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Hemoglobin: Kinetics of ligand binding and conformational change
Abstract
Functional properties of two very unusual invertebrate hemoglobins, Artemia salina, and Paramphistomum epiclitum (P.E.), have been investigated through their ligand binding kinetics and equilibria. The conformational change of dimeric Artemia hemoglobin ($\alpha\beta,$ 9 heme domains per chain) is best described by a three-state model, $\rm R\rightarrow S\rightarrow T,$ revealed from CO binding kinetics. The rate constant for $\rm R\rightarrow S$ was too fast to be measured, whereas $\rm S\rightarrow T$ is very slow, 3.5 sec$\sp{-1},$ about 10,000 times slower than the $\rm R\rightarrow T$ change for human hemoglobin. The nanosecond geminate reactions for O$\sb2$ and NO are both biphasic; for CO the reaction was not detectable. In terms of the MWC allosteric model for oxygen binding, c = 0.046, L = 5.9 x 10$\sp5,$ and n = 6 sites. P.E. hemoglobin has extraordinarily high affinity for oxygen, K$\rm\sb{d}=0.5$ nM, and a low CO/O$\sb2$ partition constant (0.062) at $20\sp\circ$C. The protein appears to have very different conformations for bound CO and O$\sb2.$ Oxygen binding is favored over CO as the temperature decreases. The effects of the 2,4 vinyl groups on the heme periphery of horse Hb for geminate CO recombination and for the kinetics of the $\rm R\rightarrow T$ conformational change were compared with corresponding kinetics for horse deutero-, meso- and dibromohemoglobins. In the nanosecond time regime, the immediate CO recombination rate constant, k$\sb2,$ for protoHb is much smaller than for the modified Hbs, but there were no large differences among the other rate constants. For each Hb, Arrhenius parameters were used in a global fitting for four rate constants to data at five different temperatures. The observed $\rm R\rightarrow T$ conformational change in the native and modified horse Hbs was very well simulated by an allosteric model together with linear free energy relations for the $\rm R\ \sbsp{\to}{\gets}\ T$ equilibria. The modifications on the heme decrease the energy barrier for $\rm R\rightarrow T$ since protoHb has the highest activation energy. Except for protoHb, the transition state equilibrium constant, K$\sp\ddagger,$ correlates well with the sizes of the substituents. The $\rm R\rightarrow T$ transition states of the modified Hbs appear very nearly R-like.
Subject Area
Biophysics
Recommended Citation
Gu, YiRen, "Hemoglobin: Kinetics of ligand binding and conformational change" (1995). ETD collection for University of Nebraska-Lincoln. AAI9611052.
https://digitalcommons.unl.edu/dissertations/AAI9611052