Functional and structural roles of the N-terminal extension in Methanosarcina acetivorans protoglobin

Abstract

Functional and structural properties of protoglobin from Methanosarcina acetivorans, whose Cys(101)E20 residue was mutated to Ser (MaPgb*), and of mutants missing either the first 20 N-terminal amino acids (MaPgb*-ΔN20 mutant), or the first 33 N-terminal amino acids [N-terminal loop of 20 amino acids and a 13-residue Z-helix, preceding the globin fold A-helix; (MaPgb*-ΔN20Z mutant)] have been investigated. In keeping with the MaPgb*-ΔN20 mutant crystal structure, here reported at 2.0Å resolution, which shows an increased exposure of the haem propionates to the solvent, the analysis of ligand binding kinetics highlights high accessibility of ligands to the haem pocket in ferric MaPgb*-ΔN20. CO binding to ferrous MaPgb*-ΔN20 displays a marked biphasic behavior, with a fast binding process close to that observed in MaPgb* and a slow carbonylation process, characterized by a rate-limiting step. Conversely, removal of the first 33 residues induces a substantial perturbation of the overall MaPgb* structure, with loss of α-helical content and potential partial collapse of the protein chain. As such, ligand binding kinetics are characterized by very slow rates that are independent of ligand concentration, this being indicative of a high energy barrier for ligand access to the haem, possibly due to localized misfolding. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Keywords: 1,3-bis(tris(hydroxymethyl)methylamino)propane; BIS–TRIS propane; CD; GCS; Hb; Ligand binding property; MaPgb; MaPgb*; MaPgb*-Fe(II)-CO; MaPgb*-Fe(II)-O(2); MaPgb*-Fe(III)-cyanide; MaPgb*-Fe(ΙΙ); MaPgb*-Fe(ΙΙΙ); MaPgb*-ΔN20; MaPgb*-ΔN20-Fe(II); MaPgb*-ΔN20-Fe(II)-CO; MaPgb*-ΔN20-Fe(III); MaPgb*-ΔN20-Fe(III)-cyanide; MaPgb*-ΔN20Z; MaPgb*-ΔN20Z-Fe(II); MaPgb*-ΔN20Z-Fe(II)-CO; MaPgb*-ΔN20Z-Fe(III); MaPgb*-ΔΝ20-Fe(ΙΙΙ)-formate; Mb; Methanosarcina acetivorans protoglobin; Methanosarcina acetivorans protoglobin whose 33 N-terminal amino acids have been removed; Methanosarcina acetivorans protoglobin whose Cys(101)E20 is replaced by Ser; Methanosarcina acetivorans protoglobin whose first 20 amino acids at the N-terminal have been deleted; N-terminal trimming; Pgb; Spectroscopic property; Three-dimensional structure; carbonylated MaPgb*-Fe(II); carbonylated MaPgb*-ΔN20-Fe(II); carbonylated MaPgb*-ΔN20Z-Fe(II); circular dichroism; cyanide-bound MaPgb*-Fe(III); cyanide-bound MaPgb*-ΔN20-Fe(III); ferric MaPgb*; ferric MaPgb*-ΔN20; ferric MaPgb*-ΔN20Z; ferrous MaPgb*; ferrous MaPgb*-ΔN20; ferrous MaPgb*-ΔN20Z; formate-bound MaPgb*-ΔN20-Fe(ΙΙΙ); globin coupled sensors; hemoglobin; myoglobin; oxygenated MaPgb*-Fe(II); protoglobin.