Fourth stable radical species in X-ray irradiated sucrose

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EPR analysis of cyanide complexes of wild-type human neuroglobin and mutants in comparison to horse heart myoglobin

Electron paramagnetic resonance (EPR) data reveal large differences between the ferric (C-)cyanide complexes of wild-type human neuroglobin (NGB) and its H64Q and F28L point mutants and the cyanide complexes of mammalian myo-and haemoglobin. The point mutations, which involve residues comprising the distal haem pocket in NGB, induce smaller, but still significant changes, related to changes in the stabilization of the cyanide ligand. Furthermore, for the first time, the full C hyperfine tensor of the cyanide carbon of cyanide-ligated horse heart myoglobin (hhMb) was determined using Davies ENDOR (electron nuclear double resonance). Disagreement of these experimental data with earlier predictions based on C NMR data and a theoretical model reveal significant flaws in the model assumptions. The same ENDOR procedure allowed also partial determination of the corresponding C hyperfine tensor of cyanide-ligated NGB and H64QNGB. These C parameters differ significantly from those of cyanide-ligated hhMb and challenge our current theoretical understanding of how the haem environment influences the magnetic parameters obtained by EPR and NMR in cyanide-ligated haem proteins

Characterization of the MCRred2 form of methyl-coenzyme M reductase: a pulse EPR and ENDOR study

: Methyl-coenzyme M reductase (MCR), which catalyses the reduction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (H-S-CoB) to CH4 and CoM-S-S-CoB, contains the nickel porphinoid F430 as prosthetic group. The active enzyme exhibits the Ni(I)-derived axial EPR signal MCRred1 both in the absence and presence of the substrates. When the enzyme is competitively inhibited by coenzyme M (HS-CoM) the MCRred1 signal is partially converted into the rhombic EPR signal MCRred2. To obtain deeper insight into the geometric and electronic structure of the red2 form, pulse EPR and ENDOR spectroscopy at X- and Q-band microwave frequencies was used. Hyperfine interactions of the four pyrrole nitrogens were determined from ENDOR and HYSCORE data, which revealed two sets of nitrogens with hyperfine couplings differing by about a factor of two. In addition, ENDOR data enabled observation of two nearly isotropic 1H hyperfine interactions. Both the nitrogen and proton data indicate that the substrate analogue coenzyme M is axially coordinated to Ni(I) in the MCRred2 stat

Compound I formation and reactivity in dimeric chlorite dismutase – Impact of pH and the dynamics of the catalytic arginine

The heme enzyme chlorite dismutase (Cld) catalyzes the degradation of chlorite to chloride and dioxygen. Many questions about the molecular reaction mechanism of this iron protein have remained unanswered, including the electronic nature of the catalytically relevant oxoiron(IV) intermediate and its interaction with the distal, flexible, and catalytically active arginine. Here, we have investigated the dimeric Cld from Cyanothece sp. PCC7425 (CCld) and two variants having the catalytic arginine R127 (i) hydrogen-bonded to glutamine Q74 (wild-type CCld), (ii) arrested in a salt bridge with a glutamate (Q74E), or (iii) being fully flexible (Q74V). Presented stopped-flow spectroscopic studies demonstrate the initial and transient appearance of Compound I in the reaction between CCld and chlorite at pH 5.0 and 7.0 and the dominance of spectral features of an oxoiron(IV) species (418, 528, and 551 nm) during most of the chlorite degradation period at neutral and alkaline pH. Arresting the R127 in a salt bridge delays chlorite decomposition, whereas increased flexibility accelerates the reaction. The dynamics of R127 does not affect the formation of Compound I mediated by hypochlorite but has an influence on Compound I stability, which decreases rapidly with increasing pH. The decrease in activity is accompanied by the formation of protein-based amino acid radicals. Compound I is demonstrated to oxidize iodide, chlorite, and serotonin but not hypochlorite. Serotonin is able to dampen oxidative damage and inactivation of CCld at neutral and alkaline pH. Presented data are discussed with respect to the molecular mechanism of Cld and the pronounced pH dependence of chlorite degradation

Resolving the relative tilting of hyperfine and g tensors for vanadyl complexes in the Al-metal-organic framework MIL-53 by a multifrequency (S, X, Q, and W-band) EPR approach

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