ENDOR Signals Research Articles

Müge Kasanmascheff.

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Müge Kasanmascheff

Müge Kasanmascheff

Cross-polarisation ENDOR for spin-1 deuterium nuclei

Efficient transfer of spin polarisation from electron to nuclear spins is emerging as a common target of several advanced spectroscopic experiments, ranging from sensitivity enhancement in nuclear magnetic resonance (NMR) and methods for the detection of single molecules based on optically detected magnetic resonance (ODMR) to hyperfine spectroscopy. Here, we examine the feasibility of electron-nuclear cross-polarisation at a modified Hartmann-Hahn condition (called eNCP) for applications in ENDOR experiments with spin-1 deuterium nuclei, which are important targets in studies of hydrogen bonds in biological systems and materials. We have investigated a two-spin model system of deuterated malonic acid radicals in a single crystal. Energy matching conditions as well as ENDOR signal intensities were determined for a spin Hamiltonian under the effect of microwave and radiofrequency irradiation. The results were compared with numerical simulations and 94-GHz ENDOR experiments. The compelling agreement between theoretical predictions and experimental results demonstrates that spin density operator formalism in conjunction with suitable approximations in regard to spin relaxation provides a satisfactory description of the polarisation transfer effect. The results establish a basis for future numerical optimizations of polarisation transfer experiments using multiple-pulse sequences or shaped pulses and for moving from model systems to real applications in disordered systems.

A magnetic look into the protecting layer of Au25 clusters.

The field of molecular metal clusters protected by organothiolates is experiencing a very rapid growth. So far, however, a clear understanding of the fine interactions between the cluster core and the capping monolayer has remained elusive, despite the importance of the latter in interfacing the former to the surrounding medium. Here, we describe a very sensitive methodology that enables comprehensive assessment of these interactions. Pulse electron nuclear double resonance (ENDOR) was employed to study the interaction of the unpaired electron with the protons of the alkanethiolate ligands in four structurally related paramagnetic Au25(SR)018 clusters (R = ethyl, propyl, butyl, 2-methylpropyl). Whereas some of these structures were known, we present the first structural description of the highly symmetric Au25(SPr)018 cluster. Through knowledge of the structural data, the ENDOR signals could be successfully related to the types of ligand and the distance of the relevant protons from the central gold core. We found that orbital distribution affects atoms that can be as far as 6 Å from the icosahedral core. Simulations of the spectra provided the values of the hyperfine coupling constants. The resulting information was compared with that provided by 1H NMR spectroscopy, and molecular dynamics calculations provided useful hints to understanding differences between the ENDOR and NMR results. It is shown that the unpaired electron can be used as a very precise probe of the main structural features of the interface between the metal core and the capping ligands.

High-frequency pulsed ENDOR spectroscopy of the NV− centre in the commercial HPHT diamond

This work reports direct 94GHz ENDOR spectroscopy of the 14N nuclei in the NV− centre in single-crystal diamond. Roadmaps of ENDOR frequencies were measured and hyperfine/quadrupole interaction parameters were obtained, with AX,Y=−2.7MHz, AZ=−2.2MHz and P=−4.8MHz. The sign and value of each parameter was calculated using spin Hamiltonian matrix diagonalization, first and second order perturbation theory and confirmed experimentally. Magnetic field magnitude was measured by 13C ENDOR signal with 0.02% precision or 0.5mT. The orientation of quadrupole, hyperfine and fine structure tensors are the same within error of experiment, g-factor is isotropic.

