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

Abstract

The abilities of two bacterial active heme transporters, HmbR of Neisseria meningitidis and HemR of Yersinia enterocolitica, to use different heme sources were compared. While HmbR-expressing cells used only hemoglobin (Hb) and 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 allowed Escherichia coli cells to respond to heme-containing peptides, microperoxidases 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 heme receptors identified several highly conserved histidine residues as well as two conserved amino acid motifs, the FRAP and NPNL boxes. A site-directed mutagenesis approach was used to investigate the roles of His128, His192, His352, and His461 residues in HemR function. The HemR receptor with histidine changed to lysine at position 128 (HemR(H128K)), HemR(H461L), HemR(H461A), and HemR(H128A,H461A) mutant receptors were unable to use Hb, human serum albumin-heme, and myoglobin as sources of porphyrin and iron. Utilization of free heme was also severely affected, with some residual heme uptake in cells expressing HemR(H128K), HemR(H461A), and HemR(H461L). Conversely, the HemR(H192T), HemR(H352A), HemR(H352K), and HemR(H192T,H352K) mutant receptors were fully functional. All mutant HemR proteins were expressed in the outer membrane at levels similar to that of the wild-type HemR receptor. Nonfunctional HemRs were able to bind heme- and Hb-agarose. A hypothetical model of the HemR function in which two conserved histidine residues, His128 and His461, participate in the transport of heme through the receptor pore is postulated.

FIG. 1

Comparison of the abilities of heme and Hb receptors, HemR and HmbR, to use different heme-protein complexes as sources of iron and porphyrin in the E. coli heme biosynthesis mutant IR1532. HemR and HmbR receptors were expressed from low-copy-number plasmids pWKS130 and pWKS30, respectively (49). The expression of HmbR was done in the presence of N. meningitidis tonB exbB exbD genes (47). Bars represent the surface areas of bacterial growth around the discs on NBD plates (error bars show the standard deviations). Discs were soaked with equimolar amounts of heme-containing compounds (except for heme, see Material and Methods). Each measurement was repeated at least three times. Abbreviations: HB, hemoglobin; HPX, human hemopexin; HPT-HB, human haptoglobin-Hb complex; MY-M, skeletal muscle myoglobin; MY-H, heart muscle myoglobin.

FIG. 2

Growth-inhibitory or -stimulatory activity of microperoxidases on Y. enterocolitica and E. coli (top) and structures of microperoxidases MP-9 and MP-11 (bottom). Wild-type E. coli hemA aroB (E. coli^wt) and E. coli IR1532 (E. coli-1532), a DH5α heme biosynthesis mutant (47), were two of the strains used. Growth-inhibitory or -stimulatory activity of microperoxidases MP-8, MP-9, and MP-11, cytochrome c (cyt. c), and heme are shown as follows: R, resistant to the inhibitory activity of microperoxidases; (S, 22), sensitive to inhibitory activity of microperoxidases, with a 22-mm-diameter inhibition zone; ND, not done; +, stimulation of growth under iron-limiting conditions; −, no stimulation of growth under iron-limiting conditions. Only bacteria that possess functional HemR can be stimulated or inhibited by microperoxidases. The same results were obtained with the native and denatured forms of cytochrome c. MP-8 is one amino acid (Lys) shorter than MP-9 (not shown). Note a covalent link between the peptide and the heme moiety.

FIG. 3

Amino acid comparisons of the conserved domains of the heme and Hb receptors. (A) A highly conserved receptor domain containing an invariant histidine residue, FRAP and NPNL amino acid boxes, present in all receptors that transport heme into the periplasm. Siderophore, vitamin B₁₂, and some Tf and Lf receptors lack either the complete domain or have only the FRAP box and distal glutamic amino acid residues relatively well conserved. Highly conserved residues (indicated by asterisks), aromatic residue (Aro), and gaps introduced to maximize alignment (indicated by dashes) are shown. Hpt, haptoglobin. (B) Amino acid comparison of Hb and heme receptor domains around conserved histidine residues (conserved histidine residues are shown in bold). Y. enterocolitica HemR (HEMR), Y. pestis HmuR (HMUR), E. coli ChuA (CHUA), S. dysenteriae SduA (SHUA), H. influenzae HxuC (HXUC), E. coli FepA (FEPA), and E. coli FhuA (FHUA) are shown. Amino acid residues of all receptors except FepA and FhuA are numbered from the first methionine.

