Axial Ligand Replacement Mechanism in Heme Transfer from Streptococcal Heme-Binding Protein Shp to HtsA of the HtsABC Transporter

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The heme-binding protein Shp of Group A Streptococcus rapidly transfers its heme to HtsA, the lipoprotein component of the HtsABC transporter, in a concerted two-step process with one kinetic phase. Heme axial residue-to-alanine replacement mutant proteins of Shp and HtsA (ShpM66A, ShpM153A, HtsAM79A, and HtsAH229A) were used to probe the axial displacement mechanism of this heme transfer reaction. Ferric ShpM66A at high pH and ShpM153A have a pentacoordinate heme iron complex with a methionine axial ligand. ApoHtsAM79A efficiently acquires heme from ferric Shp but alters the reaction mechanism to two kinetic phases from a single phase in the wild-type protein reactions. In contrast, apoHtsAH229A cannot assimilate heme from ferric Shp. The conversion of pentacoordinate holoShpM66A into pentacoordinate holoHtsAH229A involves an intermediate, whereas holoHtsAH229A is directly formed from pentacoordinate holoShpM153A. Conversely, apoHtsAM79A reacts with holoShpM66A and holoShpM153A in mechanisms with one and two kinetic phases, respectively. These results imply that the Met79 and His229 residues of HtsA displace the Met66 and Met153 residues of Shp, respectively. Structural docking analysis supports this mechanism of the specific axial residue displacement. Furthermore, the rates of the cleavage of the axial bond in Shp in the presence of a replacing HtsA axial residue are greater than that in the absence of a replacing HtsA axial residue. These findings reveal a novel heme transfer mechanism of the specific displacement of the Shp axial residues with the HtsA axial residues and the involvement of the HtsA axial residues in the displacement.




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