title: Spectroscopic identification of heme axial ligands in HtsA that are involved in heme acquisition by Streptococcus pyogenes.
abstract: The heme-binding proteins Shp and HtsA of Streptococcus pyogenes are part of the heme acquisition machinery in which Shp directly transfers its heme to HtsA. Mutagenesis and spectroscopic analyses were performed to identify the heme axial ligands in HtsA and to characterize axial mutants of HtsA. Replacements of the M79 and H229 residues, not the other methionine and histidine residues, with alanine convert UV-vis spectra of HtsA with a low-spin, hexacoordinate heme iron into spectra of high-spin heme complexes. Ferrous M79A and H229A HtsA mutants possess magnetic circular dichroism (MCD) spectra that are similar with those of proteins with pentacoordinate heme iron. Ferric M79A HtsA displays UV-vis, MCD, and resonance Raman (RR) spectra that are typical of a hexacoordinate heme iron with histidine and water ligands. In contrast, ferric H229A HtsA has UV-vis, MCD, and RR spectra that represent a pentacoordinate heme iron complex with a methionine axial ligand. Imidazole readily forms a low-spin hexacoordinate adduct with M79A HtsA with a K(d) of 40.9 muM but not with H229A HtsA, and cyanide binds to M79A and H229A with K(d) of 0.5 and 19.1 microM, respectively. The ferrous mutants rapidly bind CO and form simple CO complexes. These results establish the H229 and M79 residues as the axial ligands of the HtsA heme iron, indicate that the M79 side is more accessible to the solvent than the H229 side of the bound heme in HtsA, and provide unique spectral features for a protein with pentacoordinate, methionine-ligated heme iron. These findings will facilitate elucidation of the molecular mechanism and structural basis for rapid and direct heme transfer from Shp to HtsA.
authors.affiliation: Department of Veterinary Molecular Biology, Montana State University, Bozeman,Montana 59717, USA.
authors.forename: Yanchao
authors.initials: Y
authors.lastname: Ran
authors.forename: Mengyao
authors.initials: M
authors.lastname: Liu
authors.forename: Hui
authors.initials: H
authors.lastname: Zhu
authors.forename: Tyler K
authors.initials: TK
authors.lastname: Nygaard
authors.forename: Doreen E
authors.initials: DE
authors.lastname: Brown
authors.forename: Marian
authors.initials: M
authors.lastname: Fabian
authors.forename: David M
authors.initials: DM
authors.lastname: Dooley
authors.forename: Benfang
authors.initials: B
authors.lastname: Lei
mesh_terms: D001426:Bacterial Proteins; D002248:Carbon Monoxide / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D020125:Mutation, Missense; D013057:Spectrum Analysis; D013297:Streptococcus pyogenes / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D008024:Ligands; D006418:Heme; D002248:Carbon Monoxide