Use of heme-protein complexes by the Yersinia enterocolitica HemR receptor: Histidine residues are essential for receptor function

The abilities of two bacterial active heme transporters, HmbR of Neisseria meningitidis and HemR of Yersinia enterocolitica, to use different heme sou rces were compared. While HmbR-expressing cells used only hemoglobin (Hb) a nd heme, HemR-expressing bacteria were able to grow on Hb, heme, myoglobin, hemopexin, catalase, human and bovine serum albumin-heme, and haptoglobin- hemoglobin complexes as sources of iron. Expression of functional HemR allo wed Escherichia coli cells to respond to heme-containing peptides, microper oxidases MP-8, MP-9, and MP-11, suggesting the ability of HemR to transport heme covalently linked to other molecules. Comparison of HemR with other h eme receptors identified several highly conserved histidine residues as wel l as two conserved amino acid motifs, the FRAP and NPNL boxes. A site-direc ted mutagenesis approach was used to investigate the roles of His128, His19 2, His352, and His461 residues in HemR function. The HemR receptor with his tidine changed to lysine at position 128 (HemR(H128K)), HemR(H461L), HemR(H 461A), and HemR(H128A,H461A) mutant receptors were unable to use Hb, human serum albumin-heme, and myoglobin as sources of porphyrin and iron. Utiliza tion of free heme was also severely affected, with some residual heme uptak e in cells expressing HemRH128K, HemRH461A, and HemRH461L. Conversely, the HemR(H192T), HemR(H352A), HemR(H352K), and HemR(H192T,H352K) mutant recepto rs were fully functional. All mutant HemR proteins were expressed in the ou ter membrane at levels similar to that of the wild-type HemR receptor. Nonf unctional HemRs were able to bind heme- and Hb-agarose. A hypothetical mode l of the HemR function in which two conserved histidine residues, His128 an d His461, participate in the transport of heme through the receptor pore is postulated.