Patricia C. Harrington

EPR spectroscopy of semi-methemerythrin

EPR spectra of semi-met forms of octameric hemerythrin from Themiste zostericola, prepared by one electron reduction of methemerythrin or by one electron oxidation of deoxyhemerythrin, have been visualized at liquid helium temperatures. The spectrum of that prepared by one electron reduction has principal g-values of 1.96 +/- 0.01, 1.88 +/- 0.01, and 1.67 +/- 0.02 while that obtained by one electron oxidation has g = 1.95 +/- 0.01, 1.72 +/- 0.01, and 1.68 +/- 0.02. The amplitude of either spectrum decreases with time on incubation at room temperature according to a first order rate with t 1/2 = 5-8 min, apparently because of an intramolecular disproportionation. Similar EPR spectra have been obtained with semi-metmyohemerythrin of T. zostericola and with the octameric semi-met form of Phascolopsis gouldii. However, these forms disproportionate to a much lesser degree. The azide adduct of the octameric semi-met form of T. zostericola has g-values of 1.94 +/- 0.01, 1.85 +/- 0.01, and 1.57 +/- 0.02. Its EPR spectrum differs somewhat from those of the azide adducts of the octamer of P. gouldii and the monomer of T. zostericola although all are resistant to disproportionation. Methemerythrin and deoxyhemerythrin have no EPR spectra even at liquid helium temperature.

Reduction of methemerythrin by dithionite ion

Abstract The reduction of methemerythrin (Hr + ) by dithionite produces deoxyhemerythrin (Hr o ) in multi, possibly three, stages. The kinetics were examined at pH 8·2 and 25 °C. The first stage is reduction of methemerythrin to an intermediate A by SO 2 - (k = 1.3 × 10 5 m −1 s −1 ). The much slower second and third stages have rates independent of dithionite concentrations. Reaction is completed after about 10 h. The kinetics of reactions of A with N 3 - , H 2 O 2 , and O 2 were examined, as well as the conversion of A to intermediate B (k = 4·4 × 10 −4 s −1 ). It is concluded that A is an (Fe(II)Fe(III)) 8 species, and that in B the unit (Fe(II)Fe(II)) 8 is well developed, judging by its unreactivity towards N 3 − , its reaction with H 2 O 2 , and its reversible uptake of O 2 (85–90% of the final product). There is little effect of adjusting the pH to 6·3 on the rates of the processes examined.

Kinetics of the equilibration of oxygen with monomeric and octameric hemerythrin from Themiste zostericola

Single relaxations for the equilibration of O2 with monomeric and octameric deoxy forms of hemerythrin from Themiste zostericola have been observed at 25°C using the temperature-jump technique. At 25°C, pH 8.2 (Tris/H2SO4 and I = 0.10 M (Na2SO4), formation rate constants kon are 7.8 · 107 M−1 · s−1 and 7.5 · 106 M−1 s−1, respectively. The procedure used does not give a precise measure (small intercepts) of dissociation rate constants, koff. These were determined instead by the stopped-flow method using dithionite to induce dissociation of the oxy protein. Values of koff for the monomer (3.1 · 102 s−1) and octamer (82 s−1), in association with kon values, lead to equilibrium constants for the formation of oxyhemerythrin of 2.5 · 105 M−1 and 0.9 · 105 M−1, respectively, at 25°C, pH 8.2 and I = 0.10 M (Na2SO4). These latter are in reasonable agreement with values (1.5 105 M−1 and 1.3 · 105 M−1) determined spectrally on the equilibrated solutions. Using the octameric protein, it was shown that replacement of SO42− by ClO4− or Cl− ions (at a constant I = 0.10 M) led to an approximately 2-fold enhancement of kon but had little effect on koff. The addition of Ca2+ or Mg2+ ions (0.01 M), with or without 0.50 M NaCl, also gives up to 4-fold increases in kon, but unchanged koff values. Oxygen pulse experiments on the octamer show no effect on koff of the degree of oxygenation of the protein. A comparison is made with similar data for hemoglobin, myoglobin and hemocyanin.

Reduction potentials of various hemerythrin oxidation states

Abstract The reduction potentials of the couple methemerythrin/(semi-met) r have been determined spectrally, using 2,6-dichlorophenolindophenol as a redox partner, for monomer and octamer from Themiste zostericola and for octamer protein from Phascolopsis gouldii. The reduction potentials of the couple (semi-met)o/deoxy have been determined spectrally, using Fe(III) cyanocomplexes, for monomer and octamer protein from Themiste zostericola. The values of other potentials, (met/(semi-met)o and (semi-met) r /deoxy), have thus been deduced as well as equilibrium constants for the (semi-met) r α (semi-met)o conformational changes for hemerythrins from Themiste zostericola.

Substitution and Electron Transfer Processes in Hemerythrin

The kinetics and mechanisms of reactions at the binuclear iron site of hemerythrin are discussed. These include substitution, redox and configuration changes involving deoxy, oxy, semi-met and met forms of the protein. Emphasis will be on results with the octameric and monomeric forms of the protein from Themiste zostericola.

Catalysed hydrolysis of S-p-nitrophenyl thioacetate by human carbonic anhydrase

Sigmoidal kenz–pH profiles are observed for catalysis of the hydrolysis of S-p-nitrophenyl thioacetate by native, cobalt(II), and copper(II) forms of human carbonic anhydrase B and by native C.