intermediate ZFS values, and show that these shifts can be used to estimate the values of the ZFS tensors.

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by 15N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using 1H and 2H ENDOR. In addition, 2H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary 1H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.

Lipoxygenase (LOX) enzymes produce important cell-signaling mediators, yet attempts to capture and characterize LOX-substrate complexes by X-ray co-crystallography are commonly unsuccessful, requiring development of alternative structural methods. We previously reported the structure of the complex of soybean lipoxygenase, SLO, with substrate linoleic acid (LA), as visualized through the integration of 13C/1H electron nuclear double resonance (ENDOR) spectroscopy and molecular dynamics (MD) computations. However, this required substitution of the catalytic mononuclear, nonheme iron by the structurally faithful, yet inactive Mn2+ ion as a spin probe. Unlike canonical Fe-LOXs from plants and animals, LOXs from pathogenic fungi contain active mononuclear Mn2+ metallocenters. Here, we report the ground-state active-site structure of the native, fully glycosylated fungal LOX from rice blast pathogen Magnaporthe oryzae, MoLOX complexed with LA, as obtained through the 13C/1H ENDOR-guided MD approach. The catalytically important distance between the hydrogen donor, carbon-11 (C11), and the acceptor, Mn-bound oxygen, (donor-acceptor distance, DAD) for the MoLOX-LA complex derived in this fashion is 3.4 ± 0.1 Å. The difference of the MoLOX-LA DAD from that of the SLO-LA complex, 3.1 ± 0.1 Å, is functionally important, although is only 0.3 Å, despite the MoLOX complex having a Mn-C11 distance of 5.4 Å and a "carboxylate-out" substrate-binding orientation, whereas the SLO complex has a 4.9 Å Mn-C11 distance and a "carboxylate-in" substrate orientation. The results provide structural insights into reactivity differences across the LOX family, give a foundation for guiding development of MoLOX inhibitors, and highlight the robustness of the ENDOR-guided MD approach to describe LOX-substrate structures.

The kinetics of the transfer of the chelate, ethylenediamine tetraacetate (EDTA), from Calcium(II) to Copper(II) in imidazole (Im) buffers near neutral pH, corresponding to the conversion, [Cu(II)Im4]2+→ [Cu(II)EDTA]2-, are characterized with stopped-flow absorption spectroscopy and implemented as a tool for calibrating the interval between mixing and freezing, the freeze-quench time (tQ ), of a rapid freeze-quench (RFQ) apparatus. The kinetics of this reaction are characterized by monitoring changes in UV-visible spectra (300 nm) due to changes in the charge-transfer band associated with the Cu2+ ions upon EDTA binding. Stopped-flow measurements show that the rates of conversion of the Cu2+ ions exhibit exponential kinetics on millisecond time scales at pH values less than 6.8. In parallel, we have developed a simple but precise method to quantitate the speciation of frozen solution mixtures of [Cu(II)(EDTA)]2- and tetraimidazole Cu(II) ([Cu(Im)4]2+) in X-band EPR spectra. The results are implemented in a simple high-precision 'recipe' for determining tQ . These procedures are more accurate and precise than the venerable reaction of aquometmyoglobin with azide for calibrating RFQ apparatus, with the benefit of avoiding high-concentrations of toxic azide solutions.

In nature, methane is oxidized to methanol by two enzymes, the iron-dependent soluble methane monooxygenase (sMMO) and the copper-dependent particulate MMO (pMMO). While sMMO's diiron metal active site is spectroscopically and structurally well-characterized, pMMO's copper sites are not. Recent EPR and ENDOR studies have established the presence of two monocopper sites, but the coordination environment of only one has been determined, that within the PmoB subunit and denoted CuB. Moreover, this recent work only focused on a type I methanotrophic pMMO, while previous observations of the type II enzyme were interpreted in terms of the presence of a dicopper site. First, this report shows that the type II Methylocystis species strain Rockwell pMMO, like the type I pMMOs, contains two monocopper sites and that its CuB site has a coordination environment identical to that of type I enzymes. As such, for the full range of pMMOs this report completes the refutation of prior and ongoing suggestions of multicopper sites. Second, and of primary importance, EPR/ENDOR measurements (a) for the first time establish the coordination environment of the spectroscopically observed site, provisionally denoted CuC, in both types of pMMO, thereby (b) establishing the assignment of this site observed by EPR to the crystallographically observed metal-binding site in the PmoC subunit. Finally, these results further indicate that CuC is the likely site of biological methane oxidation by pMMO, a conclusion that will serve as a foundation for proposals regarding the mechanism of this reaction.