Proton Matrix ENDOR Studies on Ca2+-depleted and Sr2+-substituted Manganese Cluster in Photosystem II

Proton matrix ENDOR spectra were measured for Ca(2+)-depleted and Sr(2+)-substituted photosystem II (PSII) membrane samples from spinach and core complexes from Thermosynechococcus vulcanus in the S2 state. The ENDOR spectra obtained were similar for untreated PSII from T. vulcanus and spinach, as well as for Ca(2+)-containing and Sr(2+)-substituted PSII, indicating that the proton arrangements around the manganese cluster in cyanobacterial and higher plant PSII and Ca(2+)-containing and Sr(2+)-substituted PSII are similar in the S2 state, in agreement with the similarity of the crystal structure of both Ca(2+)-containing and Sr(2+)-substituted PSII in the S1 state. Nevertheless, slightly different hyperfine separations were found between Ca(2+)-containing and Sr(2+)-substituted PSII because of modifications of the water protons ligating to the Sr(2+) ion. Importantly, Ca(2+) depletion caused the loss of ENDOR signals with a 1.36-MHz separation because of the loss of the water proton W4 connecting Ca(2+) and YZ directly. With respect to the crystal structure and the functions of Ca(2+) in oxygen evolution, it was concluded that the roles of Ca(2+) and Sr(2+) involve the maintenance of the hydrogen bond network near the Ca(2+) site and electron transfer pathway to the manganese cluster.

Pulsed EPR and ENDOR Study of SiC Nanopowders

The great potential of the micro(mp) nano-particle (np) silicon carbide (SiC) for future applications in spintronics stimulates detailed studies of the effects of impurities and defects in their electronic properties. In particular, mpand np-SiC doped with nitrogen (N) atoms is an important issue to be investigated, since N is a common contaminant in SiC bulk, acting as n-type dopant. Although N has been found to be present in the nanomaterials, there is no experimental evidence indicating for the formation of the N shallow donor state in np-SiC. The np-SiC samples with R 50 nm which was identified by pulsed ENDOR as the N in quasi-cubic (Nk2) site. As the size of the np-SiC decrease, the signal from VC in 6H SiC phase appeared in the FS ESE spectrum, which gives rise to the compensation of the N donors and as a result the FS ESE spectrum intensity of N decrease significantly. Only two signals due to the VC located in 3C and 6H SiC phase were observed in the FS ESE spectrum of the np-SiC samples with the R < 50 nm. The fact that the ENDOR signal centered at the hydrogen (H) nuclear Larmor frequency (n) was observed in the np-SiC with R < 100 nm indicates that H atoms are localized in the vicinity of the VC and can form the H-related defect like (VC+H). As a conclusion, two phases (and -SiC crystalline phases) are presented in np-SiC. The work was supported by GA CR 13-06697P and SAFMAT project CZ.2.16/3.1.00/22132.

Free H₂ rotation vs Jahn-Teller constraints in the nonclassical trigonal (TPB)Co-H₂ complex.

Proton exchange within the M-H2 moiety of (TPB)Co(H2) (Co-H2; TPB = B(o-C6H4P(i)Pr2)3) by 2-fold rotation about the M-H2 axis is probed through EPR/ENDOR studies and a neutron diffraction crystal structure. This complex is compared with previously studied (SiP(iPr)3)Fe(H2) (Fe-H2) (SiP(iPr)3 = [Si(o-C6H4P(i)Pr2)3]). The g-values for Co-H2 and Fe-H2 show that both have the Jahn-Teller (JT)-active (2)E ground state (idealized C3 symmetry) with doubly degenerate frontier orbitals, (e)(3) = [|mL ± 2>](3) = [x(2) - y(2), xy](3), but with stronger linear vibronic coupling for Co-H2. The observation of (1)H ENDOR signals from the Co-HD complex, (2)H signals from the Co-D2/HD complexes, but no (1)H signals from the Co-H2 complex establishes that H2 undergoes proton exchange at 2 K through rotation around the Co-H2 axis, which introduces a quantum-statistical (Pauli-principle) requirement that the overall nuclear wave function be antisymmetric to exchange of identical protons (I = 1/2; Fermions), symmetric for identical deuterons (I = 1; Bosons). Analysis of the 1-D rotor problem indicates that Co-H2 exhibits rotor-like behavior in solution because the underlying C3 molecular symmetry combined with H2 exchange creates a dominant 6-fold barrier to H2 rotation. Fe-H2 instead shows H2 localization at 2 K because a dominant 2-fold barrier is introduced by strong Fe(3d)→ H2(σ*) π-backbonding that becomes dependent on the H2 orientation through quadratic JT distortion. ENDOR sensitively probes bonding along the L2-M-E axis (E = Si for Fe-H2; E = B for Co-H2). Notably, the isotropic (1)H/(2)H hyperfine coupling to the diatomic of Co-H2 is nearly 4-fold smaller than for Fe-H2.