FIG. 3

Amino acid comparisons of the conserved domains of the heme and Hb receptors. (A) A highly conserved receptor domain containing an invariant histidine residue, FRAP and NPNL amino acid boxes, present in all receptors that transport heme into the periplasm. Siderophore, vitamin B₁₂, and some Tf and Lf receptors lack either the complete domain or have only the FRAP box and distal glutamic amino acid residues relatively well conserved. Highly conserved residues (indicated by asterisks), aromatic residue (Aro), and gaps introduced to maximize alignment (indicated by dashes) are shown. Hpt, haptoglobin. (B) Amino acid comparison of Hb and heme receptor domains around conserved histidine residues (conserved histidine residues are shown in bold). Y. enterocolitica HemR (HEMR), Y. pestis HmuR (HMUR), E. coli ChuA (CHUA), S. dysenteriae SduA (SHUA), H. influenzae HxuC (HXUC), E. coli FepA (FEPA), and E. coli FhuA (FHUA) are shown. Amino acid residues of all receptors except FepA and FhuA are numbered from the first methionine.

FIG. 4

(A) A schematic representation of mutant HemRs and their phenotypes after expression in the E. coli heme biosynthesis mutant. The testing for growth stimulation was done on LB and NBD plates essentially as described in the legend to Fig. 1. Growth stimulation with four different (DIF.) heme sources, myoglobin (MY), hemoglobin (HB), heme (HM), and HSA (HSA-heme complex, is indicated as follows: +, stimulation of growth; −, no stimulation; ∗ and −/+, very light or small zone of stimulation. HemR^W.T., wild-type HemR. (B) Growth zones of HemR histidine mutants around discs soaked in 10 μl of 10 mM heme (NBD plates). WT, wild type.

FIG. 4

(A) A schematic representation of mutant HemRs and their phenotypes after expression in the E. coli heme biosynthesis mutant. The testing for growth stimulation was done on LB and NBD plates essentially as described in the legend to Fig. 1. Growth stimulation with four different (DIF.) heme sources, myoglobin (MY), hemoglobin (HB), heme (HM), and HSA (HSA-heme complex, is indicated as follows: +, stimulation of growth; −, no stimulation; ∗ and −/+, very light or small zone of stimulation. HemR^W.T., wild-type HemR. (B) Growth zones of HemR histidine mutants around discs soaked in 10 μl of 10 mM heme (NBD plates). WT, wild type.

FIG. 5

OMs of E. coli expressing different HemR histidine mutants from a pWKS130 plasmid. Lanes: 1, HemR^STOP (pNEO1391 [see Table 1]); 2, E. coli DH5α; 3, HemR^H352K; 4, HemR^H461L; 5, HemR^H192T; 6, HemR^H128K; 7, wild-type HemR; M, molecular mass markers (30, 46, 69, and 94 kDa).

FIG. 6

Ability of E. coli EB53 (hemA aroB) cells expressing HemR mutant receptors to use BSA-heme complexes (10 μM BSA and 1 μM heme) (A) and HSA-heme complexes (50 μM heme) as sources of heme. HemR^W.T, wild-type HemR; HemR^STOP, pNEO1391. Two to three independent growth measurements were conducted, producing essentially identical results.

FIG. 7

Binding of mutant HemRs and wild-type HemR (HemR^WT) to heme- and Hb-agarose. Lanes: M, molecular size markers; 1, OMs of E. coli expressing HemR^WT; 2, Hb-agarose-bound HemR^WT; 3, heme-agarose-bound HemR^WT; 4, Hb-agarose-bound HemR^H128K; 5, heme-agarose-bound HemR^H128K; 6, Hb-agarose-bound HemR^H461L; 7, heme-agarose-bound HemR^H461L. Differences in the amount of material bound to heme and Hb-agarose are due to the 30-fold-higher binding capacity of the heme- over Hb-agarose.