By low temperature electron paramagnetic resonance we have detected the formation of a free radical signal during incubation of partially oxygenated hemoglobin at 235 K. The observed signal has g parallel = 2.0565 and g perpendicular = 2.0043, consistent with the previously reported values for superoxide. The presence of additional EPR signals for oxygen-17 bound hemoglobin, with (017-017)A perpendicular = 63 G and (017-016)A perpendicular = 94 G under identical conditions, confirms the presence of a radical containing two nonequivalent oxygens as required for a superoxide in magnetically inequivalent environments. The superoxide radical has not previously been directly detected during hemoglobin autoxidation because of its rapid dismutation. Our ability to follow the formation of superoxide for more than 15 min is attributed to its production in the hydrophobic heme pocket where dismutation is slow. The enhanced production of this free radical at intermediate oxygen pressures is shown to coincide with enhanced rates of hemoglobin autoxidation for partially oxygenated intermediates. The formation of superoxide in the heme pocket under these conditions is attributed to enhanced heme pocket flexibility. Greater flexibility facilitates distal histidine interactions which destabilize the iron-oxygen bond resulting in the release of superoxide radical into the heme pocket.
The EPR spectroscopic parameters of a series of Mo(V) oxyhalide anions, [MoOX5 - n (H2O) n ](2- n )- (X = F, Br, n = 0; X = Cl, n = 1), were obtained in fluid solutions and frozen glasses or (X = Cl, Br) doped into single crystals of a diamagnetic host lattice. The electronic structures of the complexes were approximated by optimizing the electronic structural parameters of a LCAO model to reproduce the experimentally observed EPR parameters. The results indicate quantitatively that the most important contribution to deviations from g = g e in the complexes is metal−ligand covalency. Charge transfer excited state mixing and ligand spin−orbit coupling (for X = Cl, Br) provide significant but smaller contributions to the EPR parameters. The isotropic molybdenum hyperfine coupling constants are also shown to be dominated by Fermi contact interactions. A number of implications with respect to the EPR spectroscopy of molybdenum oxidoreductases are noted. The crystal and molecular structure of a diamagnetic Nb(V) lattice is also reported. (H2dafone)[NbOCl4(H2O)]Cl (dafone = 4,5-diazafluoren-9-one) crystallizes in the monoclinic space group C2/c with a = 16.0043(2) Å, b = 24.8021(3) Å, c = 10.0162(2) Å, β = 121.048(1)°, and Z = 8.
The role of the beta-93 cysteine residue in the hemoglobin autoxidation process has been delineated by electron paramagnetic resonance. At low temperatures (8 K) after incubation at 235 K, free radical signals were detected. An analysis of the free radical spectrum produced implies that, besides the superoxide radical expected to be formed during autoxidation, an isotropic free radical is produced with a giso of 2.0133. This g value is consistent with that expected for a sulfur radical. Blocking the beta-93 sulfhydryl group with N-ethylmaleimide was found to eliminate the formation of the isotropic radical, but not the superoxide. This finding confirms the assignment of the isotropic radical as a thiyl radical originating from the oxidation of the cysteine SH group. A kinetic analysis of the time course for the formation of both the superoxide and thiyl radicals is consistent with a reversible electron transfer process between superoxide in the heme pocket of the beta-chains and the cysteine residue. This reaction is expected to produce both a thiyl radical and a peroxide. Direct evidence for peroxide production comes from the detection of a transient Fe(III) heme peroxide complex. The significance of the electron transfer process producing a thiyl radical is discussed. It is shown that the formation of the thiyl radical decreases the rate of autoxidation for the beta-chain and reduces heme degradation attributed to the reaction of superoxide with the heme. The insights gained from these low-temperature studies are believed to be relevant to room-temperature autoxidation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.