Highly resolved proton matrix ENDOR of oriented photosystem II membranes in the S2 state

Proton matrix ENDOR was performed to investigate the protons close to the manganese cluster in oriented samples of photosystem II (PS II). Eight pairs of ENDOR signals were detected in oriented PS II membranes. At an angle of θ=0° between the membrane normal vector n and the external field H0, five pairs of ENDOR signals were exchangeable in D2O medium and three pairs were not exchangeable in D2O medium. The hyperfine splitting of 3.60MHz at θ=0° increased to 3.80MHz at θ=90°. The non-exchangeable signals with 1.73MHz hyperfine splitting at θ=0°, which were assigned to a proton in an amino acid residue, were not detected at θ=90° in oriented PS II or in non-oriented PS II. Highly resolved spectra show that only limited numbers of protons were detected by CW-ENDOR spectra, although many protons were located near the CaMn4O5 cluster. The detected exchangeable protons were proposed to arise from the protons belonging to the water molecules, labeled W1-W4 in the 1.9Å crystal structure, directly ligated to the CaMn4O5 cluster, and nearby amino-acid residue.

ENDOR Evidence of Electron–H2Interaction in a Fulleride Embedding H2

An endofulleropyrrolidine, with H2 as a guest, has been reduced to a paramagnetic endofulleride radical anion. The magnetic interaction between the electron delocalized on the fullerene cage and the guest H2 has been probed by pulsed ENDOR. The experimental hyperfine couplings between the electron and the H2 guest were measured, and their values agree very well with DFT calculations. This agreement provides clear evidence of magnetic communication between the electron density of the fullerene host cage and H2 guest. The ortho-H2/para-H2 interconversion is revealed by temperature-dependent ENDOR measurements at low temperature. The conversion of the paramagnetic ortho-H2 to the diamagnetic para-H2 causes the ENDOR signal to decrease as the temperature is lowered due to the spin catalysis by the paramagnetic fullerene cage of the radical anion fulleride.

ChemInform Abstract: Radical Anions of Cyclic Azoalkanes: An ESR, ENDOR, and TRIPLE- Resonance Study

Radical anions often monocyclic and bicyclic azoalkanes containing the azo group in (Z)-conformation, have been fully characterized by their hyperfine data with the use of ESR, ENDOR, and general-TRIPLE-resonance spectroscopy. These azoalkanes are represented by 3,3,5,5-tetramethyl-1-pyrazoline (1), 2,3-diaza-bicyclo[2.2.1]hept-2-ene (4), and 2,3-diazabicyclo[2.2.2]oct-2-ene (9), as well as by their derivatives 2,3, 5–8, and 10. For all radical anions 1″–10″, the 14N-coupling constant, aN, is in the range of +0. 83 to +0. 97 mT; this finding indicates that the spin population is essentially restricted to the π system of the azo group. The 14N-hyperfine anisotropy largely affects the width of ESR lines, particularly at low temperatures. Substantial coupling constants of 7Li-, 23Na-, 39K-, and 133Cs-nuclei point to a close association of the radical anions with their alkali-metal counterions. With the exception of 39K, these nuclei give rise to readily observable ENDOR signals which appear along with those stemming from protons. The prominent hyperfine features of 1″–10″ are discussed.

