The electronic structure of the 5-coordinate quantum-mechanically mixed-spin (sextet-quartet) heme center in cytochrome c‘ was investigated by electron nuclear double resonance (ENDOR), a technique not previously applied to this mixed-spin system. Cytochrome c‘ was obtained from overexpressing variants of Rhodobacter sphaeroides 2.4.3. ENDOR for this study was done at the g|| = 2.00 extremum where single-crystal-like, well-resolved spectra prevail. The heme meso protons of cytochrome c‘ showed a contact interaction that implied spin delocalization arising from the heme (dz2) orbital enhanced by iron out-of-planarity. An exchangeable proton ENDOR feature appeared from the proximal His123 Nδ hydrogen. This Nδ hydrogen, which crystallographically has no hydrogen-bonding partner and thus belongs to a neutral imidazole, showed a larger hyperfine coupling than the corresponding hydrogen-bonded Nδ proton from metmyoglobin. The unique residue Phe14 occludes binding of a sixth ligand in cytochrome c‘, and ENDOR from a proton of the functionally important Phe14 ring, 3.3 Å away from the heme iron, was detected. ENDOR of the nitrogen ligand hyperfine structure is a direct probe into the σ-antibonding (dz2) and (dx2-y2) orbitals whose energies alter the relative stability and admixture of sextet and quartet states and whose electronic details were thus elucidated. ENDOR frequencies showed for cytochrome c‘ larger hyperfine couplings to the histidine nitrogen and smaller hyperfine couplings to the heme nitrogens than for high-spin ferric hemes. Both of these findings followed from the mixed-spin ground state, which has less (dx2-y2) character than have fully high-spin ferric heme systems.
JACS, 2005, V.127, No.26, pp.9485-9494

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. Light was shown to trigger ABLM attack on DNA in liquid at −30 °C, so ABLM was irradiated (at its 350 nm ligand-to-metal charge-transfer transition) at 77 K to stabilize possible intermediates. ABLM photolysis (quantum yield, Φ = 0.005) generates two kinds of product: Fe(III)·BLM (with no detectable intermediate) and one or more minor (1−2%) radical O−Fe−BLM byproduct, photostable at 77 K. Adding DNA, even without its target H4‘, increases the quantum yield of ABLM conversion >10-fold while suppressing the observed radical yield. Since cryogenic solid-phase reactions can entail only constrained local rearrangement, the reaction(s) converting ABLM to Fe(III)·BLM must be similarly constrained.
J.Phys.Chem. B, 2006, V.110, No.41, pp. 20702-20709

The five-coordinate NO-bound heme in cytochrome c‘ from an overexpressing variant of denitrifying R. sphaeroides 2.4.3 was investigated by proton, nitrogen, and deuterium Q-band ENDOR (electron nuclear double resonance). ENDOR was a direct probe of the unpaired electron density on the nitrogen of NO and, as measured across the EPR line shape, showed a hyperfine coupling range from 36 to 44 MHz for 14NO and 51 to 63 MHz for 15NO. The smallest NO coupling occurred at an electronic g-tensor axis perpendicular to the FeNO plane, and the largest hyperfine coupling occurred in the FeNO plane where the highest nitrogen valence spin density is located. The isotropic component of the NO hyperfine coupling indicated that the electron spin on the NO is not simply in a π* orbital having only 2p character but is in an orbital having 2s and 2p character in a 1:2 ratio. ENDOR frequencies from heme meso-protons, assigned with reference to porphyrin models, were determined to result from an anisotropic hyperfine tensor. This tensor indicated the orientation of the heme with respect to the FeNO plane and showed that the FeNO plane bisects the heme N−Fe−N 90° angle. ENDOR provided additional structural information through dipolar couplings, as follows: (1) to the nearest proton of the Phe14 ring, 3.1 Å away from the heme iron, where Phe14 is positioned to occlude binding of NO as a 6th (distal) ligand; (2) to exchangeable deuterons assigned to Arg127 which may H-bond with the proximal NO ligand.
JACS, 2006, V.128, No. 15, pp.5021-5032

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. 14N- and 15N-nitrite substrates gave rise to characteristic 14NO and 15NO EPR hyperfine features indicating NO involvement, and enrichment of NiR with 63Cu 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 50% spin on the N of NO and 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 Cδ1 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 Cδ1 protons of I289 and electron spin on the NO, where the NO would be located 3.0−3.7 Å from these protons. Such a distance positions the NO of Cu(I)NO as an axial ligand to Type 2 Cu(I).
JACS, 2006, V.128, No. 40. pp.13102-13111

Di-μ-oxo MnIII−MnIV dimers are models for coupled, mixed-valence manganese in the oxygen-evolving centers of green plants. Using a recently reported method of exchanging water oxygen into the di-μ-oxo cross-bridges , we have incorporated 17O into the μ-oxo cross bridges of the MnIII−MnIV bipyridyl dimer for study of oxygen electron-spin hyperfine couplings by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR). The ENDOR evidence was for a di-μ-oxo 17O hyperfine coupling of 12.8 ± 1.0 MHz. Narrow and highly resolved EPR features from dimers exchanged with H216O (I = 0) became broadened when the dimer was prepared by exchanging the cross bridging oxygens with H217O (I = 5/2). The EPR broadening due to 17O was quantitatively reproduced by a model where the dimer has two equivalent di-μ-oxo cross-bridging 17Ο, and the 17O hyperfine coupling was highly consistent with that determined by ENDOR. This work explicitly points out evidence for covalent spin transfer to the cross-bridging di-μ-oxo oxygens which provide chemical bonds and antiferromagentic coupling between the mixed-valence manganese centers.
JACS, 2007. V.129, No.39, pp. 11886-11887.

