Nygaard Tyler K, Blouin George C, Liu Mengyao, Fukumura Maki, Olson John S, Fabian Marian, Dooley David M, Lei Benfang
Departments of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59718.
Department of Biochemistry and Cell Biology and the W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005.
J Biol Chem. 2006 Jul 28;281(30):20761-20771. doi: 10.1074/jbc.M601832200. Epub 2006 May 22.
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.
血红素结合蛋白Shp和HtsA是化脓性链球菌中血红素获取机制的一部分。六配位血红素（Fe(II)-原卟啉IX）或全Shp（血红素Shp）的高铁血红素形式在pH 8.0的Tris-HCl缓冲液中在空气中稳定，以120±18 μM的解离常数（K(d)）与脱辅基HtsA结合，并在25℃、pH 8.0时以28±6 s⁻¹的速率常数将其血红素转移至脱辅基HtsA。然后，血红素HtsA产物自动氧化为六配位高铁血红素（Fe(III)-原卟啉IX）或高铁血红素形式（高铁血红素HtsA），表观速率常数为0.017±0.002 s⁻¹。高铁血红素Shp也通过高铁血红素Shp.脱辅基HtsA复合物（K(d)=48±7 μM）快速将高铁血红素转移至脱辅基HtsA，其速率比高铁血红素从高铁血红素Shp简单解离进入溶剂的速率大约高40000倍（转移速率常数k(transfer)=43±3 s⁻¹，而高铁血红素解离速率常数k(-hemin)=0.0003±0.00006 s⁻¹）。高铁血红素与HtsA结合和解离的速率常数（k'(hemin)约为80 μM⁻¹ s⁻¹，k(-hemin)=0.0026±0.0002 s⁻¹）分别比Shp相应的速率常数大50倍和10倍（k(hemin)约为1.6 μM⁻¹ s⁻¹，k(-hemin)=0.0003 s⁻¹），这意味着HtsA具有更易接近的活性位点。然而，脱辅基HtsA对高铁血红素的亲和力（k(hemin)约为31000 μM⁻¹）比脱辅基Shp对高铁血红素的亲和力（k(hemin)约为5300 μM⁻¹）大约高5倍，这解释了从Shp到HstA的净转移。这些结果支持了一种从细胞表面受体Shp到ATP结合盒转运体系统的血红素和高铁血红素直接、快速且由亲和力驱动的转移机制。