ENDOR study of CO2− radicals in hydroxyapatite of γ-irradiated bone

ENDOR spectra and ENDOR-induced EPR spectra (dependences of ENDOR signals vs. the value of magnetic field B ) of γ-irradiated bone plates and powders are studied. Typical ENDOR spectrum consisted of two signals centred at the Larmor frequencies of 1 H and 31 P nuclei, respectively. The ENDOR signals were found to be the superposition of the signals of axial (oriented) and orthorhombic (disordered) CO 2 − radicals. Changes of the ratio between the ENDOR signal intensities for g ‖ and g ⊥ at the rotation of the plate in the magnetic field is explained by the orientation-dependent redistribution of the EPR spectral density. EI-EPR spectra satisfactorily coincide with the spectra of conventional EPR absorption. Changes of ENDOR spectrum at the annealing of a sample before γ-irradiation ( T ann = 400 °C) are explained by Larmor ENDOR mechanism. Larmor ENDOR mechanism is associated with diffusive polarization of distant (far from a paramagnetic center) “free” nuclei under the conditions of EPR signal saturation and their depolarization by radio-frequency field in ENDOR.

Trapping an Intermediate of Dinitrogen (N2) Reduction on Nitrogenase

Nitrogenase reduces dinitrogen (N2) by six electrons and six protons at an active-site metallocluster called FeMo cofactor, to yield two ammonia molecules. Insights into the mechanism of substrate reduction by nitrogenase have come from recent successes in trapping and characterizing intermediates generated during the reduction of protons as well as nitrogenous and alkyne substrates by MoFe proteins with amino acid substitutions. Here, we describe an intermediate generated at a high concentration during reduction of the natural nitrogenase substrate, N2, by wild-type MoFe protein, providing evidence that it contains N2 bound to the active-site FeMo cofactor. When MoFe protein was frozen at 77 K during steady-state turnover with N2, the S = 3/2 EPR signal (g = [4.3, 3.64, 2.00]) arising from the resting state of FeMo cofactor was observed to convert to a rhombic, S = 1/2, signal (g = [2.08, 1.99, 1.97]). The intensity of the N2-dependent EPR signal increased with increasing N2 partial pressure, reaching a maximum intensity of approximately 20% of that of the original FeMo cofactor signal at > or = 0.2 atm N2. An almost complete loss of resting FeMo cofactor signal in this sample implies that the remainder of the enzyme has been reduced to an EPR-silent intermediate state. The N2-dependent EPR signal intensity also varied with the ratio of Fe protein to MoFe protein (electron flux through nitrogenase), with the maximum signal intensity observed with a ratio of 2:1 (1:1 Fe protein:FeMo cofactor) or higher. The pH optimum for the signal was 7.1. The N2-dependent EPR signal intensity exhibited a linear dependence on the square root of the EPR microwave power in contrast to the nonlinear response of signal intensity observed for hydrazine-, diazene-, and methyldiazene-trapped states. 15N ENDOR spectroscopic analysis of MoFe protein captured during turnover with 15N2 revealed a 15N nuclear spin coupled to the FeMo cofactor with a hyperfine tensor A = [0.9, 1.4, 0.45] MHz establishing that an N2-derived species was trapped on the FeMo cofactor. The observation of a single type of 15N-coupled nucleus from the field dependence, along with the absence of an associated exchangeable 1H ENDOR signal, is consistent with an N2 molecule bound end-on to the FeMo cofactor.

