title: The mechanism of direct heme transfer from the streptococcal cell surface protein Shp to HtsA of the HtsABC transporter.
abstract: The heme-binding proteins Shp and HtsA are part of the heme acquisition machinery found in Streptococcus pyogenes. The hexacoordinate heme (Fe(II)-protoporphyrin IX) or hemochrome form of holoShp (hemoShp) is stable in air in Tris-HCl buffer, pH 8.0, binds to apoHtsA with a K(d) of 120 +/- 18 microm, and transfers its heme to apoHtsA with a rate constant of 28 +/- 6s(-1) at 25 degrees C, pH 8.0. The hemoHtsA product then autoxidizes to the hexacoordinate hemin (Fe(III)-protoporphyrin IX) or hemichrome form (hemiHtsA) with an apparent rate constant of 0.017 +/- 0.002 s(-1). HemiShp also rapidly transfers hemin to apoHtsA through a hemiShp.apoHtsA complex (K(d) = 48 +/- 7 microM) at a rate approximately 40,000 times greater than the rate of simple hemin dissociation from hemiShp into solvent (k(transfer) = 43 +/- 3s(-1) versus k(-hemin) = 0.0003 +/- 0.00006 s(-1)). The rate constants for hemin binding to and dissociation from HtsA (k'(hemin) approximately 80 microm(-1) s(-1), k(-hemin) = 0.0026 +/- 0.0002 s(-1)) are 50- and 10-fold greater than the corresponding rate constants for Shp (k(hemin) approximately 1.6 microM(-1) s(-1), k(-hemin) = 0.0003 s(-1)), which implies that HtsA has a more accessible active site. However, the affinity of apoHtsA for hemin (k(hemin) approximately 31,000 microm(-1)) is roughly 5-fold greater than that of apoShp (k(hemin) approximately 5,300 microM(-1)), accounting for the net transfer from Shp to HstA. These results support a direct, rapid, and affinity-driven mechanism of heme and hemin transfer from the cell surface receptor Shp to the ATP-binding cassette transporter system.
authors.affiliation: Departments of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59718.
authors.forename: Tyler K
authors.initials: TK
authors.lastname: Nygaard
authors.affiliation: Department of Biochemistry and Cell Biology and the W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005.
authors.forename: George C
authors.initials: GC
authors.lastname: Blouin
authors.affiliation: Departments of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59718.
authors.forename: Mengyao
authors.initials: M
authors.lastname: Liu
authors.affiliation: Departments of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59718.
authors.forename: Maki
authors.initials: M
authors.lastname: Fukumura
authors.affiliation: Department of Biochemistry and Cell Biology and the W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005.
authors.forename: John S
authors.initials: JS
authors.lastname: Olson
authors.affiliation: Department of Biochemistry and Cell Biology and the W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005.
authors.forename: Marian
authors.initials: M
authors.lastname: Fabian
authors.affiliation: Departments of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59718.
authors.forename: David M
authors.initials: DM
authors.lastname: Dooley
authors.affiliation: Departments of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59718. Electronic address: blei@montana.edu.
authors.forename: Benfang
authors.initials: B
authors.lastname: Lei
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000737:chemistry*; D006420:Hemeproteins / Q000235:genetics / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry; D008956:Models, Chemical; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013297:Streptococcus pyogenes / Q000378:metabolism*
chemical_list: D006420:Hemeproteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D006418:Heme; D000255:Adenosine Triphosphate; D010100:Oxygen