Is superoxide important in oxygen poisoning?

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24039/1/0000288.pd

Electron–nuclear double resonance on copper (II) tetraimidazole

We have investigated the electron–nuclear double resonance (ENDOR) from frozen aqueous solutions of 65Cu++(imidazole)4, 65Cu++ (imidazole–15N)4, and 65Cu++(imidazole–Dn)4, where n = 1, 2, 3, and 4 for selectively deuterated imidazole. We have observed ENDOR associated with the imidazole protons and the two imidazole nitrogens. The selective deuteration has allowed us to attempt identification of the weakly coupled protons responsible for the ENDOR spectrum, and a comparison of the overall line shape of that spectrum taken at two extreme points of the EPR spectrum suggests that some of the imidazole planes are tilted with respect to the plane of the complex. The ENDOR arising from the nitrogen nearest the copper is primarily isotropic with A(g⊥) = 41.6±1.5 MHz and A(g∥) = 39.8±1.5 MHz. The resonance shows little structure and seems consistent with a picture that requires some inequivalence among the various imidazoles. The remote nitrogen ENDOR reveals both hyperfine and quadrupole effects with approximately isotropic A(14N) = 1.79 MHz, Qz′z′?0.360 MHz, and Qx′x′y′x′?0.349 MHz. These values are in agreement with the results of the nuclear modulation effect [J. Chem. Phys. 69, 4921 (1978)]. The values for the quadrupole constants are thought to be accurate within 10% and are the same as are found in free imidazole. It is also demonstrated that, in this instance, ENDOR and the nuclear modulation effect are complementary in that they have each provided different parts of the same hyperfine spectrum.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/70838/2/JCPSA6-75-5-2098-1.pd

The properties of parsley ferredoxin and its selenium-containing homolog

The ferredoxin from parsley has been purified to a high degree. The protein contains 2.0 iron atoms per molecule, has an iron to labile sulfide ratio of unity and consumes 9 moles of mercurial, leading to calculated molecular weights of 10800, 10750 and 10960, respectively. These compare well with the results of amino acid analysis which gave a molecular weight of 10660. The dithionite-reduced material showed an EPR spectrum with the experimental g values: 1.899, 1.959, 2.061. CL- was found to have a specific broadening effect upon this spectrum.Selenium has been substituted for the labile sulfur of the native protein to yield a biologically active homolog. A new method for this reaction has been developed. The optical and circular dichroism spectra of the oxidized and reduced forms of the selenium protein have been recorded and are compared with the corresponding state of the native protein.The following isotopic derivatives have been prepared and studied by EPR spectroscopy: 32S56Fe (native), 32S57Fe, 30Se56Fe, 77Se56Fe and 80Se57Fe. The 32S57Fe derivative shows a broadened EPR spectrum but no hyperfine structure is resolved, this is presumably due to the broad line of the native material. The 80Se56Fe spectrum tended toward axial symmetry with apparent g values of 1.937, 1.965, 2.062. This derivative also showed a substantially narrower line width in the z-region. Hyperfine structure is resolved in the z-region of the 77Se56Fe homolog, and the EPR spectrum has been analyzed to determine the number of I = 1/2 hyperfine centers contributing to the line widths. Hyperfine structure was not resolved in the 80Se57Fe species; analysis of the line shape of the low field resonance suggests that the broadening observed is consistent with two iron (I = 1/2) centers with similar effective hyperfine splitting constants.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33582/1/0000085.pd

Potentiometric study of cytochrome c1aa3 from thermus thermophilus

We have examined the redox behavior of the cytochrome c1aa3 complex from Thermus thermophilus. In potentiometric titrations the cytochrome c behaves as an independent center having n = 1 and E = 205 mV (NHE). Under the assumption that the individual centers equilibrate independently in this experiment, changes in the absorption band at 603 nm have been resolved into two components: cytochrome a (n = 1, Em = 270 mV, 60% spectral contribution) and cytochrome a3 (n = 2, Em= 360 mV, 40% spectral contribution). The n = 2 process was attributed to strong chemical coupling between cytochrome a3 and CuB. The enzyme was also titrated with a mixture of NADH and PMS, and the results are shown not to conform to a model of intramolecular equilibrium according to the equilibrium constants obtained from the potentiometric titration. It is suggested that a conformational equilibrium within the complex may control electron transfer between cytochromes a and a3.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25750/1/0000310.pd

The oxidation-reduction potentials of parsley ferredoxin and its selenium-containing homolog

We have measured the oxidation-reduction potential of parsley ferredoxin and its derivative in which the two atoms of labile sulfide have been replaced by selenide. The values are -0.416 V (25[deg], pH 7.94) and -0.378 V (25[deg], pH 8.14) for the sulfur and selenium derivatives, respectively. Both values show a slight negative dependence on pH.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33583/1/0000086.pd

Spectral analysis of cytochrome c: effect of heme conformation, axial ligand, peripheral substituents and local electric fields,

We present in this work low-temperature visible absorption spectra for recombinant Thermus thermophilus cytochrome c 552 . The Q-band presents a remarkable splitting at low temperature. We performed quantum chemical calculations to evaluate quantitatively the effect of heme conformation, axial ligand, peripheral substituents and local electric fields on the electronic spectra. In an attempt to find correlation between protein structure and spectral splitting, we carried out the same calculations on three other cytochrome c's: horse heart, tuna heart, and yeast. The quantum chemical calculations were performed at the INDO level with extensive configuration interaction. The electric field at the heme pocket was included in the calculations through a set of point charges fitting the actual electric field. The results obtained show clearly that all mentioned effects contribute to the observed spectral splitting in a complex nonadditive way

Iron-ethylenediaminetetraacetic acid (EDTA)-catalyzed superoxide dismutation revisited: An explanation of why the dismutase activity of Fe-EDTA cannot be detected in the cytochrome c/xanthine oxidase assay system

The recent assertion of [1.] that Fe-EDTA does not catalyze superoxide dismutation is disputed. By directly observing superoxide generated during pulse radiolysis, we have confirmed the results of a previous study ( [2.]) which concluded that Fe-EDTA catalyzed superoxide dismutation. We also demonstrate that the reaction of Fe(II)-EDTA, formed during catalyzed superoxide dismutation, with cytochrome c, the probe molecule in the cytochrome c/xanthine oxidase/xanthine assay system for superoxide dismutase activity, is sufficiently rapid ([12.]) to obscure the weak catalysis of superoxide dismutation by Fe-EDTA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24002/1/0000251.pd

Saturation behavior of superoxide dismutation catalyzed by the iron containing superoxide dismutase of E. coli B

SummaryThe iron containing superoxide dismutase from E. coli B is shown to catalyze superoxide dismutation by a mechanism which exhibits saturation kinetics. This behavior is quite different from that observed previously with bovine Zn/Cu- and iron-containing superoxide dismutase from P. leiognathi. Two parameters of catalysis were measured in the pH range 7.2 to 10.4: kcat was found to be independent of pH and Km varied with the function Km = Km(low pH) [1 + exp(pH -- 8.8)]. These results implicate a group in the catalytic mechanism which ionizes with pKa = 8.8.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24375/1/0000644.pd