Expression and Characterization of a Novel Bacterial Heme Transport Protein

Iron, an essential nutrient for living organisms, plays crucial biological roles ranging from oxygen transport, energy generation, signal transduction to bacterial virulence. Certain pathogenic bacteria have developed highly specific strategies to acquire heme (iron‐protoporphyrine IX complex) from the host environment as a valuable iron source. A BLAST search has implicated that numerous Gram‐negative pathogenic bacteria may use a novel heme‐acquisition system to steal iron from hosts. This system involves specific cell surface receptors for host hemeproteins, a periplasmic heme‐transport protein and inner membrane proteins typical for ABC transporters. We have cloned the gene coding for a putative periplasmic heme‐transport protein (HTP) from the human pathogen Pseudomonas aeruginosa , and over‐expressed the recombinant protein in E. coli . A 33 kDa His‐tagged HTP has been purified by Ni‐NTA affinity chromatography. UV‐visible spectra of the purified protein show soret, α, β and charge transfer bands, which are typical for a heme protein, which is further supported by native PAGE followed by heme‐staining assays. Heme titration monitored by UV‐vis and fluorescence spectroscopy suggests that apo‐HTP binds heme in vitro at 1:1 ratio with high affinity. Protoporphyrine IX also binds to apo‐HTP at 1:1 ratio but with a lowered affinity. HTP can be reduced by dithionite but not ascorbate. Circular dichroism spectra reveal a conformational change upon Heme binding to apo‐HTP, and this is further evidenced by limited proteolysis assays.