Proton ENDOR Research Articles

Exchangeable proton ENDOR as a probe of the redox-active iron center in activated bleomycin and ferric bleomycin.

Activated bleomycin (ABLM) is a drug-Fe(iii)-hydroperoxide complex kinetically competent in DNA attack (via H4' abstraction). This intermediate is relatively stable, but its spontaneous conversion to ferric bleomycin (Fe(iii)·BLM) is poorly characterized because no observable intermediate product accumulates. The Fe(iii)·BLM formed cryophotolytically from ABLM and kept at 77 K was remarkably similar by EPR and ENDOR criteria to Fe(iii)·BLM formed from Fe(iii) + BLM solution. The notable ENDOR criteria were the ENDOR frequencies and features of orientation-selected, strongly hyperfine-coupled, exchangeable protons associated with the environs of the iron within <3.5 Å of paramagnetic Fe(iii) in Fe(iii)·BLM and ABLM. Cryophotolytic conversion of activated bleomycin to its ferric bleomycin product in the frozen solid is a sign that the reaction requires only constrained local proton rearrangements. We have characterized the metal-proton distances and orientations of the protons in that rearrangement, especially noting that these protons are of mechanistic importance in the ambient temperature conversion of ABLM to Fe(iii)·BLM in concert with a directed radical-forming attack on DNA.

ENDOR Spectroscopy Reveals Light Induced Movement of the H-Bond from Ser-L223 upon Forming the Semiquinone (QB-•) in Reaction Centers fromRhodobacter sphaeroides

Proton ENDOR spectroscopy was used to monitor local conformational changes in bacterial reaction centers (RC) associated with the electron-transfer reaction DQB --> D+*QB-* using mutant RCs capable of photoreducing QB at cryogenic temperatures. The charge separated state D+*QB-* was studied in mutant RCs formed by either (i) illuminating at low temperature (77 K) a sample frozen in the dark (ground state protein conformation) or (ii) illuminating at room temperature prior to and during freezing (charge separated state protein conformation). The charge recombination rates from the two states differed greatly (>10(6) fold) as shown previously, indicating a structural change (Paddock et al. (2006) Biochemistry 45, 14032-14042). ENDOR spectra of QB-* from both samples (35 GHz, 77 K) showed several H-bond hyperfine couplings that were similar to those for QB-* in native RCs indicating that in all RCs, QB-* was located at the proximal position near the metal site. In contrast, one set of hyperfine couplings were not observed in the dark frozen samples but were observed only in samples frozen under illumination in which the protein can relax prior to freezing. This flexible H-bond was assigned to an interaction between the Ser-L223 hydroxyl and QB-* on the basis of its absence in Ser L223 --> Ala mutant RCs. Thus, part of the protein relaxation, in response to light induced charge separation, involves the formation of an H-bond between the OH group of Ser-L223 and the anionic semiquinone QB-*. These results show the flexibility of the Ser-L223 H-bond, which is essential for its function in proton transfer to reduced QB.

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.

EPR−ENDOR of the Cu(I)NO Complex of Nitrite Reductase

With limited reductant and nitrite under anaerobic conditions, copper-containing nitrite reductase (NiR) of Rhodobacter sphaeroides yielded endogenous NO and the Cu(I)NO derivative of NiR. (14)N- and (15)N-nitrite substrates gave rise to characteristic (14)NO and (15)NO EPR hyperfine features indicating NO involvement, and enrichment of NiR with (63)Cu isotope caused an EPR line shape change showing copper involvement. A markedly similar Cu(I)NONiR complex was made by anaerobically adding a little endogenous NO gas to reduced protein and immediately freezing. The Cu(I)NONiR signal accounted for 60-90% of the integrated EPR intensity formerly associated with the Type 2 catalytic copper. Analysis of NO and Cu hyperfine couplings and comparison to couplings of inorganic Cu(I)NO model systems indicated approximately 50% spin on the N of NO and approximately 17% spin on Cu. ENDOR revealed weak nitrogen hyperfine coupling to one or more likely histidine ligands of copper. Although previous crystallography of the conservative I289V mutant had shown no structural change beyond the 289 position, this mutation, which eliminates the Cdelta1 methyl of I289, caused the Cu(I)NONiR EPR spectrum to change and proton ENDOR features to be significantly altered. The proton hyperfine coupling that was significantly altered was consistent with a dipolar interaction between the Cdelta1 protons of I289 and electron spin on the NO, where the NO would be located 3.0-3.7 A from these protons. Such a distance positions the NO of Cu(I)NO as an axial ligand to Type 2 Cu(I).

