Axial ligand replacement mechanism in heme transfer from streptococcal heme-binding protein Shp to HtsA of the HtsABC transporter.

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 (Shp(M66A), Shp(M153A), HtsA(M79A), and HtsA(H229A)) were used to probe the axial displacement mechanism of this heme transfer reaction. Ferric Shp(M66A) at high pH and Shp(M153A) have a pentacoordinate heme iron complex with a methionine axial ligand. ApoHtsA(M79A) 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, apoHtsA(H229A) cannot assimilate heme from ferric Shp. The conversion of pentacoordinate holoShp(M66A) into pentacoordinate holoHtsA(H229A) involves an intermediate, whereas holoHtsA(H229A) is directly formed from pentacoordinate holoShp(M153A). Conversely, apoHtsA(M79A) reacts with holoShp(M66A) and holoShp(M153A) 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.