The role of diffusion in limiting the rate of ligand binding to hemoglobin

Abstract

The Root effect hemoglobin from Thunnus thynnus, the Atlantic bluefin tuna, remains in the T state at acid pH even when liganded. There are two components in the binding of CO to the T state with rates of 0.01 and 1.0 X 10(6) M-1 S-1 at 20 degrees C, 0.1 M KPi, pH 6.0, and two components in binding to the R state with rates of 1.7 and 8.5 X 10(6) M-1 S-1 as measured by partial flash photolysis at 20 degrees C, 0.05 M Tris-HCl, pH 8.0. A single rate of NO binding of 1.8 X 10(7) M-1 S-1 is found at both pH 6 and pH 8. The rate of binding of NO to dimethyldeuterohemedisulfonate is much higher still at 5 X 10(8) M-1 S-1. The observed rate of NO binding to the protein is not affected by chain differences or by the R to T transition. It may measure the rate of diffusion of ligand to the vicinity of the heme site. The T state components have different NO dissociation velocity constants of 0.004 and 0.07 S-1. The apparent quantum yields for the T state components are approximately 0.035 and 0.0008; the high affinity component has the lower yield. A two-step scheme is used to explain the results. The presence of chain differences and allosteric states is reflected in the intrinsic binding rates of both NO and CO to the heme group.