The mechanism of direct heme transfer from the streptococcal cell surface protein shp to HtsA of the HtsABC transporter

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 mu M, and transfers its heme to apoHtsA with a rate constant of 28 +/- 6 s(-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 mu M) at a rate 40,000 times greater than the rate of simple hemin dissociation from hemiShp into solvent ( k(transfer) = 43 +/- 3 s(-1) versus k (-) (hemin) = 0.0003 +/- 0.00006 s(-1)). The rate constants for hemin binding to and dissociation from HtsA ( k'(hemin) approximate to 80 mu M-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) approximate to 1.6 mu M-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 approximate to 31,000 mu M-1) is roughly 5-fold greater than that of apoShp ( K-hemin approximate to 5,300 mu M-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.