Hydrogen tunneling in enzymatic C-H activation requires a dynamical sampling among ground-state enzyme-substrate (E-S) conformations, which transiently generates a tunneling-ready state (TRS). The TRS is characterized by a hydrogen donor-acceptor distance (DAD) of 2.7 Å, ∼0.5 Å shorter than the dominant DAD of optimized ground states. Recently, a high-resolution, 13C electron-nuclear double-resonance (ENDOR) approach was developed to characterize the ground-state structure of the complex of the linoleic acid (LA) substrate with soybean lipoxygenase (SLO). The resulting enzyme-substrate model revealed two ground-state conformers with different distances between the target C11 of LA and the catalytically active cofactor [Fe(III)-OH]: the active conformer "a", with a van der Waals DAD of 3.1 Å between C11 and metal-bound hydroxide, and an inactive conformer "b", with a distance that is almost 1 Å longer. Herein, the structure of the E-S complex is examined for a series of six variants in which subtle structural modifications of SLO have been introduced either at a hydrophobic side chain near the bound substrate or at a remote residue within a protein network whose flexibility influences hydrogen transfer. A remarkable correlation is found between the ENDOR-derived population of the active ground-state conformer a and the kinetically derived differential enthalpic barrier for D versus H transfer, ΔEa, with the latter increasing as the fraction of conformer a decreases. As proposed, ΔEa provides a "ruler" for the DAD within the TRS. ENDOR measurements further corroborate the previous identification of a dynamical network coupling the buried active site of SLO to the surface. This study shows that subtle imperfections within the initial ground-state structures of E-S complexes are accompanied by compromised geometries at the TRS.

Activation of the diferrous center of the β2 (R2) subunit of the class 1a Escherichia coli ribonucleotide reductases by reaction with O2 followed by one-electron reduction yields a spin-coupled, paramagnetic Fe(III)/Fe(IV) intermediate, denoted X, whose identity has been sought by multiple investigators for over a quarter of a century. To determine the composition and structure of X, the present study has applied (57)Fe, (14,15)N, (17)O, and (1)H electron nuclear double resonance (ENDOR) measurements combined with quantitative measurements of (17)O and (1)H electron paramagnetic resonance line-broadening studies to wild-type X, which is very short-lived, and to X prepared with the Y122F mutant, which has a lifetime of many seconds. Previous studies have established that over several seconds the as-formed X(Y122F) relaxes to an equilibrium structure. The present study focuses on the relaxed structure. It establishes that the inorganic core of relaxed X has the composition [(OH(-))Fe(III)-O-Fe(IV)]: there is no second inorganic oxygenic bridge, neither oxo nor hydroxo. Geometric analysis of the (14)N ENDOR data, together with recent extended X-ray absorption fine structure measurements of the Fe-Fe distance (Dassama, L. M.; et al. J. Am. Chem. Soc. 2013, 135, 16758), supports the view that X contains a "diamond-core" Fe(III)/Fe(IV) center, with the irons bridged by two ligands. One bridging ligand is the oxo bridge (OBr) derived from O2 gas. Given the absence of a second inorganic oxygenic bridge, the second bridging ligand must be protein derived, and is most plausibly assigned as a carboxyl oxygen from E238.

