Confirmation of a Unique Intra-Dimer Cooperativity in the
Human Hemoglobin
1
1
Half-Oxygenated Intermediate
Supports the Symmetry Rule Model of Allosteric Regulation
Gary K. Ackers,
*
Jo M. Holt, Yingwen Huang, Yelena Grinkova, Alexandra L. Klinger, and Ilia Denisov
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri
ABSTRACT The contribution of the
1
1
half-
oxygenated tetramer [:O
2
O
2
] (species 21) to
human hemoglobin cooperativity was evaluated us-
ing cryogenic isoelectric focusing. The cooperative
free energy of binding, reflecting O
2
-driven protein
structure changes, was measured as
21
G
c
5.1
0.3 kcal for the Zn/FeO
2
analog. For the Fe/FeCN
analog,
21
G
c
was estimated as 4.0 kcal after correc-
tion for a CN ligand rearrangement artifact, demon-
strating that ligand rearrangement does not invali-
date previous conclusions regarding this species. In
the context of the entire Hb cooperativity cascade,
which includes eight intermediate species, the 21
tetramer is highly abundant relative to the other
doubly-ligated species, providing strong support for
the previously determined consensus partition func-
tion of O
2
binding and for the Symmetry Rule model
of hemoglobin cooperativity (Ackers et al., Science
1992;255:54 – 63). Cooperativity of normal human he-
moglobin is shown to depend on site-configuration,
and not solely the number of O
2
bound, nor the
occupancy of vs. subunits. Verification of a
unique contribution from the
1
1
doubly-oxygen-
ated species to the equilibrium O
2
binding curve
strongly reinforces the Symmetry Rule interpreta-
tion that the
1
1
dimer acts both as a structural and
functional element in cooperative O
2
binding.
Proteins 2000;Suppl 4:23– 43. © 2000 Wiley-Liss, Inc.
Key words: thermodynamic linkage; free energy;
subunit coupling; oxygen binding; mo-
lecular code
INTRODUCTION
The remarkable positive cooperativity that defines hu-
man hemoglobin (Hb) as an archetype of complex systems
in biology also serves, ironically, to mask the site-specific
contributions of its partially ligated intermediates, or
microstates (Fig. 1), which hold essential clues to the
molecular mechanism of allostery. These combinatorial
arrangements of oxy- and deoxy-hemes comprise a func-
tional cascade of 16 binding steps in which the microstate
intermediates have very low equilibrium concentrations.
Consequently, their energetic and structural properties
are irresolvable by conventional techniques (reviewed in
Ackers
1
). However, it has been possible to prepare stable
analogs of each unique microstate species that are ame-
nable to study by capitalizing on the thermodynamic
linkages between hemesite binding and dimer 3
2
2
tetramer assembly as illustrated in Figure 2. This ap-
proach has required the ability to resolve small differences
in free energies of the microstate intermediates, necessitat-
ing the development and optimization of techniques
2–4
capable of measuring their cooperative free energies of
binding (see Fig. 2 legend) to within 0.2– 0.5 kcal/mol.
2–4
Pursuing a consensus strategy, the cooperative free
energy
ij
G
c
was measured for the ten microstates (ij =
01 to 41), over a series of Hb tetramers containing
previously characterized hemesite analogs.
5
For each ana-
log, the nine
ij
G
c
energies reflect the tetramer’s tertiary
and quaternary structural responses to ligation in all
combinatorial forms (Fig. 1). In initial studies, the cooper-
ative free energies
ij
G
c
were measured for three analog
systems: Fe
2+
/Fe
3+
CN (CNmet), Fe
2+
/Mn
3+
, and Co
2+
/
Fe
2+
CO. From this database, a common pattern of
ij
G
c
partitioning emerged, which was independent of analog
substitution effects, and a consensus free energy distribu-
tion for native HbO
2
microstates (herein referred to as
Z
Hb
) was proposed in 1992.
5
In the consensus distribution Z
Hb
, each of the ten
microstate tetramers was observed to fall into one of 5 free
energy levels, as reflected in Figure 1. A notable split was
evident among
ij
G
c
values of the four doubly ligated
intermediates. The microstate tetramer with both ligands
on the same
1
1
dimer, species 21, had greater stability
than intermediates having only partially ligated
1
1
dimers, i.e., species 22, 23, and 24. The overall pattern of
the ten microstate
ij
G
c
values was, therefore, indicative
of a specific pairwise coupling between the subunits of the
1
1
(and the equivalent
2
2
) dimeric unit within the
tetramer. In contrast, no such coupling was indicated by
the consensus distribution for
1
2
subunit pairs of the
species 22 tetramer,
5
even though it has the same composi-
tion of ligated vs. unligated and subunits. This pattern
was interpreted in terms of the
1
1
dimer acting as a
Grant sponsor: National Institutes of Health; Grant sponsor: Na-
tional Science Foundation.
Yelena Grinkova’s present address is Beckman Institute, Univer-
sity of Illinois, Urbana-Champaign, IL.
Ilia Denisov’s present address is Beckman Institute, University of
Illinois, Urbana-Champaign, IL.
*Correspondence to: G.K. Ackers, Department of Biochemistry and
Molecular Biophysics, Washington University School of Medicine, St.
Louis, MO 63110. E-mail: ackers@biochem.wustl.edu
Received 17 March 2000; Accepted 28 June 2000
PROTEINS: Structure, Function, and Genetics Suppl 4:23– 43 (2000)
© 2000 WILEY-LISS, INC.