Monomer-dimer equilibrium and oxygen binding properties of ferrous Vitreoscilla hemoglobin

The monomer-dimer equilibrium and the oxygen binding properties of ferrous recombinant Vitreoscilla hemoglobin (Vitreoscilla Hb) have been investigate d. Sedimentation equilibrium data indicate that the ferrous deoxygenated an d carbonylated derivatives display low values of equilibrium dimerization c onstants, 6 x 10(2) and 1 x 10(2) M-1, respectively, at pH 7.0 and 10 degre esC. The behavior of the oxygenated species, as measured in sedimentation v elocity experiments, is superimposable to that of the carbonylated derivati ve. The kinetics of OZ combination, measured by laser photolysis at pH 7.0 and 20 degreesC, is characterized by a second-order rate constant of 2 x 10 (8) M-1 s(-1) whereas the kinetics of O-2 release at pH 7.0 is biphasic bet ween 10 and 40 degreesC, becoming essentially monophasic below 10 degreesC. Values of the first-order rate constants (at 20 degreesC) and of the activ ation energies for the fast and slow phases of the Vitreoscilla Hb deoxygen ation process are 4.2 s(-1) and 19.2 kcal mol(-1) and 0.15 s(-1) and 24.8 k cal mol(-1), respectively. Thus the biphasic kinetics of Vitreoscilla Hb de oxygenation is unrelated to the association state of the protein. The obser ved biphasic oxygen release may be accounted for by the presence of two dif ferent conformers in thermal equilibrium within the monomer. The two confor mers may be assigned to a structure in which the heme-iron-bound ligand is stabilized by direct hydrogen bonding to TyrB 10 and a structure in which s uch interaction is absent. The slow interconversion between the two conform ers may reflect a very large conformational rearrangement in the disordered distal pocket segment connecting helices degreesC and E.