When are Two Waters Worse Than One? Doubling the Hydration Number of a Gd–DTPA Derivative Decreases Relaxivity

The synthesis of a novel ligand, based on N-methyl-diethylenetriaminetetraacetate and containing a diphenylcyclohexyl serum albumin binding group (L1) is described and the coordination chemistry and biophysical properties of its Gd(III) complex Gd-L1 are reported. The Gd(III) complex of the diethylenetriaminepentaacetate analogue of the ligand described here (L2) is the MRI contrast agent MS-325. The effect of converting an acetate to a methyl group on metal-ligand stability, hydration number, water-exchange rate, relaxivity, and binding to the protein human serum albumin (HSA) is explored. The complex Gd-L1 has two coordinated water molecules in solution, that is, [Gd(L1)(H2O)2]2- as shown by D-band proton ENDOR spectroscopy and implied by 1H and 17O NMR relaxation rate measurements. The Gd-H(water) distance of the coordinated waters was found to be identical to that found for Gd-L2, 3.08 A. Loss of the acetate group destabilizes the Gd(III) complex by 1.7 log units (log K(ML) = 20.34) relative to the complex with L2. The affinity of Gd-L1 for HSA is essentially the same as that of Gd-L2. The water-exchange rate of the two coordinated waters on Gd-L1 (k(ex) = 4.4x10(5) s(-1)) is slowed by an order of magnitude relative to Gd-L2. As a result of this slow water-exchange rate, the observed proton relaxivity of Gd-L1 is much lower in a solution of HSA under physiological conditions (r1(obs) = 22.0 mM(-1) s(-1) for 0.1 mM Gd-L1 in 0.67 mM HSA, HEPES buffer, pH 7.4, 35 degrees C at 20 MHz) than that of Gd-L2 (r1(obs) = 41.5 mM(-1) s(-1)) measured under the same conditions. Despite having two exchangeable water molecules, slow water exchange limits the potential efficacy of Gd-L1 as an MRI contrast agent.

Proton ENDOR study of the primary donor P740 +, a special pair of chlorophyll d in photosystem I reaction center of Acaryochloris marina

Oxidized primary electron donor P740, a special pair of chlorophyll (Chl) d in photosystem (PS) I reaction center of a newly identified cyanobacterium Acaryochloris marina was studied by EPR and proton ENDOR spectroscopy. EPR and ENDOR spectra of P740 + were compared with those of P700 + in PS I reaction center of spinach. The g-factors of the purified Chl a + and Chl d + in CH 2Cl 2/THF, P700 + and P740 + in PS I reaction centers were determined to be 2.0025, 2.0032, 2.0027 and 2.0028, respectively. Hyperfine coupling constants of 1.9, 2.8 and 3.8 MHz that were detected in the ENDOR spectrum of P700 + were absent in the ENDOR spectrum of P740 +. These features of P740 + were mainly ascribed to the difference between the chemical structures of Chl a and Chl d.

EPR and ENDOR characterization of intermediates in the cryoreduced oxy-nitric oxide synthase heme domain with bound L-arginine or N(G)-hydroxyarginine.