Copper nitrite reductase contains a T1 and T2 Cu center and catalyzes the one electron reduction of nitrite to NO. This study determines that reaction of the reduced enzyme with excess NO generates the T1 reduced T2 oxidized-nitrite bound form and not the η2-NO·Cu+ species observed in the crystal structure. Reaction intermediates have been trapped and N2O gas detected during this reaction, allowing elucidation of the mechanism by which both the T2 and NO are oxidized by the reaction of NO with the fully reduced enzyme. Density functional theory calculations provide insight into factors which favor the η2-NO·Cu+ complex in the crystal structure.
JACS, 2007, v. 129, No.34, pp. 10310-10311

Cytochrome c' is a heme protein from a denitrifying variant of Rhodobacter sphaeroides which may serve to store and transport metabolic NO while protecting against NO toxicity. Its heme site bears resemblance through its 5-coordinate NO-binding capability to the regulatory site in soluble guanylate cyclase.Aconserved arginine (Arg-127) abuts the 5-coordinate NO-heme binding site, and the alanine mutant R127A provided insight into the role of the Arg-127 in establishing the electronic structure of the heme-NO complex and in modifying the heme-centered redox potential and NO-binding affinity. By comparison to R127A, the wild-type Arg-127 was determined to increase the heme redox potential, diminish the NO-binding affinity, perturb and diminish the 14NO hyperfine coupling determined by ENDOR (electron nuclear double resonance), and increase the maximal electronic g-value. The larger isotropic NO hyperfine and the smaller maximal g-value of the R127A mutant together predicted that the Fe-N-O bond angle in the mutant is larger than that of the Arg-127-containing wild-type protein. Deuterium ENDOR provided evidence for exchangeable H/D consistent with hydrogen bonding of Arg-127, but not Ala-127, to theOof the NO. Proton ENDOR features previously assigned to Phe-14 on the distal side of the heme were unperturbed by the proximal side R127A mutation, implying the localized nature of that mutational perturbation at the proximal, NO-binding side of the heme. From this work two functions of positively charged Arg-127 emerged: the first was to maintain the KD of the cytochrome c' in the 1 μM range, and the second was to provide a redox potential that enhances the stability of the ferrous heme.
Biochemistry, 2009, v.48, No. 38, pp. 8985–8993

Nonstoichiometric b-SiC nanoparticles (np-SiC) have been studied by electron paramagnetic resonance (EPR) and pulsed magnetic resonance methods including field swept electron spin echo (FS ESE), pulsed electron nuclear double resonance (ENDOR) and hyperfine sublevel correlation spectroscopy (HYSCORE). Four ESE signals related to the paramagnetic centers labeled D1, D2, D3, D4 with g = 2.0043, g = 2.0029, g = 2.0031, g = 2.0037 were resolved in FS ESE spectrum due to their different spin relaxation times. As deduced from the study of the superhyperfine structure of the D2 defect by FS ESE, pulse ENDOR and HYSCORE methods the dominant paramagnetic center is a carbon vacancy (VC) localized in b-SiC crystalline phase of the np-SiC. The parameters of the D2 center coincide with those found for the VC in np-SiC obtained by laser pyrolysis method. Three other defects were identified by comparison of their EPR parameters with the microstructure of the np-SiC. The D1 defect was attributed to the VC vacancy located in b-SiC crystalline phase. The D3 defect is identified with the carbon dangling bonds located in the carbon excess phase. The D4 defect was assigned to a threefold-coordinated Si atom bonded with one nitrogen atom, resulting in the formation of the local bonding Si-Si2N configuration in a-Si3N4 phase.

The X-, Q-band field-sweep electron spin echo (FS ESE) study of n-type 6H SiC wafers shows besides the well known hyperfine (hf) triplet lines of 14N on two quasi-cubic (Nc1, Nc2) and hex-agonal (Nh) sites additional triplet lines (Nx) of comparatively low intensity with a g-tensor of about the average value of the Nc2 and Nh spectrum at 4.2 K. The Nx triplet has half of the hf splitting with respect to the nitrogen residing at cubic sites Nc2. Pulsed electron nuclear double resonance (ENDOR) measure-ments performed on the Nx EPR triplet line demonstrate that there is an efficient spin-coupling between Nh and Nx nitrogen re-lated centers. The Nx triplet lines were attributed to distant donor Nc–Nh pairs between nitrogen atoms residing at quasi-cubic and hexagonal sites having a total electron spin S = 1, which were found before in n-type 4H SiC. Based on Q-band FS ESE and X-band pulsed ENDOR measurements the values of the hf interac-tion for Nc1, Nc2 were reassigned with respect to the assignment accepted hitherto. The super hyperfine (shf) interaction with neighboring atoms of nitrogen on the quasi-cubic lattice sites was studied by FS ESE and pulsed ENDOR. The new shf lines due to 29Si and 13C atoms with large shf interaction which were not observed previously were found in nitrogen FS ESE and ENDOR spectra. One of them, with the largest shf splitting (23.12 MHz), were attributed to the 29Si atoms along the c-axis in nearest-neighbor positions of nitrogen on the quasi-cubic sites which unambiguously confirms that nitrogen substitute carbon lattice site in 6H SiC.