Variations in the internal conformational dynamics of supramolecular nanostructures may be important for their function, yet such dynamics have been difficult to probe experimentally. Now, the molecular motion through a nanofibre cross-section at subnanometre resolution has been quantified using site-directed spin labelling and electron paramagnetic resonance spectroscopy. A large variety of functional self-assembled supramolecular nanostructures have been reported over recent decades1. The experimental approach to these systems initially focused on the design of molecules with specific interactions that lead to discrete geometric structures1,2,3,4, and more recently on the kinetics and mechanistic pathways of self-assembly5,6. However, there remains a major gap in our understanding of the internal conformational dynamics of these systems and of the links between their dynamics and function. Molecular dynamics simulations have yielded information on the molecular fluctuations of supramolecular assemblies5,6,7, yet experimentally it has been difficult to obtain analogous data with subnanometre spatial resolution. Using site-directed spin labelling and electron paramagnetic resonance spectroscopy, we measured the conformational dynamics of a self-assembled nanofibre in water through its 6.7 nm cross-section. Our measurements provide unique insight for the design of supramolecular functional materials.

We have employed EPR and a set of recently developed electron nuclear double resonance (ENDOR) spectroscopies to characterize a suite of [2Fe–2S] ferredoxin clusters from Aquifex aeolicus (Aae Fd1, Fd4, and Fd5). Antiferromagnetic coupling between the FeII, S = 2, and FeIII, S = 5/2, sites of the [2Fe–2S]+ 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 Mössbauer 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 underlying causes of asymmetric intensities in Davies pulsed ENDOR spectra that are associated with the signs of the hyperfine interaction are reinvestigated. The intensity variations in these asymmetric ENDOR patterns are best described as shifts in an apparent baseline intensity that occurs dynamically following on-resonance ENDOR transitions. We have developed an extremely straightforward multi-sequence protocol that is capable of giving the sign of the hyperfine interaction by probing a single ENDOR transition, without reference to its partner transition. This technique, Pulsed ENDOR Saturation and Recovery (PESTRE) monitors dynamic shifts in the ‘baseline’ following measurements at a single RF frequency (single ENDOR peak), rather than observing anomalous ENDOR intensity differences between the two branches of an ENDOR response. These baseline shifts, referred to as dynamic reference levels (DRLs), can be directly tied to the electron-spin manifold from which that ENDOR transition arises. The application of this protocol is demonstrated on 57Fe ENDOR of a 2Fe–2S ferredoxin. We use the 14N ENDOR transitions of the S = 3/2[Fe(II)NO]2+ center of the non-heme iron enzyme, anthranilate dioxygenase (AntDO) to examine the details of the relaxation model using PESTRE.

N(2) binds to the active-site metal cluster in the nitrogenase MoFe protein, the FeMo-cofactor ([7Fe-9S-Mo-homocitrate-X]; FeMo-co) only after the MoFe protein has accumulated three or four electrons/protons (E(3) or E(4) states), with the E(4) state being optimally activated. Here we study the FeMo-co (57)Fe atoms of E(4) trapped with the α-70(Val→Ile) MoFe protein variant through use of advanced ENDOR methods: 'random-hop' Davies pulsed 35 GHz ENDOR; difference triple resonance; the recently developed Pulse-Endor-SaTuration and REcovery (PESTRE) protocol for determining hyperfine-coupling signs; and Raw-DATA (RD)-PESTRE, a PESTRE variant that gives a continuous sign readout over a selected radiofrequency range. These methods have allowed experimental determination of the signed isotropic (57)Fe hyperfine couplings for five of the seven iron sites of the reductively activated E(4) FeMo-co, and given the magnitude of the coupling for a sixth. When supplemented by the use of sum-rules developed to describe electron-spin coupling in FeS proteins, these (57)Fe measurements yield both the magnitude and signs of the isotropic couplings for the complete set of seven Fe sites of FeMo-co in E(4). In light of the previous findings that FeMo-co of E(4) binds two hydrides in the form of (Fe-(μ-H(-))-Fe) fragments, and that molybdenum has not become reduced, an 'electron inventory' analysis assigns the formal redox level of FeMo-co metal ions in E(4) to that of the resting state (M(N)), with the four accumulated electrons residing on the two Fe-bound hydrides. Comparisons with earlier (57)Fe ENDOR studies and electron inventory analyses of the bio-organometallic intermediate formed during the reduction of alkynes and the CO-inhibited forms of nitrogenase (hi-CO and lo-CO) inspire the conjecture that throughout the eight-electron reduction of N(2) plus 2H(+) to two NH(3) plus H(2), the inorganic core of FeMo-co cycles through only a single redox couple connecting two formal redox levels: those associated with the resting state, M(N), and with the one-electron reduced state, M(R). We further note that this conjecture might apply to other complex FeS enzymes.

