Hexacoordination in the algal hemoglobin THB1: A combined computer simulation and experimental study

CAPECE, LUCIANA; JULIÓ PLANA, L.; MARTINEZ GRUNDMAN, J. E.; ESTRIN, DARÍO A.; LECOMTE, JULIETTE T.J.

Conferencia; Oxygen Binding and Sensing Proteins 2022; 2022

Universita Roma Tre

THB1 is a monomeric truncated hemoglobin from the green alga Chlamydomonas reinhardtii. In the absence of exogenous ligands and at neutral pH, the heme group of THB1 is coordinated by two protein residues, Lys53 and His77. THB1 is thought to function as a nitric oxide dioxygenase, and the distal binding of O2 requires the cleavage of the Fe-Lys53 bond accompanied by protonation and expulsion of the lysine from the heme cavity into the solvent. Nuclear magnetic resonance spectroscopy and crystallographic data have provided dynamic and structural insights of the process, but the details of the mechanism were not fully elucidated. We applied a combination of computer simulations and site-directed mutagenesis experiments to shed light on this issue. Molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations were performed to explore the nature of the transition between the decoordinated and lysine-bound states of the ferrous heme in THB1. Lys49 and Arg52, which form ionic interactions with the heme propionates in the X-ray structure of lysine-bound THB1, were observed to assist in maintaining Lys53 inside the protein cavity and play a key role in the transition. Lys49Ala, Arg52Ala and Lys49Ala/Arg52Ala THB1 variants were prepared, and the consequences of the replacements on the Lys (de)coordination equilibrium were characterized experimentally. Additionally, the "Lys-off" X-ray structure, represented by the cyanide adduct of the Fe(III) protein, allowed to hypothesize that interactions that differ between the known "Lys-on" structure and the Lys-off structure participate in the control of Lys53 affinity for the heme iron. The results reinforced the dynamic role of protein-propionate interactions and strongly suggested that cleavage of the Fe-Lys53 bond and ensuing conformational rearrangement is facilitated by protonation of the amino group inside the distal cavity.