Reconstitution of the endothelial nitric oxide synthase heme domain (NOS) with the catalytically noncompetent 4-aminotetrahydrobiopterin has allowed us to prepare at -40 degrees C the oxyferrous-NOS-substrate complexes of both L-arginine (Arg) and N(G)-hydroxyarginine (NOHA). We have radiolytically cryoreduced these complexes at 77 K and used EPR and ENDOR spectroscopies to characterize the initial products of reduction, as well as intermediates that arise during stepwise annealing to higher temperatures. Peroxo-ferri-NOS is the primary product of 77 K cryoreduction when either Arg or NOHA is the substrate. Proton ENDOR spectra of this state suggest that the peroxo group is H-bonded to a [guanidinium-water] network that forms because the binding of O2 to the ferroheme of NOS recruits H2O. At no stage of reaction/annealing does one observe an EPR signal from a hydroperoxo-ferri state with either substrate. Instead, peroxo-ferri-NOS-substrate complexes convert to a product-state intermediate at the extremely low temperature of 165-170 K. EPR and proton ENDOR spectra of the intermediate formed with Arg as substrate support the suggestion that the reaction involves the formation and attack of Compound I. Within the time/temperature resolution of the present experiments, samples with Arg and NOHA as substrate behave the same in the initial steps of cryoreduction/annealing, despite the different acid/base characteristics of the two substrates. This leads us to discuss the possibility that ambient-temperature catalytic conversion of both substrates is initiated by reduction of the oxy-ferroheme to the hydroperoxo-ferriheme through a coupled proton-electron transfer from a heme-pocket reductant, and that Arg may provide the stoichiometrically second proton of catalysis.

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.

EPR and Angle-Selected ENDOR Study of 5f-Ligand Interactions in the [U(η7-C7H7)2]- Anion, an f1 Analogue of Uranocene

A frozen solution of [U(η7-C7H7)2]- is studied by EPR and angle-selected ENDOR spectroscopy. EPR confirms the anticipated f 1 configuration of this compound. Analysis of the g tensor indicates that the ground state molecular orbital is principally made of 5fπ (51%) and 5fσ (39%) uranium orbitals. This composition results from the combined effect of the spin−orbit interaction and the strong fδ-ligand interactions. The small admixture of 5fφ (9%) uranium orbitals could result from a low-symmetry effect imposed by the solid-state packing forces at low temperature. Starting from the ground-state orbital, the f-level ordering consistent with the g tensor is found to be (fπ,fσ) < fφ < fπ < fφ, (fσ,fπ) < fδ (antibonding). Proton ENDOR spectroscopy shows that there is a positive spin density ρπ ≥ 4 × 10-2 in 2pπ carbon orbitals, which manifests the covalent character of the ground-state orbital.

Paramagnetic States of Four Iron−Four Sulfur Clusters. 2. Proton ENDOR Study of a 1+ State in an Asymmetrical Cluster

Proton ENDOR spectroscopy on γ-irradiated single crystals of the synthetic model compound (Et4N)2[Fe4S4(SC6H4-o-OH)4] has been used to study a [4Fe−4S]+ center that has properties similar to those of the reduced states of some particular ferredoxins and of the enzyme aconitase. The interesting point about this model compound is that one iron atom is five-coordinate, with an extra phenolic oxygen attached to one of its thiolate ligand. The complex thus represents an example of an asymmetrical [4Fe−4S] cluster in the solid crystalline state. From the study of the angular dependence of the proton ENDOR lines, it has been possible to derive eight hyperfine tensors of protons occupying different positions in the near vicinity of the iron atoms, the main bearers of the spin population of the paramagnetic anion. Some protons belong to thiolate ligands of the anion whereas others belong to close (Et4N)+ counterions. From the anisotropic parts of the tensorsand by using a multicentric point-dipole modelit has been...

Confirmation of syn—anti isomerism of 2,5-dimethoxydicyanoquinonediimine radical anions in solution

The conformational behaviour of 2,5-dimethoxydicyanoquinonediimine radical anions has been studied in polar solvents with ESR and proton ENDOR spectroscopy. Both syn and anti isomers of the radical anions have been identified. The two conformers differ in molecular symmetry and hyperfine coupling between the unpaired electron with nearby protons. Separate spectra of the conformers have been obtained with ENDOR-induced ESR. Finally, from electronic spin relaxation, the activation energy for the interconversion process between the syn and anti conformers was estimated at 28.5 kJ/mol (6.8 kcal/mol).

