Unusually Fast bis-Histidyl Coordination in a Plant Hemoglobin

Abstract

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 10⁸ M^-1s^-1 range. Binding of the distal histidine to the heme competes with CO rebinding with extremely high rates (k_h ~ 10⁵ s^-1). Histidine dissociation from the heme occurs with comparable rates, thus resulting in moderate equilibrium binding constants (K_H ~ 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.

Keywords: CO rebinding kinetics; Medicago truncatula; iron/heme hexacoordination; plant hemoglobins; ultrafast spectroscopy.

Figure 1

CO rebinding kinetics after nanosecond laser flash photolysis (LFP) of (a) WT1 and (b) WT4 solutions equilibrated with 1 atm CO (solid lines) and 0.2 atm CO (dotted lines) at 10 °C (black), 20 °C (red), 30 °C (green) and 40 °C (blue). Data are reported as the progress curve representing the fraction of deoxy molecules, N(t), as a function of time after photolysis. The absorbance change was monitored at 436 nm, following laser photolysis at 532 nm.

Figure 2

CO rebinding kinetics for WT1 (gray dots) solution equilibrated with 1 atm CO and at room temperature collected with the hybrid femtosecond–nanosecond pump–probe setup. The corresponding kinetics measured after nanosecond LFP is reported as a gray solid line. For comparison, the rebinding kinetics to single and double His_d mutants are reported (orange, 238; magenta, 74; olive, 74/238; see Section 2.4 for definitions).

Scheme 1

Minimal kinetic scheme proposed for the reaction of CO with Glbs. Deoxy (pentacoordinate) and bis-histidyl (hexacoordinate) species are indicated by Glb_P and Glb_H, respectively. Immediately after photodissociation of GlbCO, the ligand is in a primary docking site (Glb:CO) within the distal pocket. From this site, the ligand can be rebound (rate k₋₁), migrate to a nearby temporary docking site (tr)₁ with rate k_c (reverse rate k_−c), or exit to the solvent with rate k_out. Ligands are rebound by Glb_P from the solvent with rate k_in, whereas binding and dissociation of His_d occur with rates k_h and k_−h.

Figure 3

Analysis of the complete course of CO rebinding kinetics to WT1, WT4 and their mutants. The progress curve for the CO rebinding reaction after the hybrid pump–probe experiment is plotted as concentration of deoxy heme versus time. (a) Comparison between WT1 and WT4. The experimental data are superimposed on the fitted curves obtained using Scheme 1. (b,c) Replacement of His_d by Leu affects the time evolution of the CO rebinding kinetics, as demonstrated by the comparison between WT1 and its mutant 74/238 in (b) and between WT4 and its mutant 109 in c. The fitting curves are superimposed on the experimental data (circles). In (b,c), the time courses of the other relevant species are shown using the same color code as for the kinetic model reported in Scheme 1. (d) Comparison between the kinetics of WT1 and its 74, 238 and 74/238 mutants.

Figure 4

Analysis of CO rebinding kinetics to the (a) 238 and (b) 74 mutants at room temperature, 1 atm CO. The fitting curves are superimposed on the experimental data (open circles). The time course of the relevant species shown in the kinetic scheme (Scheme 1) are reported: Glb:CO (violet), (tr)₁ (light blue), Glb_H (red) and Glb_P (green).