Distal lysine (de)coordination in the algal hemoglobin THB1: A combined computer simulation and experimental study

• Published in: Journal of inorganic biochemistry 2021-07, Vol.220, p.111455, Article 111455
• Main Authors: Julió Plana, Laia, Martinez Grundman, Jaime E., Estrin, Darío A., Lecomte, Juliette T.J., Capece, Luciana
• Format: Article
• Language: English
• Subjects: Algal Proteins - chemistry; Algal Proteins - genetics; Algal Proteins - metabolism; Chlamydomonas reinhardtii - chemistry; Density Functional Theory; Hexacoordination; Iron - chemistry; Iron - metabolism; Lysine - chemistry; Lysine - metabolism; Lysine ionization; Models, Chemical; Molecular dynamics; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Mutation; Protein Binding; Protein Conformation; QM/MM calculation; THB1; Truncated hemoglobins; Truncated Hemoglobins - chemistry; Truncated Hemoglobins - genetics; Truncated Hemoglobins - metabolism
• ISSN: 0162-0134; 1873-3344
• DOI: 10.1016/j.jinorgbio.2021.111455

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 have not been 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 restrained optimizations 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 for comparison with computational prediction. 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. Molecular dynamics simulations, hybrid quantum mechanics/molecular mechanics calculations, and site directed mutagenesis were used to study the influence of propionate interactions and distal lysine protonation in the transition from hexacoordinate to pentacoordinate heme in hemoglobin THB1 from Chlamydomonas reinhardtii. Protonation appears to be energetically favored inside the heme cavity. [Display omitted] •The heme iron in the green alga hemoglobin THB1 has a lysine distal ligand.•Critical heme-protein interactions were disrupted with alanine replacements.•Lys decoordination at neutral pH was explored by computational methods.•pH titrations probed Lys affinity for the heme in the Fe(II) and Fe(III) states.•Protonation of the Lys NH2 is a key step for conformational rearrangeme