Terminal Hydride Complex of High-Spin Mn.

The iron-molybdenum cofactor of nitrogenase (FeMoco) catalyzes fixation of N 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 Fe configurations, synthetically accessible hydride complexes featuring d or d electron counts are almost exclusively low-spin.

Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC.

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.

C Electron Nuclear Double Resonance Spectroscopy-Guided Molecular Dynamics Computations Reveal the Structure of the Enzyme-Substrate Complex of an Active, -Linked Glycosylated Lipoxygenase.

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 C/H 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 Mn ion as a spin probe.

A New Reaction for Improved Calibration of EPR Rapid-Freeze Quench Times: Kinetics of Ethylene Diamine Tetraacetate (EDTA) Transfer from Calcium(II) to Copper(II).

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)Im]→ [Cu(II)EDTA], are characterized with stopped-flow absorption spectroscopy and implemented as a tool for calibrating the interval between mixing and freezing, the freeze-quench time ( ), 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 Cu ions upon EDTA binding. Stopped-flow measurements show that the rates of conversion of the Cu ions exhibit exponential kinetics on millisecond time scales at pH values less than 6.

Coordination of the Copper Centers in Particulate Methane Monooxygenase: Comparison between Methanotrophs and Characterization of the Cu Site by EPR and ENDOR Spectroscopies.

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 Cu.

Temporal and anatomic relationship between superficial and deep vein thromboses in hospitalized children.

Background: Recent publications have increasingly demonstrated a link between superficial-vein thrombosis (SVT) and deep-vein thrombosis (DVT) in the adult population and have led to changes in SVT treatment considerations. A similar relationship between SVT and DVT in pediatric populations, however, is not currently well established.

Objectives: We sought to evaluate the temporal and anatomic relationship between SVT and DVT among pediatric inpatients in order to determine to what degree SVT is associated with DVT.

The Soybean Lipoxygenase-Substrate Complex: Correlation between the Properties of Tunneling-Ready States and ENDOR-Detected Structures of Ground States.

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.

Composition and Structure of the Inorganic Core of Relaxed Intermediate X(Y122F) of Escherichia coli Ribonucleotide Reductase.

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.

Internal dynamics of a supramolecular nanofibre.

A large variety of functional self-assembled supramolecular nanostructures have been reported over recent decades. The experimental approach to these systems initially focused on the design of molecules with specific interactions that lead to discrete geometric structures, and more recently on the kinetics and mechanistic pathways of self-assembly. 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.

EPR and (57)Fe 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-2S] 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-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.

57Fe ENDOR spectroscopy and 'electron inventory' analysis of the nitrogenase E4 intermediate suggest the metal-ion core of FeMo-cofactor cycles through only one redox couple.

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.

Combining steady-state and dynamic methods for determining absolute signs of hyperfine interactions: pulsed ENDOR Saturation and Recovery (PESTRE).

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.

Simulating suppression effects in Pulsed ENDOR, and the 'hole in the middle' of Mims and Davies ENDOR Spectra.

All pulsed 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 has 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 (Section I).

Identification of protonated oxygenic ligands of ribonucleotide reductase intermediate X.

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.

An organometallic intermediate during alkyne reduction by nitrogenase.

Nitrogenase is the metalloenzyme that catalyzes the nucleotide-dependent reduction of N(2), as well as reduction of a variety of other triply bonded substrates, including the alkyne, acetylene. Substitution of the alpha-70(Val) residue in the nitrogenase MoFe protein by alanine expands the range of substrates to include short-chain alkynes not reduced by the unaltered protein. Rapid freezing of the alpha-70(Ala) nitrogenase MoFe protein during reduction of the alkyne propargyl alcohol (HC triple bond CH(2)OH; PA) traps an S = (1)/(2) intermediate state of the active-site metal cluster, the FeMo-cofactor.

Oxygen and the copper chaperone CCS regulate posttranslational activation of Cu,Zn superoxide dismutase.

Oxidative stress leads to the up-regulation of many antioxidant enzymes including Cu,Zn superoxide dismutase (SOD1) via transcriptional mechanisms; however, few examples of posttranslational regulation are known. The copper chaperone for SOD1 (CCS) is involved in physiological SOD1 activation, and its primary function is thought to be delivery of copper to the enzyme. Data presented here are consistent with a previously uncharacterized function for CCS in the SOD1 pathway, namely mediating enzyme activation in response to increases in oxygen tension.

EPR and ENDOR evidence for a 1-His, hydroxo-bridged mixed-valent diiron site in Desulfovibrio vulgaris rubrerythrin.

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.

Rh(II) and Rh(I) two-legged piano-stool complexes: structure, reactivity, and electronic properties.

The ligand 1,4-bis[4-(diphenylphosphino)butyl]-2,3,5,6-tetramethylbenzene, 3, was used to synthesize a mononuclear Rh(II) complex [(eta(1):eta(6):eta(1)-1,4-bis[4-(diphenylphosphino)butyl]-2,3,5,6-tetramethylbenzene)Rh][PF(6)](2), 6+, in a two-legged piano-stool geometry. The structural and electronic properties of this novel complex including a single-crystal EPR analysis are reported. The complex can be cleanly interconverted with its Rh(I) form, allowing for a comparison of the structural properties and reactivity of both oxidation states.

Purified particulate methane monooxygenase from Methylococcus capsulatus (Bath) is a dimer with both mononuclear copper and a copper-containing cluster.

Particulate methane monooxygenase (pMMO) is a membrane-bound enzyme that catalyzes the oxidation of methane to methanol in methanotropic bacteria. Understanding how this enzyme hydroxylates methane at ambient temperature and pressure is of fundamental chemical and potential commercial importance. Difficulties in solubilizing and purifying active pMMO have led to conflicting reports regarding its biochemical and biophysical properties, however.

Spectroscopy of Cu(II)-PcoC and the multicopper oxidase function of PcoA, two essential components of Escherichia coli pco copper resistance operon.

The plasmid-encoded pco copper resistance operon in Escherichia coli consists of seven genes that are expressed from two pco promoters in response to elevated copper; however, little is known about how they mediate resistance to excess environmental copper. Two of the genes encode the soluble periplasmic proteins PcoA and PcoC. We show here that inactivation of PcoC, and PcoA to a lesser extent, causes cells to become more sensitive to copper than wild-type nonresistant strains, consistent with a tightly coupled detoxification pathway.

Electro-nuclear double resonance spectroscopic evidence for a hydroxo-bridge nucleophile involved in catalysis by a dinuclear hydrolase.

Despite the current availability of several crystal structures of purple acid phosphatases, to date there is no direct evidence for solvent-derived ligands occupying terminal positions in the active enzyme. This is of central importance, because catalysis has been shown to proceed through the direct attack on a metal-bound phosphate ester by a metal-activated solvent-derived moiety, which has been proposed to be either (i) a hydroxide ligand terminally bound to the ferric center or (ii) a bridging hydroxide. In this work we use (2)H Q-band (35 GHz) pulsed electron-nuclear double resonance (ENDOR) spectroscopy to identify solvent molecules coordinated to the active mixed-valence (Fe(3+)Fe(2+)) form of the dimetal center of uteroferrin (Uf), as well as to its complexes with the anions MoO(4), AsO(4), and PO(4).