2065-Pos Board B51 Locating the Phosphate Moiety of an Annular Lipid Near the KCSA Potassium Channel Using Pulsed EPR (ESEEM and ENDOR) Spectroscopy and X-Ray Crystallography John A. Cieslak, Peter Doan, William Liu, Brian Hoffman, Adrian Gross. Recent studies on voltage-dependent potassium channels have demonstrated the importance of the lipid bilayer in maintaining the native structure and packing of the voltage-sensor (Cuello, 2004; Starace and Bezanilla, 2004; Lee, 2005; Ramu, 2006; Schmidt, 2006; Vamvouka, 2008). It has also been suggested that lipids play an integral role in the actual gating mechanism of these channels (Ramu, 2006; Schmidt, 2006; Jogini and Roux 2007; Vamvouka, 2008). However, annular lipids are frequently lost during solubilization and/or are poorly ordered in crystal lattices. Additionally, annular lipids may shift position in the absence of a bilayer’s constraining forces. New tools are therefore needed to detect and locate annular lipids under native conditions. In published work (Cieslak, 2010), we have described a weak (~5 A) ESEEM interaction between a nitroxide spin label (V48R1) in KcsA under native conditions and (presumably) a single 31P nucleus of a well-ordered annular lipid. These results highly constrain the possible locations of the interacting phosphate moiety based on a crystal structure of KcsA-V48R1 in detergent. ENDOR is a superior experiment compared to ESEEM in this case because it is more sensitive to weak interactions and provides additional angular information. Furthermore, ENDOR is a frequency rather than an intensity measurement and thus no assumptions regarding the number of interacting nuclei are made. Mims ENDOR at Q-band of KcsA-V48R1 under native conditions both confirms the previous ESEEM data and further constrains the location of the phosphate moiety. The ENDOR data locate the interacting 31P nucleus at a distance of ~6 A from the nitroxide in a direction near normal to the nitroxide plane. This information provides essentially atomic coordinates for the 31P nucleus of the annular lipid.

All pulsed electron-nuclear double resonance (ENDOR) techniques, and in particular the Mims and Davies sequences, suffer from detectability biases (‘blindspots’) that are directly correlated to the size of the hyperfine interactions of coupled nuclei. Our efforts at ENDOR ‘crystallography’ and ‘mechanism determination’ with these techniques have led our group to refine our simulations of pulsed ENDOR spectra to take into account these biases, and we here describe the process and illustrate it with several examples. We first focus on an issue whose major significance is not widely appreciated, the ‘hole in the middle’ of pulsed ENDOR spectra caused by the n = 0 suppression hole in Mims ENDOR and by the analogous A → 0 suppression in Davies ENDOR for I = ½ and for 2 H ( I = 1). We then discuss the general treatment of suppression effects for I = 1, illustrating it with a treatment of Mims suppression for 14 N.

