Cloning and characterization of a novel periplasmic heme-transport protein from the human pathogen Pseudomonas aeruginosa.
Successful iron acquisition plays a crucial role in bacterial virulence. Numerous Gram-negative pathogenic bacteria have developed a novel heme-acquisition system to steal iron from hosts. This system involves a cell-surface heme receptor, a periplasmic heme-transport protein (HTP) and inner-membrane proteins typical for ATP binding cassette transporters. We have cloned the gene encoding a periplasmic HTP from Pseudomonas aeruginosa, overexpressed it in Escherichia coli and purified it as a 33-kDa His-tagged protein. Heme-staining and heme-content assays reveal that the isolated HTP contains approximately 50% heme-bound and apo forms. The heme is noncovalently attached and can be transferred to apomyoglobin in vitro. Electron paramagnetic resonance and UV-vis spectroscopies indicate a five-coordinate, high-spin, ferric heme in HTP. HTP is reduced by dithionite but not by either dithiothreitol or ascorbate. Fluorescence and circular dichroism spectroscopies indicate a well-ordered structure for the HTP and a conformational change upon heme binding to apo-HTP. This was confirmed by limited proteolysis assays. Apo-HTP binds heme or protoporphyrin IX at 1:1 ratio with high affinity (K (d) approximately 1.2 and 14 nM, respectively). A BLASTP search revealed approximately 52 putative bacterial periplasmic heme transporters, which can be grouped into six classes, most of which are associated with pathogenic bacteria. Multiple sequence alignment reveals that these HTPs share low sequence similarity and no conserved common binding motif for heme ligation. However, a tyrosine residue (Y71) is highly conserved in the HTP sequences, which is likely an axial heme ligand in HTPs. Mutagenesis studies support Y71-heme iron ligation in the recombinant HTP.
Tong
Yong
Y
Department of Chemistry and Biochemistry, University of Massachusetts, Dartmouth, MA 02747-2300, USA.
Guo
Maolin
M
D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D006418:Heme / Q000378:metabolism*; D006801:Humans; D033903:Periplasmic Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011524:Protoporphyrins; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D016415:Sequence Alignment; D013057:Spectrum Analysis
D001426:Bacterial Proteins; D002352:Carrier Proteins; D033903:Periplasmic Proteins; D011524:Protoporphyrins; D006418:Heme; C028025:protoporphyrin IX