EPR, ESE and pulsed ENDOR study of the nitrogen donor pairs on quasi-cubic lattice sites in 6H SiC

Abstract D-band EPR, X-band FS ESE and pulsed ENDOR studies of the additional nitrogen (N) related centers observed in highly compensated n-type 6H-SiC wafers are presented. The D-band EPR and X-band FS ESE spectrum consists of the 14N hyperfine (hf) triplet lines of the N donors incorporated at the two quasi-cubic (Nc1, Nc2) sites with the intensity ratio of INc1/INc2=0.7 and three additional triplet lines. X-band pulsed ENDOR spectra have shown intense 14N ENDOR signals of Nc1, Nc2 centers and new 14N lines due to the N related centers N1, N2, N3 with the isotropic hf splitting: 21.04, 26.43, 29.77 MHz, respectively. It was found that the g-tensors of the N2 and N3 triplet lines coincide with those of N on quasi-cubic Nc2 site. The g-tensor of the third N1 triplet lines corresponds to the average value of the Nc1 and Nc2 spectrum: g ( N 1 ) ≈ 1 2 [ g ( N c 1 ) + g ( N c 2 ) ] . It was suggested that N1 center with Aiso=21.04 MHz is due to the spin coupling between N on two quasi-cubic Nc1 and Nc2 sites while two others are tentatively attributed to the N pairs formed between N atoms on one quasi-cubic Nc2 site.

EPR and ENDOR Studies of Cryoreduced Compounds II of Peroxidases and Myoglobin. Proton-Coupled Electron Transfer and Protonation Status of Ferryl Hemes

The nature of the [Fe(IV)-O] center in hemoprotein Compounds II has recently received considerable attention, as several experimental and theoretical investigations have suggested that this group is not necessarily the traditionally assumed ferryl ion, [Fe(IV)=O]2+, but can be the protonated ferryl, [Fe(IV)-OH]3+. We show here that cryoreduction of the EPR-silent Compound II by gamma-irradiation at 77 K produces Fe(III) species retaining the structure of the precursor [Fe(IV)=O]2+ or [Fe(IV)-OH]3+, and that the properties of the cryogenerated species provide a report on structural features and the protonation state of the parent Compound II when studied by EPR and 1H and 14N ENDOR spectroscopies. To give the broadest view of the properties of Compounds II we have carried out such measurements on cryoreduced Compounds II of HRP, Mb, DHP and CPO and on CCP Compound ES. EPR and ENDOR spectra of cryoreduced HRP II, CPO II and CCP ES are characteristic of low-spin hydroxy-Fe(III) heme species. In contrast, cryoreduced "globins", Mb II, Hb II, and DHP II, show EPR spectra having lower rhombicity. In addition the cryogenerated ferric "globin" species display strongly coupled exchangeable (1)H ENDOR signals, with A max approximately 20 MHz and a iso approximately 14 MHz, both substantially greater than for hydroxide/water ligand protons. Upon annealing at T > 180 K the cryoreduced globin compounds II relax to the low-spin hydroxy-ferric form with a solvent kinetic isotope effect, KIE > 6. The results presented here together with published resonance Raman and Mossbauer data suggest that the high-valent iron center of globin and HRP compounds II, as well as of CCP ES, is [Fe(IV)=O]2+, and that its cryoreduction produces [Fe(III)-O]+. Instead, as proposed by Green and co-workers, CPO II contains [Fe(IV)-OH]3+ which forms [Fe(III)-OH]2+ upon radiolysis. The [Fe(III)-O]+ generated by cryoreduction of HRP II and CCP ES protonate at 77 K, presumably because the heme is linked to a distal-pocket hydrogen bonding/proton-delivery network through an H-bond to the "oxide" ligand. The data also indicate that Mb and HRP compounds II exist as two major conformational substates.

