Unusually Fast bis-Histidyl Coordination in a Plant Hemoglobin

The recently identified nonsymbiotic hemoglobin gene MtGlb1-2 of the legume Medicago truncatula possesses unique properties as it generates four alternative splice forms encoding proteins with one or two heme domains. Here we investigate the ligand binding kinetics of MtGlb1-2.1 and MtGlb1-2.4, bearing two hemes and one heme, respectively. Unexpectedly, the overall time-course of ligand rebinding was unusually fast. Thus, we complemented nanosecond laser flash photolysis kinetics with data collected with a hybrid femtosecond-nanosecond pump-probe setup. Most photodissociated ligands are rebound geminately within a few nanoseconds, which leads to rates of the bimolecular rebinding to pentacoordinate species in the 10(8) M(-1)s(-1) range. Binding of the distal histidine to the heme competes with CO rebinding with extremely high rates (k(h) similar to 10(5) s(-1)). Histidine dissociation from the heme occurs with comparable rates, thus resulting in moderate equilibrium binding constants (K-H similar to 1). The rate constants for ligation and deligation of distal histidine to the heme are the highest reported for any plant or vertebrate globin. The combination of microscopic rates results in unusually high overall ligand binding rate constants, a fact that contributes to explaining at the mechanistic level the extremely high reactivity of these proteins toward the physiological ligands oxygen, nitric oxide and nitrite.

Automated summary

The MtGlb1-2 gene produces four different protein splice forms, with very fast ligand binding rates and high reactivity. The hemoglobin encoded by this gene has remarkably high rates of binding to oxygen, nitric oxide, and nitrite.