Purification and Characterization of an Extracellular Heme-Binding Protein, HasA, Involved in Heme Iron Acquisition

Abstract

Many bacterial hemoproteins involved in heme acquisition have been isolated recently, comprising outer membrane receptors and extracellular heme-binding protein. The mechanisms by which these proteins extract heme have not been described up to now. One such protein, HasA, which can bind free heme as well as capture it from hemoglobin, is secreted by the Gram-negative bacteria Serratia marcescens under iron deficiency conditions. The fact that HasA does not present sequence similarities with other known hemoproteins suggests that it possesses a new type of heme binding site. This work describes the main physicochemical properties of HasA, essential for understanding its function. HasA is a monomer of 19 kDa that binds one b heme per molecule with high affinity. The electron paramagnetic resonance spectra indicate that the heme iron is in a low-spin ferric state and that the two iron axial ligands are His and His-. The low oxidation-reduction potential value (-550 mV vs standard hydrogen electrode) of the heme bound to HasA suggests that heme could be exposed to the solvent. According to circular dichroism data, the binding of heme does not seem to modify the conformation of HasA.

Deciphering the Structural Role of Histidine 83 for Heme Binding in Hemophore HasA

“…Uniformly 15 N-and 15 N/ 13 C-labeled proteins were produced at 303 K in M9 minimal medium containing 15 NH 4 Cl and 13 C-glycerol as the sole nitrogen and carbon sources and were purified as described previously (9,21). The purity of the proteins was checked by SDS-PAGE.…”

“…marcescens wild-type HasA (HasA WT ) is a 19-kDa monomeric protein that binds b-heme with a very high affinity (K d ϭ 1.9 ϫ 10 Ϫ11 M) and a 1:1 stoichiometry (8,9). In the heme loaded hemophore holoHasA WT , the iron ion is in the oxidized form with the lowest redox potential (Ϫ550 mV) reported so far for heme-binding proteins (9). The x-ray structure of holoHasA WT shows an unprecedented heme-binding site (10).…”

The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125

“…The band corresponding to g 2 of form 3 is likely hidden by the bands of the other two species at g * 2.2 or by the spurious band. The g tensor anisotropy of these LS forms is typical of N and O ligation to the heme (Table 3) [5,20,[35][36][37][38][39][40][41]. The Blumberg-Peisach diagram [36] can provide some empirical guidance to obtain an indication of possible LS heme iron axial ligands, although it should be kept in mind that a number of pitfalls have been found in the use of this diagram, and it should not be used as the only criterion for ligand assignment [42].…”

“…A similar assignment to the H group of the Blumberg-Peisach diagram was also reported for the HasA proteins from S. marcescens and P. aeruginosa at neutral pH (g = 2.86, 2.21, 1.71), which are characterized by HisFe-Tyr axial coordination. It is noted that the original EPR study of S. marcescens HasA, prior to the determination of the crystal structure, mistakenly assigned a bis-His axial heme coordination [39,40]. Furthermore, the rhombicity and tetragonality crystal parameters determined from the g values of Chlamydomonas Hb, which is assigned to HisFe-Tyr axial coordination, place it in the H structural group [20].…”

The Crystal Structure of the Secreted Dimeric Form of the Hemophore HasA Reveals a Domain Swapping with an Exchanged Heme Ligand

“…It is secreted under iron deficiency condition by an ABC-type pathway via a C-terminal secretion signal. This 19kDa protein binds one b heme molecule with an affinity of 5.3 10 10 M -1 (2,3). HasA is able to bind free heme or to capture it from other hemoproteins, like hemoglobin.…”