TABLE II.

Rate and Equilibrium Constants for Hemin Binding to apoShp and apoHtsA

Kinetic parameter	apoShp	apoHtsA
$\frac{k_2}{k_1}$ or Kd (hemin binding)a	22 ± 2 μM	8 ± 0.7 μM
kcoordinatioina	35 ± 4 s−1	655 ± 47 s−1
k′hemin ≈ kcoordination/Kd, apparent bimolecular rate constant at low[Protein],	1.6 μM−1s−1	80 μM−1s−1
k-heminb	0.0003 ± 0.00006 s−1	0.0026 ± 0.0002 s−1
Khemin≈k′hemin/k-hemin	5,300 μM−1	31,000 μM−1

^aThe hemin binding reaction at 25°C in 20 mM Tris-HCl at pH8.0 appears to occur by a two-step process involving an initial hemin binding step followed by first order iron coordination. In this case, values for k₂/k₁ and k_coordination were obtained from fits of the dependence of the observed rates of transfer on [apoprotein] to Equation 4.

^bThe hemin dissociation rate constants from hemiShp and hemiHtsA were determined by the H64Y/V68F apomyoglobin assay (17).