Electronic structure of [Ga 2(tren) 2(CA sq,cat)](BPh 4) 2(BF 4): An EPR, ENDOR, and density functional study

The bimetallic [M 1M 2(tren) 2(CA n− )] m+ series, where M = Ga III or Cr III and CA is the chloranilate ligand which can take on diamagnetic (CA cat,cat) 4− or paramagnetic (CA sq,cat) 3− forms, comprises an electronically diverse series of compounds ranging from the closed-shell [Ga 2(tren) 2(CA cat,cat)] 2+ to the S = 5/2 ground state of [Cr 2(tren) 2(CA sq,cat)] 3+. This report deals with the interpretation of the EPR and ENDOR spectra of [Ga 2(tren) 2(CA sq,cat)](BPh 4) 2(BF 4) ( 2) and the related derivative [Ga 2(tren) 2(DHBQ)](BPh 4) 2(BF 4) ( 2a) (where DHBQ is the fully deprotonated trianionic form of 2,5-dihydroxy-1,4-benzoquinone) in an effort to further characterize the electronic structure of this radical species. The X-band (∼9.5 GHz) EPR spectrum of complex 2 acquired in a butyronitrile/propionitrile glass at 4 K reveals a rhombic g-tensor with g xx = 2.0100, g yy = 2.0097, and g zz = 2.0060 with hyperfine interactions due to spin delocalization onto the two Ga nuclei ( a xx = 4.902 G, a yy = 4.124 G, a zz = 3.167 G); the origin of the hyperfine coupling was confirmed by analysis of the room temperature spectra of complexes 2 and 2a. The low-temperature spectrum of complex 2 also indicates the presence of a triplet electronic state characterized by a g-value of 2.009 and axial zero-field splitting of D = 150 G (0.012 cm −1) as determined from measurements carried out at both X- and W-band (∼95 GHz) frequencies. This triplet state is believed to arise due to a weak intermolecular Heisenberg exchange interaction between two aggregating complexes. ENDOR measurements on complex 2a at 20 K allowed for a determination of the magnitude of hyperfine coupling to the protons associated with the radical bridge as well as providing a rare example of an ENDOR signal arising from coupling to a gallium nucleus. Finally, these results were combined with literature data on the free semiquinone form of the bridging ligand in order to assess the extent to which density functional theory can predict unpaired spin density distribution in a complex molecule of this type. Although differences between theory and experiment were noted, DFT was able to provide a reasonably accurate picture of the electronic structure of this system as well as provide insight into the spin polarization mechanism(s) responsible for the observed hyperfine interactions.

Protons bound to the Mn cluster in photosystem II oxygen evolving complex detected by proton matrix ENDOR

Protons in the vicinity of the oxygen-evolving manganese cluster in photosystem II were studied by proton matrix ENDOR. Six pairs of proton ENDOR signals were detected in both the S 0 and S 2 states of the Mn-cluster. Two pairs of signals that show hyperfine constants of 2.3/2.2 and 4.0 MHz, respectively, disappeared after D 2O incubation in both states. The signals with 2.3/2.2 MHz hyperfine constants in S 0 and S 2 state multiline disappeared after 3 h of D 2O incubation in the S 0 and S 1 states, respectively. The signal with 4.0 MHz hyperfine constants in S 0 state multiline disappeared after 3 h of D 2O incubation in the S 0 state, while the similar signal in S 2 state multiline disappeared only after 24 h of D 2O incubation in the S 1 state. The different proton exchange rates seem to be ascribable to the change in affinities of water molecules to the variation in oxidation state of the Mn cluster during the water oxidation cycle. Based on the point dipole approximation, the distances between the center of electronic spin of the Mn cluster and the exchangeable protons were estimated to be 3.3/3.2 and 2.7 Å, respectively. These short distances suggest the protons belong to the water molecules ligated to the manganese cluster. We propose a model for the binding of water to the manganese cluster based on these results.

