Metmyoglobin/azide: the effect of heme-linked ionizations on the rate of complex formation.

The research studied the formation and dissociation rate of horse metmyoglobin/azide complex under various experimental conditions, revealing the impact of heme-linked ionizations. It also elucidated the roles of specific protective groups in the process and the effects of differing ionic strengths.

Study of complex formation and dissociation

• The investigation involved studying the kinetics – or rates – of formation and dissociation of the metmyoglobin/azide complex derived from horses, ranging from a pH of 3.5 to 11.5.
• They observed the rate constant for the binding of hydrazoic acid (HN3) to metmyoglobin is 3.8 +/- 1.0 x 10(5) M-1 s-1.
• Upon protonation of a group (with an apparent pKa of 4.0 +/- 0.3), the rate of HN3 binding increased 6.5-fold, suggesting that a more acidic environment enhances the complex formation.

Role of distal histidine and ionizable groups

• The researchers attribute the ionizable group that facilitates the HN3 binding to the distal histidine, specifically histidine-64.
• The binding of the azide anion (N-3) to metmyoglobin has a slower rate constant, approximately 100 times less than that of HN3.
• When aquometmyoglobin is converted to hydroxymetmyoglobin, the binding process for azide slows down significantly. In fact, HN3 binding to hydroxymetmyoglobin was not detected in the experiment.

Effects of protonation and ionic strength

• Protonation – or the addition of a proton – of the distal histidine eases the dissociation of HN3 from the complex.
• The dissociation rate constant of the HN3 from the complex was found to be significantly faster than that of the azide anion.
• Research demonstrated that the observed association rate constant is affected by both primary and secondary kinetic salt effects, revealing the intricate relationship between ionic strength and the rate of complex formation and dissociation.
• An increased ionic strength beyond metmyoglobin’s isoelectric point – a pH where the overall charge of the protein is neutral – showed a decrease in the rate of N-3 binding. This primary kinetic salt effect is described as “anomalous”.
• The dissociation rate constant’s ionic strength dependence could be accounted for solely by the changes in the acid dissociation constant for His-64 in the complex.