We previously used a combination of continuous-wave (CW) and pulsed electron-nuclear double resonance (ENDOR) protocols to identify the types of protonated oxygen (OH(x)) species and their disposition within the Fe(III)/Fe(IV) cluster of intermediate X, the direct precursor of the essential diferric-tyrosyl radical cofactor of the beta2 subunit of Escherichia coli ribonucleotide reductase (RNR). We concluded that X contains the [(H(x)O)Fe(III)OFe(IV)] fragment (T model), and does not contain a mu-hydroxo bridge. When combined with a subsequent (17)O ENDOR study of X prepared with H(2)(17)O and with (17)O(2), the results led us to suggest that this fragment is the entire inorganic core of X. This has been questioned by recent reports, but these reports do not themselves agree on the core of X. One concluded that X possesses a di-mu-oxo Fe(III)/Fe(IV) core plus a terminal (H(2)O) bound to Fe(III) [e.g., Han, W.-G.; Liu, T.; Lovell, T.; Noodleman, L. J. Am. Chem. Soc. 2005, 127, 15778-15790]. The other [Mitic, N.; Clay, M. D.; Saleh, L.; Bollinger, J. M.; Solomon, E. I. J. Am. Chem. Soc. 2007, 129, 9049-9065] concluded that X contains only a single oxo bridge and postulated the presence of an additional hydroxo bridge plus a terminal hydroxyl bound to Fe(III). In this report we take advantage of improvements in 35 GHz pulsed ENDOR performance to reexamine the protonation state of oxygenic ligands of the inorganic core of X by directly probing the exchangeable proton(s) with (2)H pulsed ENDOR spectroscopy. These (2)H ENDOR measurements confirm that X contains an Fe(III)-bound terminal aqua ligand (H(x)O), but the spectra contain none of the features that would be required for the proton of a bridging hydroxyl. Thus, we confirm that X contains a terminal aqua (most likely hydroxo) ligand to Fe(III) in addition to one or two mu-oxo bridges but does not contain a mu-hydroxo bridge. The (2)H ENDOR measurements further demonstrate that this conclusion is applicable to both wild type and Y122F-beta2 mutant, and in fact we detect no difference between the properties of protons on the terminal oxygens in the two variants; likewise, (14)N ENDOR measurements of histidyl ligands bound to Fe show no difference between the two variants.

Key features differentiating the coordination environment of the two irons in the mixed-valent (Fe(2+),Fe(3+)) diiron site of Desulfovibrio vulgaris rubrerythrin (Rbr(mv)) were determined by continuous wave (CW) and pulsed ENDOR spectroscopy at 35GHz. (14)N ENDOR evidence indicates that a nitrogen is bound only to the Fe(2+) ion of the mixed-valent site. Assuming that this nitrogen is from His131Ndelta, the same one that furnishes an iron ligand in the crystal structure of the diferric site, the ENDOR data allow us to specify the Fe(2+) and Fe(3+) positions within the molecular reference frame. In addition, the (1,2)H ENDOR on Rbr(mv) indicates the presence of a solvent-derived aqua/hydroxo ligand bound either terminally or in a bridging mode to Fe(3+) in the mixed-valent site. The relatively large g anisotropy of Rbr(mv) and weak antiferromagnetic coupling, J approximately -8 cm(-)(1) (in the 2JS(1)*S(2) formalism), between the irons is more consistent with a bridging than terminal hydroxo ligand. gamma-Irradiation was used to cryoreduce Rbr at 77 K, thereby producing a mixed-valent diiron site [(Rbr(ox))(mv)] that retains the structure of the diferric site. The EPR spectrum of (Rbr(ox))(mv) was nearly identical to that of the as-isolated or chemically reduced samples. This near identity implies that the structure of the mixed-valent Rbr diiron site is essentially identical to that of the diferric site, except for protonation of the oxo bridge, which apparently occurred via a proton jump from hydrogen-bonded solvent at 77 K. The EPR spectrum of (Rbr(ox))(mv) thus supports the (14)N ENDOR-assigned His131 ligation to Fe(2+) and assignment of the solvent-derived ligand observed in the (1,2)H ENDOR to a hydroxo bridge between the irons of the mixed-valent diiron site.