Kinetic evidence for the existence of a rate-limiting step in the reaction of ferric hemoproteins with anionic ligands

The kinetics of azide and fluoride binding to various monomeric and tetrameric ferric hemoproteins (sperm whale Mb, isolated alpha and beta chains of human Hb reacted with p-chloromercuribenzoate, dromedary, ox and human Hb) has been investigated (at pH 6.5 and 20 degrees C) over a large range (20 mu M to 2 M) of ligand concentration. It has been observed that the pseudo-first-order rate constant for azide binding to the hemoproteins investigated does not increase linearly with ligand concentration, but tends to level off toward an asymptotic concentration-independent value typical for each hemoprotein. This behaviour, which has been detected only by an investigation covering an unusually large range of ligand concentrations, appears to be independent of the ionic strength, and it underlies the existence of a rate-limiting step in the dynamic pathway of azide binding to ferric hemoproteins, which is detectable whenever the observed pseudo-first-order rate constant becomes faster than a given value characteristic of the specific hemoprotein. Such a behaviour is not observed in the case of fluoride binding probably because the pseudo-first-order rate constant for this ligand is much slower and never attains a value faster than that of the rate-limiting step. In general terms, this feature should involve a conformational equilibrium between at least two forms (possibly related to the interaction of H2O with distal histidine and its exchange with the bulk solvent) which modulates the access of the anionic ligand into the heme pocket and its reaction with the ferric heme iron.