Role of Ionic Strength and the Bohr Effect in Modulating Thermodynamic Profiles Associated with Co Escape in Rice Non-Symbiotic Hemoglobin 1

Abstract

Rice hemoglobin (rHb1) is a type 1 non-symbiotic hemoglobin (nsHb1), which is found in plants and which is evolutionarily related to leghemoglobins and truncated hemoglobins. It is structurally similar to vertebrate heme proteins, containing Fe-protoporphyrin IX and the classic 3-over-3 globin fold. Unlike other hexacoordinate heme proteins found in plants, the ligand binding properties of nsHb1 proteins make it unlikely to be involved in nitrogen fixation or oxygen transport. We have characterized ligand migration upon CO photorelease from rHb1 under various conditions in order to elucidate functional role(s) of rHb1 in vivo. Using photoacoustic calorimetry, we have determined thermodynamic parameters (ΔH and ΔV) for CO photorelease from wild type rHb1 and a distal histidine to leucine (H74L) mutant taking place within ∼ 5 µs after ligand photo-dissociation. Under stripped conditions, we observe a strong temperature dependence of the observed parameters. Photorelease of CO from the wild type protein below 16°C is associated with ΔH = 28.7 ± 2.2 kcal mol−1 and ΔV = 4.3 ± 0.4 mL mol−1, while above 16°C we observe ΔH = 9.7 ± 3.4 kcal mol−1 and ΔV = 0.4 ± 0.7 mL mol−1. This temperature dependence is not observed in the H74L mutant, in the presence of 500 mM NaCl, or at pH 6. We have also determined rate constants and quantum yields for bimolecular rebinding of CO using transient absorption spectroscopy, both of which increase with temperature. The contributions of electrostriction, proton uptake by Bohr groups, oligomerization state, and the distal residue of rHb1 to the observed kinetic and thermodynamic parameters will be discussed. We will also discuss ligand migration pathways and protein dynamics upon CO photorelease in rHb1.