Making hyperfine selection in Mims ENDOR independent of deadtime

A form of remote echo-detected ENDOR can be performed by adding an additional π pulse to the detection phase of a Mims three-pulse, stimulated-echo ENDOR experiment. The resulting four-pulse experiment, denoted refocused Mims (ReMims) ENDOR, retains the hyperfine selectivity of the Mims technique, but makes it possible to obtain arbitrarily short preparation intervals, τ 1, independent of the spectrometer deadtime. As a result, without resonator redesign, it is possible to study a wider range of hyperfine values without distortions from “blind-spots”. The hyperfine selectivity of the ReMims ENDOR sequence is demonstrated on the 1H and 14N ENDOR spectra from a single crystal of Cu(glycine) 2. Use of the ReMims protocol to augment the basic Mims and Davies ENDOR sequences is shown in two different types of situation: the sign of a 9 MHz coupled proton is assigned by the use of the implicit-TRIPLE effect in the proton ENDOR spectrum of nitrile hydratase; the true lineshape of the proton ENDOR spectrum of fluorometmyoglobin is determined without distortions by blindspots.

ENDOR and pulsed ESR study of proton glass behavior in the mixed crystal (betaine phosphate)0.15(betaine phosphite)0.85.

The proton ENDOR spectra of ${\mathrm{BP}}_{0.15}$${\mathrm{BPI}}_{0.85}$ taken between 90 and 290 K allow a direct determination of the local polarization distribution W(p) of the protons in the hydrogen bonds linking the phosphate and phosphite groups to chains. The temperature dependence of the Edwards-Anderson glass order parameter can be described by a random-field dominated proton glass behavior in ${\mathrm{BP}}_{0.15}$${\mathrm{BPI}}_{0.85}$ with a glass temperature of ${\mathit{T}}_{\mathit{G}}$=30 K and a random-field induced freezing temperature of ${\mathit{T}}_{\mathit{f}}$=(95\ifmmode\pm\else\textpm\fi{}5) K. The experimental results do not permit one to discriminate between three-dimensional (3D) or quasi-one-dimensional interactions of the hydrogen bonds. The experimentally determined polarization distribution function W(p) can be described to a certain extent within the 3D random-bond random-field model if a long-range order of the protons is introduced by a non-zero mean ${\mathit{J}\mathit{\ifmmode \tilde{}\else \~{}\fi{}}}_{0}$/${\mathit{k}}_{\mathit{B}}$=160 K of the random-bond interaction. Additionally, the local field variance \ensuremath{\Delta}\ifmmode \tilde{}\else \~{}\fi{} has to be taken as a temperature-dependent parameter with a minimal value at the resulting phase transition at 144 K. However, even at lowest temperatures the maxima of the order parameter distribution function W(p) appear at values of the local proton polarization distinctly smaller than one. This is not describable within the above models. We conclude that proton tunneling is of importance in betaine phosphite-phosphate proton glasses. \textcopyright{} 1996 The American Physical Society.

Vinylogous Tetrathiafulvalene (TTF) π-Electron Donors and Derived Radical Cations: ESR Spectroscopic, Magnetic, and X-ray Structural Studies

The properties of new 2,2‘-ethanediylidene(1,3-dithiole) derivatives 5, 6, and 8−11 are reported. Cyclic voltammetric studies establish that they are efficient donor molecules, with the extended conjugation resulting in stabilization of dications, relative to tetrathiafulvalene TTF (1). Radical cations are generated by oxidation of the neutral compounds with trifluoroacetic acid or anhydrous silver perchlorate in dichloromethane, and their ESR and proton ENDOR spectra are reported. The bulk of the spin population resides in the central S2CCCCS2 part of the π-system. The X-ray crystal structure of donor 6 reveals that the 2,2‘-ethanediylidene(1,3-dithiole) framework is planar. Donor 6 forms a crystalline 1:1 charge-transfer complex with TCNQ, the X-ray crystal structure of which shows a mixed stack structure. A solution of this complex in acetonitrile exhibits ESR spectra of both radical ions, 6·+ and TCNQ·−. Static susceptibility data are reported for TCNQ complexes of some of these donors.

