Semihemoglobins, High Oxygen Affinity Dimeric Forms of Human Hemoglobin Respond Efficiently to Allosteric Effectors without Forming Tetramers

Significant reduction in oxygen affinity resulting from interactions between heterotropic allosteric effectors and hemoglobin in not only the unligated derivative but also the fully ligated form has been reported (Tsuneshige, A., Park, S. I., and Yonetani, T. (2002) Biophys. Chem. 98, 49-63; Yonetani, T., Park, S. I., Tsuneshige, A., Imai, K., and Kanaori, K. (2002) J. Biol. Chem. 277, 34508-34520). To further investigate this effect in more detail, alpha- and beta-semihemoglobins, namely, alpha(heme)beta(apo) and alpha(apo)beta(heme), respectively, were prepared and characterized with respect to the impact of allosteric effectors on both conformation and ligand binding properties. Semihemoglobins are dimers characterized by a high affinity for oxygen and lack of cooperativity. We found that, compared with stripped conditions, semihemoglobins responded to effectors (inositol hexaphosphate and L35) by decreasing the affinity for oxygen by 60- and 130-fold for alpha- and beta-semihemoglobins, respectively. 1H NMR and sedimentation velocity experiments carried out with their ligated and unligated forms in the absence and presence of effectors revealed that semihemoglobins always remain as single-heme-carrying dimers. Recombination kinetics of their photolyzed CO derivatives showed that effectors did indeed interact with their ligated forms. Measurements of the Fe-His stretching mode show that the semihemoglobins undergo a large ligand binding-induced conformational shift and that both ligand-free and ligand derivatives respond to the presence of effectors. Contradictions to the Monod-Wyman-Changeaux/Perutz allosteric model arise since 1) the modulation of ligand affinity is not achieved in semihemoglobins by the formation of a low affinity T conformation (quaternary effect) but by direct interaction with effectors, 2) effectors do interact significantly with ligated forms of high affinity semihemoglobins, and 3) modulation of the ligand affinity and the cooperativity are not necessarily linked but instead can be separated into two distinct phenomena that can be isolated

Cyclic Hydroxylamines as Monitors of Peroxynitrite and Superoxide-Revisited

There is a considerable need for methods that allow quantitative determination in vitro and in vivo of transient oxidative species such as peroxynitrite (ONOOH/ONOO−) and superoxide (HO2•/O2•−). Cyclic hydroxylamines, which upon oxidation yield their respective stable nitroxide radicals, have been suggested as spin probes of peroxynitrite and superoxide. The present study investigated this approach by following the kinetics of peroxynitrite decay in the absence and presence of various 5-membered and 6-membered ring hydroxylamines, and comparing the yield of their respective nitroxides using electron paramagnetic spectroscopy. The results demonstrate that hydroxylamines do not react directly with peroxynitrite, but are oxidized to their respective nitroxides by the radicals formed during peroxynitrite self-decomposition, namely •OH and •NO2. The accumulated nitroxides are far below their expected yield, had the hydroxylamines fully scavenged all these radicals, due to multiple competing reactions of the oxidized forms of the hydroxylamines with •NO2 and ONOO−. Therefore, cyclic hydroxylamines cannot be used for quantitative assay of peroxynitrite in vitro. The situation is even more complex in vivo where •OH and •NO2 are formed also via other oxidizing reactions systems. The present study also compared the yield of accumulated nitroxides under constant flux of superoxide in the presence of various cyclic hydroxylamines. It is demonstrated that certain 5-membered ring hydroxylamines, which their respective nitroxides are poor SOD-mimics, might be considered as stoichiometric monitors of superoxide in vitro at highest possible concentrations and pH

Matrix Photochemistry of Cycloheptatriene: Site Effects

An experimental study of CHT photolysis at 2537 Å in argon and nitrogen (N 2 ) matrices is presented. The sole IR detectable product is identified as BCHD: bicyclo[3.2.0]hepta-2,6-diene. It is found that CHT molecules trapped in spectroscopically distinct sites were converted to the product at different rates. In the initial stages of the photolysis, the population of some CHT trapping sites increased, indicating site population transfer. In the long run, however, all sites were depleted. Molecular dynamics simulations are performed to estimate the structure of the different sites, and together with a kinetic model, are used to account for the UV induced changes. A tentative energy level diagram of the system is offered, in an attempt to rationalize the total absence of toluene which is formed predominantly in the gas-phase irradiation of CHT at this wavelength. The "twin state" concept is instrumental in this rationalization

Biologically active metalindependent superoxide dismutase mimics. Biochemistry.

ABSTRACT: Superoxide dismutase (SOD) is an enzyme that detoxifies superoxide (02'-), a potentially toxic oxygen-derived species. Attempts to increase intracellular concentrations of SOD by direct application are complicated because SOD, being a relatively large molecule, does not readily cross cell membranes. We have identified a set of stable nitroxides that possess SOD-like activity, have the advantage of being low molecular weight, membrane permeable, and metal independent, and at pH 7.0 have reaction rate constants with 02'-ranging from 1.1 X IO3 to 1.3 X IO6 M-' s-l. These S O D mimics protect mammalian cells from damage induced by hypoxanthine/xanthine oxidase and H202, although they exhibit no catalase-like activity. In addition, the nitroxide SOD mimics rapidly oxidize DNA-Fe" and thus may interrupt the Fenton reaction and prevent formation of deleterious OH radicals and/or higher oxidation states of metal ions. Whether by SOD-like activity and/or interception of an electron from redox-active metal ions they protect cells from oxidative stress and may have use in basic and applied biological studies. I n an oxygen-containing environment, cellular metabolism results in the production of several potentially harmful oxygen-derived species. The first in this cascade of active oxygen metabolites is the one-electron reduction product, superoxide (02*-). The function of the superoxide-detoxifying metalloenzyme superoxide dismutase (SOD)' in protecting against oxygen-mediated biological damage is well documented EXPERIMENTAL PROCEDURES Chemicals. Desferrioxamine (DF) was a gift from Ciba Geigy; hypoxanthine (HX) was purchased from CalbiochemBoehringer Co.; 2,2,6,6-tetramethylpiperidine-1 -oxy1 (TEM-PO), 4-hydroxy-2,2,6,6-tetramethylpiperidine-I-oxy1 (TEM-POL), 4-hydroxypyrazolo[3,4,-d]pyrimidine (allopurinol), p-toluenesulfonic acid, 2-amino-2-methyl-1 -propanol, 2-butanone, and cyclohexanone were purchased from Aldrich Chemical Co.; tris(oxalato)chromat