Direct-detected 13C NMR to investigate the iron(III) hemophore HasA.
Hemophore HasA is a 19 kDa iron(III) hemoprotein that participates in the shuttling of heme to a specific membrane receptor. In HasA, heme iron has an original coordination environment with a His/Tyr pair as axial ligands. Recently developed two-dimensional protonless (13)C-detected experiments provide the sequence-specific assignment of all but three protein residues in the close proximity of the paramagnetic center, thus overcoming limitations due to the short relaxation times induced by the presence of the iron(III) center. Mono-dimensional (13)C and (15)N experiments tailored for the detection of paramagnetic signals allow the identification of resonances of the axial ligands. These experiments are used to characterize the conformational features and the electronic structure of the heme iron(III) environment. The good complementarity among (1)H-, (13)C-, and (15)N-detected experiments is highlighted. A thermal high-spin/low-spin equilibrium is observed and is related to a modulation of the strength of the coordination bond between the iron and the Tyr74 axial ligand. The key role of a neighboring residue, His82, for the stability of the axial coordination and its involvement in the heme delivery to the receptor is discussed.
Caillet-Saguy
Célia
C
Unité de RMN des Biomolécules (CNRS URA 2185), Institut Pasteur, 28 rue du Docteur Roux, 75015 Paris cedex 05, France.
Delepierre
Muriel
M
Lecroisey
Anne
A
Bertini
Ivano
I
Piccioli
Mario
M
Turano
Paola
P
D000818:Animals; D016880:Anisotropy; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002247:Carbon Isotopes; D002352:Carrier Proteins / Q000737:chemistry*; D002417:Cattle; D006418:Heme / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D002352:Carrier Proteins; C093956:HasA protein, Serratia marcescens; D006420:Hemeproteins; D008565:Membrane Proteins; D006418:Heme