Rate Constants for O2 and CO Binding to the α and β Subunits within the R and T States of Human Hemoglobin

Satoru Unzai,Raymund Eich,Naoya Shibayama,John S Olson,Hideki Morimoto

doi:10.1074/jbc.273.36.23150

Abstract

Despite a large amount of work over the past 30 years, there is still no universal agreement on the differential reactivities of the individual alpha and beta subunits in human hemoglobin. To address this question systematically, we prepared a series of hybrid hemoglobins in which heme was replaced by chromium(III), manganese(III), nickel(II), and magnesium(II) protoporphyrin IXs in either the alpha or beta subunits to produce alpha2(M)beta2(Fe)1 and alpha2(Fe)beta2(M) tetramers. None of the abnormal metal complexes react with dioxygen or carbon monoxide. The O2 affinities of the resultant hemoglobins vary from 3 microM-1 (Cr(III)/Fe(II) hybrids) to 0.003 microM-1 (Mg(II)/Fe(II) hybrids), covering the full range expected for the various high (R) and low (T) affinity quaternary conformations, respectively, of human hemoglobin A0. The alpha and beta subunits in hemoglobin have similar O2 affinities in both quaternary states, despite the fact that the R to T transition causes significantly different structural changes in the alpha and beta heme pockets. This functional equivalence almost certainly evolved to maintain high n values for efficient O2 transport.

Highlights

Despite a large amount of work over the past 30 years, there is still no universal agreement on the differential reactivities of the individual ␣ and ␤ subunits in human hemoglobin
The O2 affinities of the resultant hemoglobins vary from 3 ␮M؊1 (Cr(III)/Fe(II) hybrids) to 0.003 ␮M؊1 (Mg(II)/Fe(II) hybrids), covering the full range expected for the various high (R) and low (T) affinity quaternary conformations, respectively, of human hemoglobin A0
The results have provided two sets of kinetic parameters for ␣ and ␤ subunits thought to be in mostly T conformations, i.e. the Ni(II) and Mg(II) hybrids, and two sets of parameters for subunits in mostly R conformations, i.e. the Cr(III) and Mn(III) hybrids

Summary

Introduction

Despite a large amount of work over the past 30 years, there is still no universal agreement on the differential reactivities of the individual ␣ and ␤ subunits in human hemoglobin. A myriad of experimental approaches were developed to resolve this controversy concerning functional differences between the subunits, including the following: (i) measurements of the ligand binding properties of the isolated ␣ and ␤ chains [3]; (ii) discovery or development of spectral signals for ligation of the individual subunits within intact tetramers [11]; (iii) selective chemical modification or mutation of key residues in the ␣ and ␤ chains [12, 13]; (iv) separation of kinetic intermediates by cryogenic electrophoresis [14]; (v) x-ray diffraction analysis of partially liganded Hb crystals [15]; and (vi) construction of valency and metal hybrid Hbs in which one pair of subunits has an inert metal-porphyrin group and the other O2and CO-reactive heme groups (16 –24) The latter approach is the most definitive with respect to assigning observed rate and equilibrium constants since only one type of subunit is capable of reacting with ligands. Time courses for ligand binding to ␣(Fe)␤(inert) and ␣(inert)␤(Fe) hybrids are often biphasic, with rapid and slow phases representing very slowly interconverting R and T conformations, respectively

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Conclusion