Abstract
The surface protein Shp of Streptococcus pyogenes rapidly transfers its hemin to HtsA, the lipoprotein component of the HtsABC transporter, in a concerted two-step process with one kinetic phase. The structural basis and molecular mechanism of this hemin transfer have been explored by mutagenesis and truncation of Shp. The heme-binding domain of Shp is in the amino-terminal region and is functionally active by itself, although inclusion of the COOH-terminal domain speeds up the process approximately 10-fold. Single alanine replacements of the axial methionine 66 and 153 ligands (Shp(M66A) and Shp(M153A)) cause formation of pentacoordinate hemin-Met complexes. The association equilibrium constants for hemin binding to wild-type, M66A, and M153A Shp are 5,300, 22,000, and 38 microM(-1), respectively, showing that the Met(153)-Fe bond is critical for high affinity binding and that Met(66) destabilizes hemin binding to facilitate its rapid transfer. Shp(M66A) and Shp(M153A) rapidly bind to hemin-free HtsA (apoHtsA), forming stable transfer intermediates. These intermediates appear to be Shp-hemin-HtsA complexes with one axial ligand from each protein and decay to the products with rate constants of 0.4-3 s(-1). Thus, the M66A and M153A replacements alter the kinetic mechanism and unexpectedly slow down hemin transfer by stabilizing the intermediates. These results, in combination with the structure of the Shp heme-binding domain, allow us to propose a "plug-in" mechanism in which side chains from apoHtsA are inserted into the axial positions of hemin in Shp to extract it from the surface protein and pull it into the transporter active site.
Highlights
The Fe(II)-protoporphyrin IX complex or Fe(III)protoporphyrin IX complex exchange from one protein to another has been demonstrated biochemically in only a few bacterial systems, including transfers from hemoglobin to Serratia marcescens hemophore HasA (10); from the cell surface protein Shp to HtsA, the lipoprotein component of the HtsABC transporter, in Streptococcus pyogenes and Streptococcus equi (11, 12); from HasA to its outer membrane receptor HasR (10); and from hemoglobin to Shigella dysenteriae outer membrane receptor ShuA (5)
A detailed kinetic mechanism has only been proposed for the S. pyogenes Shp/HtsA system (13)
In order to gain insight into the structural mechanism of rapid hemin transfer from Shp to HtsA, we examined these processes for Shp mutants containing only the NH2-terminal heme-binding domain or full-length Shp in which the Met axial ligands were replaced with alanine (Ala) or histidine (His)
Summary
Protein Preparation—The preparation of the heme-binding domain of Shp containing amino acids 30 –180 (designated Shp180) has been described elsewhere. Ala and His replacement mutants of Met and Met153 of Shp (designated ShpM66A, ShpM153A, ShpM66H, and ShpM153H) were generated by site-directed mutagenesis using the Stratagene QuikChange site-directed mutagenesis kit. The sample was loaded onto a DEAE column (2.5 ϫ 10 cm), and the column was washed with 100 ml of Tris-HCl and eluted with a 100-ml linear gradient of 0 – 0.25 M NaCl. Each protein was dialyzed against 3 liters of 10 mM acetate buffer, pH 5.5, and loaded onto an SPSepharose column (1.5 ϫ 6 cm). HoloShpM153H was prepared by incubating purified apoShpM153H with excess hemin, loading the mixture onto a Sephadex G-25 column (1 ϫ 20 cm), and eluting with Tris-HCl. ApoShp and mutant proteins were prepared from inclusion bodies as described above except that hemin was absent in the refolding step. Kinetic Analyses—A stopped-flow spectrophotometer equipped with a photodiode array detector (SX20; Applied Photophysics) was used to measure the rates of hemin transfer from Shp mutants to apoHtsA and the binding of hemin to apoShp mutants. Changes in absorbance at appropriate wavelengths were fitted to a single or double exponential expression, yielding pseudo-first-order rate constants for each reaction step for further analysis as described under “Results.”
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