Structure of CuB in the Binuclear Heme-Copper Center of the Cytochrome aa3-Type Quinol Oxidase from Bacillus subtilis: An ENDOR and EXAFS Study

We have studied the structure of the CuB site in the binuclear heme-copper center of the fully oxidized form of the quinol-oxidizing cytochrome aa3-600 from Bacillus subtilis by EXAFS and ENDOR spectroscopy. This enzyme is member of the large superfamily of heme-copper respiratory oxidases, which catalyze the reduction of dioxygen to water and link it to translocation of protons across the bacterial or mitochondrial membrane. The EXAFS of the CuB site strongly suggests tetragonal coordination by two or three histidines with one or two O/N donor ligands. There are some indications that a Cl- ion might fractionally occupy substitution-labile sites, although the majority of enzyme molecules did not contain any heavy (second row) scatters, indicative of a Cl- (or S) bridge between the heme iron and CuB [cf. Powers, L., et al. (1994) Biochim. Biophys. Acta 1183, 504-512]. Proton ENDOR spectroscopy of the CuB site in 1H2O and 2H2O media showed evidence of an oxygenous copper ligand with an exchangeable proton. 14N ENDOR revealed three inequivalent nitrogenous ligands with hyperfine coupling constants consistent with histidines. Together, these results strongly suggest that the fully oxidized enzyme has a low-symmetry, tetragonal CuB site with three histidine nitrogens and one oxygen as ligands, the latter with an exchangeable proton(s). The identity and assignment of these ligands are discussed.

Hyperfine couplings of N‐alkoxy‐N‐polynitrophenylaminyl radicals determined by ENDOR and TRIPLE resonance spectroscopy

AbstractLead tetraacetate one‐electron oxidation of nine different N‐alkoxy‐2,6‐dinitroanilines substituted with trifluoromethyl, methyl and nitro groups in position 4 yielded aminyl radicals for which hyperfine couplings were measured by ENDOR and TRIPLE resonance spectroscopy. The optimum temperature range for proton ENDOR and general TRIPLE resonance measurements of aminyl radicals was 210–250 K and for nitrogen ENDOR 260 K in toluene. Further lowering for the temperature rapidly decreased the EPR intensity. The concentration of the sample and the amount of oxidant were optimized for obtaining ENDOR spectra. The relative signs of the hyperfine couplings of nitrogens, fluorines and protons were determined in the basis of general TRIPLE resonance experiments. The oxidation of N‐methoxy‐N‐2,6‐dinitrophenylamine and N‐ethoxy‐N‐2,6‐dinitrophenylamine with lead tetraacetate produced first the aminyl radical of the respective 2,6‐dinitro compound at low temperature (below 260 K) and very soon afterwards the aminyl radical of the respective 2,4,6‐trinitro compound, by a route in which hydrogen has to be removed from the system. Rotational correlation times were estimated for N‐methoxy‐and N‐ethoxy‐N‐2,4,6‐trinitrophenylaminyl radicals. Nitroxyl radicals were detected only under conditions where oxygen was present in the solvent.