A Davies/Hahn multi-sequence for studies of spin relaxation in pulsed ENDOR

We extend earlier studies of the effects of relaxation on the intensities of pulsed ENDOR signals by introducing a Davies/Hahn (D/H) pulsed ENDOR multi-sequence that corresponds to a series of Davies sequences with the preparation pulse ‘turned off’. In this pulse train, the Hahn [π/2, π] detection pulse pair of sequence n−1 both generates the echo detected for that sequence and acts as the preparation portion of sequence n, in effect replacing the π preparation pulse of the Davies sequence. We show both theoretically, through a master-equation approach, and with both 1H ( I = 1/2) and 14N ( I = 1) ENDOR experiments on the non-heme Fe enzymes, superoxide reductase (SOR) ( S = 1/2) and AntDO ( S = 3/2), that under conditions of high electron–spin polarization (high microwave frequency/low temperature) the D/H multi-sequence allows simplification of ENDOR spectra by suppression of nuclear transitions associated with the m S = +1/2 ( α) manifold. As such suppression depends on the sign of A, it allows determination of this sign. The suppression as a function of the time between individual sequences is found to exhibit behaviors that can be classified into three regimes of the ratio of cross-relaxation to spin–lattice relaxation rates: strong cross-relaxation (X-case); comparable rates (XL); negligible cross relaxation (L). Interestingly, the ENDOR behavior of the S = 1/2 SOR center indicates it is an L case, while the S = 3/2 AntDO is an L case. Overall, the D/H protocol appears to be a robust and general tool for using relaxation effects to manipulate ENDOR spectra.

The Interstitial Atom of the Nitrogenase FeMo-Cofactor: ENDOR and ESEEM Evidence That it is Not a Nitrogen

X-ray crystallographic study of the nitrogenase MoFe protein revealed electron density from an atom (denoted X) inside the active-site metal cluster, the [MoFe7S9:homocitrate] FeMo-cofactor. The electron density associated with X is consistent with a single N, O, or C atom. We now have tested whether X is an N or not by comparing the Q-band ENDOR and ESEEM signals from resting-state (S = 3/2) MoFe protein and NMF-extracted FeMo-co from bacteria grown with either 14N or 15N as the exclusive N source. All of the 14N or 15N signals associated with the protein are lost upon extraction of the FeMo-co. We interpret this as strong evidence that X is not an N.

A superoxo-ferrous state in a reduced oxy-ferrous hemoprotein and model compounds.

Cryoreduction of the [FeO2]6 (n = 6 is the number of electrons in 3d orbitals on Fe and pi* orbitals on O2) dioxygen-bound ferroheme through irradiation at 77 K generates an [FeO2]7 reduced oxy-heme. Numerous investigations have examined [FeO2]7 centers that have been characterized as peroxo-ferric centers, denoted [FeO2]per7, in which a ferriheme binds a dianionic peroxo-ligand. The generation of such an intermediate can be understood heuristically if the [FeO2]6 parent is viewed as a superoxo-ferric center and the injected electron localizes on the O-O moiety. We here report EPR/ENDOR experiments which show quite different properties for the [FeO2]7 centers produced by cryoreduction of monomeric oxy-hemoglobin (oxy-GMH3) from Glycera dibranchiata, which is unlike mammalian "globins" in having a leucine in place of the distal histidine; of frozen aprotic solutions of oxy-ferrous octaethyl porphyrin; and of the oxy-ferrous complex of the heme model, cyclidene. These [FeO2]7 centers are characterized as "superoxo-ferrous" centers ([FeO2]sup7), with nearly unit spin density localized on a superoxo moiety which is end-on coordinated to a low-spin ferrous ion. This assignment is based on their g tensors and 17O hyperfine couplings, which are characteristic of the superoxide ion coordinated to a diamagnetic metal ion, and on the absence of detectable ENDOR signals either from the in-plane 14N ligands or from an exchangeable H-bond proton. Such a center would arise if the electron that adds to the [FeO2]6 superoxo-ferric parent localizes on the Fe ion, to make a superoxo-ferrous moiety. Upon annealing to T > 150 K, the [FeO2]sup7 species converts to peroxo/hydroperoxo-ferric ([FeO2H]7) intermediates. These experiments suggest that the primary reduction product is [FeO2]sup7 and that the internal redox transition to [FeO2]per7/[FeO2H]7 states is driven at least in part by H-bonding/proton donation by the environment.