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 $\text{I = 0.10}\text{M}$ (Na₂SO₄), formation rate constants $\text{k}{}_{\mathrm{<mtext>on</mtext>}}$ 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, $\text{k}{}_{\mathrm{<mtext>off</mtext>}}$. These were determined instead by the stopped-flow method using dithionite to induce dissociation of the oxy protein. Values of $\text{k}{}_{\mathrm{<mtext>off</mtext>}}$ for the monomer (3.1 · 10² s⁻¹) and octamer (82 s⁻¹), in association with $\text{k}{}_{\mathrm{<mtext>on</mtext>}}$ 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 $\text{I = 0.10}\text{M}$ (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 $\text{SO}{}_4{}^{\mathrm{2-}}$ by $\text{ClO}{}_4{}^{\mathrm{-}}$ or Cl⁻ ions (at a constant $\text{I = 0.10}\text{M}$) led to an approximately 2-fold enhancement of k_on but had little effect on $\text{k}{}_{\mathrm{<mtext>off</mtext>}}$. 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 $\text{k}{}_{\mathrm{<mtext>on</mtext>}}$, but unchanged $\text{k}{}_{\mathrm{<mtext>off</mtext>}}$ values. Oxygen pulse experiments on the octamer show no effect on $\text{k}{}_{\mathrm{<mtext>off</mtext>}}$ of the degree of oxygenation of the protein. A comparison is made with similar data for hemoglobin, myoglobin and hemocyanin.