Anticooperative ligand binding properties of recombinant ferric Vitreoscilla homodimeric hemoglobin: A thermodynamic, kinetic and X-ray crystallographic study
M. Bolognesi et al., Anticooperative ligand binding properties of recombinant ferric Vitreoscilla homodimeric hemoglobin: A thermodynamic, kinetic and X-ray crystallographic study, J MOL BIOL, 291(3), 1999, pp. 637-650
Thermodynamics and kinetics for cyanide, azide, thiocyanate and imidazole b
inding to recombinant ferric Vitreoscilla sp. homodimeric hemoglobin (Vitre
oscilla Hb) have been determined at PH 6.4 and 7.0, and 20.0 degrees C, in
solution and in the crystalline state. Moreover, the three-dimensional stru
ctures of the diligated thiocyanate and imidazole derivatives of recombinan
t ferric Vitreoscilla Hb have been determined by X-ray crystallography at 1
.8 Angstrom (R-factor = 19.9 %) and 2.1 Angstrom (R-factor = 23.8 %) resolu
tion, respectively. Ferric Vitreoscilla Hb displays an anticooperative liga
nd binding behaviour in solution. This very unusual feature can only be acc
ounted for by assuming ligand-linked conformational changes in the monoliga
ted species, which lead to the observed 300-fold decrease in the affinity o
f cyanide, azide, thiocyanate and imidazole for the monoligated ferric Vitr
eoscilla Hb with respect to that of the fully unligated homodimer. In the c
rystalline state, thermodynamics for azide and imidazole binding to ferric
Vitreoscilla Hb may be described as a simple process with an overall ligand
affinity for the homodimer corresponding to that for diligation in solutio
n. These data suggest that the ligand-free homodimer, observed in the cryst
alline state, is constrained in a low affinity conformation whose ligand bi
nding properties closely resemble those of the monoligated species in solut
ion. From the kinetic viewpoint, anticooperativity is reflected by the 300-
fold decrease of the second-order rate constant for cyanide and imidazole b
inding to the monoligated ferric Vitreoscilla Hb With respect to that for l
igand association to the ligand-free homodimer in solution. On the other ha
nd, values of the first-order rate constant for cyanide and imidazole disso
ciation from the diligated and monoligated derivatives of ferric Vitreoscil
la Hb in solution are closely similar. As a whole, ligand binding and struc
tural properties of ferric Vitreoscilla Hb appear to be unique among all Hb
s investigated to date. (C) 1999 Academic Press.