A Kinetic Description of Dioxygen Motion within R- and -Subunits of Human
Hemoglobin in the R-State: Geminate and Bimolecular Stages of the Oxygenation
Reaction
²
Sergei V. Lepeshkevich,
‡
Jerzy Karpiuk,
§
Igor V. Sazanovich,
‡
and Boris M. Dzhagarov*
,‡
Institute of Molecular and Atomic Physics, National Academy of Sciences of Belarus, 70 F. Skaryna AVe., Minsk 220072,
Belarus, and Institute of Physical Chemistry, Polish Academy of Sciences, 01-224 Warsaw, Kasprzaka 44/52, Poland
ReceiVed May 29, 2003; ReVised Manuscript ReceiVed December 15, 2003
ABSTRACT: Laser flash photolysis technique is used to study human hemoglobin (HbA) oxygenation.
Monomolecular geminate oxygenation of triliganded R-state HbA molecules is described by a function
of three exponentials. Geminate oxygenation of the R-subunit within R-state HbA is characterized by two
components with time constants of 0.14 and 1 ns, while geminate oxygenation of the -subunit within
HbA is characterized by two components with time constants of 1 and ∼30 ns. Bimolecular oxygenation
of triliganded R-state HbA molecules is described by a biexponential law. Two observed rate constants
are assigned to oxygenation of the R- and -subunit within HbA. The bimolecular association rate constants
for O
2
rebinding with the R- and -subunit within triliganded R-state HbA are k
R
) 18.8 ( 1.3 (µM‚s)
-1
and k
) 52 ( 4(µM‚s)
-1
, respectively. The apparent quantum yields of photodissociation of the - and
R-subunit within completely oxygenated R-state HbA differ from each other by a factor of 3.6 and are
equal to 0.041 ( 0.004 and 0.0114 ( 0.0012, respectively. The apparent quantum yield of photodissociation
of completely oxygenated R-state HbA is equal to 0.026 ( 0.003.
Tetrameric human hemoglobin (HbA)
1
is an allosteric
protein that carries molecular oxygen (O
2
) in blood and
tissues. With its relatively simple structure, it serves as a
good model for studying nonlinear and cooperative interac-
tions in proteins composed of several subunits. HbA is an
ensemble of two dimers formed by a pair of R- and
-subunits, each containing heme b. This protein is known
to be able to bind four O
2
molecules, one molecule per one
heme in each subunit (1). With the use of X-ray diffraction
analysis, it has been found that there exist two conformations
of the HbA quaternary structure. Oxygenated HbA has a
high-affinity structure called R (R-state), and the deoxygen-
ated one has a low-affinity structure called T (T-state) (1).
As HbA is oxygenated, its O
2
affinity is enhanced, and
consequently, the protein itself regulates the ligand affinity
(1, 2). Since the R- and -subunits differ in structure,
knowledge of individual properties of each subunit type in
the isolated state and in the different conformational forms
of tetrameric HbA (2-4) is necessary to understand the
molecular mechanism of HbA cooperative oxygenation.
Studies of photodissociation of the oxygenated protein
(5-21) aimed to examine the dynamics of O
2
motion within
the protein. This will make it possible to define O
2
trajectories
when the molecular oxygen moves in the interior of the
protein toward the binding center and when it moves back
to the surrounding medium. The considered motion is a
complex process. It is commonly accepted that after photo-
dissociation, a free O
2
molecule moves diffusionally inside
the protein globule, successively overcoming barriers im-
posed by the protein structure (22). A considerable number
of O
2
molecules cannot overcome these barriers inside the
protein matrix or the border between the protein and the
solvent and return and rebind to heme iron atoms. Such a
reaction is referred to as geminate recombination (GR). For
those HbA subunits from which O
2
molecules succeed in
escaping into the surrounding medium, rebinding is a
bimolecular reaction (BR). Besides the oxygenation studies,
the hemoglobin reactions with other small ligands (i.e., CO
and NO) were also extensively studied (see, for example,
refs 23-25), in particular for comparison with myoglobin
(26, 27).
In a related approach to Gibson’s work (5), the flash
photolysis technique was extensively used in kinetic studies
of the oxygenated hemoproteins (5-21). The earlier studies
(5-10) dealt with bimolecular O
2
rebinding within micro-
second and longer time ranges. The laser nanosecond flash
photolysis technique has helped to identify a 50-100 ns
component in the GR kinetics (11-14). Picosecond photo-
excitation has provided a basis for GR investigation within
the time range from several picoseconds up to 1.5 ns (15,
16, 19). The fast component (200 ps) of geminate recom-
bination was detected for the first time in the work of
Chernoff et al. (15). The complex biexponential geminate
²
This work was supported by the Foundation of Basic Research of
the Republic of Belarus (Grant No. B00-176) and the Polish -
Belarusian Collaboration Program (Committee for Scientific Research,
Project 56, Warsaw).
* To whom correspondence should be addressed. Telephone: +375
172841620. Fax: +375 172840030. E-mail: bmd@imaph.bas-net.by.
‡
Institute of Molecular and Atomic Physics.
§
Institute of Physical Chemistry.
1
Abbreviations: HbA, human hemoglobin; O2, molecular oxygen;
GR, geminate recombination; BR, bimolecular recombination.
1675 Biochemistry 2004, 43, 1675-1684
10.1021/bi034928q CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/24/2004