Unusually Fast bis-Histidyl Coordination in a Plant Hemoglobin

13 Pags.- 4 Figs.- 2 Tabls- 1 Suppl File. © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license. ; 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 (kh ~ 105 s−1). Histidine dissociation from the heme occurs with comparable rates, thus resulting in moderate equilibrium binding constants (KH ~ 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. ; This research was funded by the Spanish Agencia Estatal de Investigación (AEI) grant AGL2017-85775-R, co-funded by the European Regional Development Fund, and by Government of Aragón (group A09_17R). The APC was funded by AEI and, in part, by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI-CSIC). G.C. acknowledges support from the ...