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Sedimentation equilibrium is a powerful tool for the characterization of protein self-association and heterogeneous protein interactions. Frequently, it is applied in a configuration with relatively long solution columns and with equilibrium profiles being acquired sequentially at several rotor speeds. The present study proposes computational tools,(More)
The Fe- and Mn-containing superoxide dismutases catalize the same reaction and have almost superimposable active sites. Therefore, the details of their mechanisms have been assumed to be similar. However, we now show that the pH dependence of Escherichia coli MnSOD activity reflects a different active site proton equilibrium in (oxidized) Mn(3+)SOD than the(More)
A combined spectroscopic/computational approach has been utilized to explore the chemical origins of the active-site pKs of the structurally homologous Fe- and Mn-dependent superoxide dismutases (SODs). Absorption, circular dichroism, magnetic circular dichroism, and variable-temperature, variable-field magnetic circular dichroism spectroscopic experiments(More)
Plasmodium falciparum iron regulatory-like protein (PfIRPa) has homology to both mammalian iron regulatory proteins and aconitases and is capable of binding RNA iron response elements. We examined the subcellular localization of PfIRPa and its enzymatic properties at low oxygen tension. Differential digitonin permeabilization of isolated trophozoites with(More)
Iron regulatory proteins (IRPs) regulate iron metabolism in mammalian cells. We used biophysical techniques to examine the solution properties of apo-IRP1 and apo-IRP2 and the interaction with their RNA ligand, the iron regulatory element (IRE). Sedimentation velocity and equilibrium experiments have shown that apo-IRP1 exists as an equilibrium mixture of(More)
Like many non-heme iron enzymes, reduced iron superoxide dismutase (Fe(2+)SOD) reacts with nitric oxide (NO) to yield an [Fe-NO]7 system. Electron paramagnetic resonance (EPR) data obtained for this Fe-NO adduct of FeSOD (NO-FeSOD) exhibit two rhombic S = 3/2 signals of comparable population; E/D = 0.128 (42%) and 0.154 (58%). While similar results were(More)
Plasmodium falciparum iron regulatory-like protein (PfIRPa, accession AJ012289) has homology to a family of iron-responsive element (IRE)-binding proteins (IRPs) found in different species. We have previously demonstrated that erythrocyte P. falciparum PfIRPa binds a mammalian consensus IRE and that the binding activity is regulated by iron status. In the(More)
A combination of spectroscopic and computational methods has been employed to explore the nature of the yellow and pink low-temperature azide adducts of iron(III) superoxide dismutase (N(3)-FeSOD), which have been known for more than two decades. Variable-temperature variable-field magnetic circular dichroism (MCD) data suggest that both species possess(More)
We are addressing the puzzling metal ion specificity of Fe- and Mn-containing superoxide dismutases (SODs) [see C.K.Vance, A.-F. Miller. J. Am. Chem. Soc. 120(3) (1998) 461-467]. Here, we test the significance to activity and active site integrity of the Gln side chain at the center of the active site hydrogen bond network. We have generated a mutant of(More)
The highly homologous proteins of Fe-containing superoxide dismutase (FeSOD) and MnSOD from Escherichia coli nonetheless exert very different redox tuning on the active site metal ion [Vance; Miller J. Am. Chem. Soc. 1998, 120, 461-467; Biochemistry 2001, 40, 13079-13087]. This was proposed to stem from different hydrogen bonding between the protein and the(More)