Thermodynamics of heme binding to the HasA(SM) hemophore: effect of mutations at three key residues for heme uptake.
HasA(SM) secreted by the Gram-negative bacterium Serratia marcescens belongs to the hemophore family. Its role is to take up heme from host heme carriers and to shuttle it to specific receptors. Heme is linked to the HasA(SM) protein by an unusual axial ligand pair: His32 and Tyr75. The nucleophilic nature of the tyrosine is enhanced by the hydrogen bonding of the tyrosinate to a neighboring histidine in the binding site: His83. We used isothermal titration microcalorimetry to examine the thermodynamics of heme binding to HasA(SM) and showed that binding is strongly exothermic and enthalpy driven: DeltaH = -105.4 kJ x mol(-1) and TDeltaS = -44.3 kJ x mol(-1). We used displacement experiments to determine the affinity constant of HasA(SM) for heme (K(a) = 5.3 x 10(10) M(-1)). This is the first time that this has been reported for a hemophore. We also analyzed the thermodynamics of the interaction between heme and a panel of single, double, and triple mutants of the two axial ligands His32 and Tyr75 and of His83 to assess the implication of each of these three residues in heme binding. We demonstrated that, in contrast to His32, His83 is essential for the binding of heme to HasA(SM), even though it is not directly coordinated to iron, and that the Tyr75/His83 pair plays a key role in the interaction.
Deniau
Clarisse
C
Unité de Résonance Magnétique Nucléaire des Biomolécules, CNRS URA 2185, Institut Pasteur, Paris, France.
Gilli
Robert
R
Izadi-Pruneyre
Nadia
N
Létoffé
Sylvie
S
Delepierre
Muriel
M
Wandersman
Cécile
C
Briand
Claudette
C
Lecroisey
Anne
A
D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002417:Cattle; D004926:Escherichia coli / Q000378:metabolism; D006427:Hemin / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D013816:Thermodynamics; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; C093956:HasA protein, Serratia marcescens; D008565:Membrane Proteins; D014443:Tyrosine; D006639:Histidine; D006427:Hemin