Spin dynamics in continuous wave and pulsed ENDOR spectroscopy of coals

ENDOR experiments on coals recorded using continuous wave (CW) and pulsed techniques appear to give qualitatively different spectra. A matrix proton signal dominates the ENDOR spectrum of coals recorded in the CW ENDOR experiment while both a matrix and local proton ENDOR signals with huperfine couplings of up to 20 MHz are observed in spectra recorded using pulsed excitation techniques. Analysis of these spectra lead to different implications for the structure of the molecules that host the unpaired electron. Using a combination of pulsed EPR (Electron Spin Echo, FID detected hole burning) and pulsed Electron Nuclear Multiple Resonance (Sub-level relaxation, hyperfine selective ENDOR, EPR sub-spectra) experiments, we investigate the electron and nuclear spin dynamics in order to reconcile the different signal amplitudes observed in the CW and pulsed ENDOR spectra. In the CW ENDOR experiment, the results of the FID detected hole burning experiments prove that the low ENDOR signal intensity can not be attributed to spectral diffusion mechanisms competing with ENDOR mechanisms. Instead, we find that an unfavorable ratio of the electron and nuclear spin relaxation rates results in small local ENDOR signals. The matrix line dominates the spectrum because of the large number of matrix protons. In the pulsed ENDOR experiment, the hyperfine contrast selectivity mechanism suppresses the intensity of the matrix ENDOR signal and enhances the amplitudes of the local ENDOR signals. In addition, the ENDOR signal is not a function of the ratio of the electron and nuclear relaxation rates.

Proton ENDOR from randomly oriented NO-ligated haemoglobin: approaching the structural basis for the R–T transition

EPR and ENDOR spectra of nitrosyl (NO)-ligated haemoglobin (HbNO) and its isolated α- and β-chains are presented. In comparison with the NO-ligated Fe-tetraphenylporphyrin as a model compound, with imidazole as the sixth ligand, the temperature dependence of the EPR spectra is analysed in terms of two different stereochemistries of the N(imidazole)—Fe—N(NO) bonds, with respect to the porphyrin plane. One represents an axial EPR spectrum with the bond direction coinciding with the intermediate g factor which is parallel to the normal porphyrin plane. The other comprises a rhombic EPR spectrum in which that direction is tilted from the plane normal by 30–40°. These two structures were deduced from analysis of the ENDOR response in the region of weakly coupled protons (10–20 MHz) by theoretical simulations using the low-temperature X-ray structure of met-myoglobin as the backbone. The R–T transition in HbNO was reported to involve α-chain conformational changes only comprising a partial loss of the proximal histidine in the isolated subunits and in the tetrameric HbNO. The strong tilt of the N(imidazole)—Fe—N(NO) in the α-chains with decreasing temperatures is proposed to be the precursor to the loss of the proximal histidine. It involves a sideward movement of the F-helix which is not possible in β-chains owing to a smaller haem crevice.

EPR and ENDOR detection of compound I from Micrococcus lysodeikticus catalase.

We present the first EPR and ENDOR examination of a catalase compound I (Cat I), the one formed by peracetic acid treatment of Micrococcus lysodeikticus catalase. The Cat I rapid-passage EPR signal (g perpendicular eff = 3.32; g parallel eff approximately 2) appears quite different from those reported previously for the compounds I from horseradish peroxidase (HRP I) and chloroperoxidase. Nonetheless, all three signals can be explained by the same model for exchange coupling between an S = 1 oxoferryl [Fe = O]2+ moiety and a porphyrin pi-cation radical (S' = 1/2) (Schulz, C. E., et al. (1979) FEBS Lett. 103, 102-105). The signal for Cat I is unlike those for the two peroxidases in that it reflects a ferromagnetic rather than antiferromagnetic exchange. Preliminary 1H ENDOR spectra for Cat I appear to differ from the proton (1H) ENDOR spectra of HRP I; the latter, along with the 14N ENDOR spectra, indicate that the porphyrin radical in HRP I exhibits a predominantly A2u-like state having large spin densities on porphyrin N and C(beta). The proton ENDOR spectrum of Cat I is insensitive to H/D exchange, which indicates that the [Fe = O]2+ moiety is not protonated. Consideration of the EPR results for a series of compounds I suggests that the sign and magnitude of the exchange parameter (J) is correlated with the nature of the proximal axial ligand.

Proton ENDOR identification of bridging hydroxide ligands in mixed-valent diiron centers of proteins: methane monooxygenase and semimet azidohemerythrin

Proton ENDOR identification of bridging hydroxide ligands in mixed-valent diiron centers of proteins: methane monooxygenase and semimet azidohemerythrin