Spectroscopic evidence for a 5-coordinate oxygenic ligated high spin ferric heme moiety in the Neisseria meningitidis hemoglobin binding receptor

Abstract

Background For many pathogenic microorganisms, iron acquisition represents a significant stress during the colonization of a mammalian host. Heme is the single most abundant source of soluble iron in this environment. While the importance of iron assimilation for nearly all organisms is clear, the mechanisms by which heme is acquired and utilized by many bacterial pathogens, even those most commonly found at sites of infection, remain poorly understood. Methods An alternative protocol for the production and purification of the outer membrane hemoglobin receptor (HmbR) from the pathogen Neisseria meningitidis has facilitated a biophysical characterization of this outer membrane transporter by electronic absorption, circular dichroism, electron paramagnetic resonance, and resonance Raman techniques. Results HmbR co-purifies with 5-coordinate high spin ferric heme bound. The heme binding site accommodates exogenous imidazole as a sixth ligand, which results in a 6-coordinate, low-spin ferric species. Both the 5- and 6-coordinate complexes are reduced by sodium hydrosulfite. Four HmbR variants with a modest decrease in binding efficiency for heme have been identified (H87C, H280A, Y282A, and Y456C). These findings are consistent with an emerging paradigm wherein the ferric iron center of bound heme is coordinated by a tyrosine ligand. Conclusion In summary, this study provides the first spectroscopic characterization for any heme or iron transporter in Neisseria meningitidis, and suggests a coordination environment heretofore unobserved in a TonB-dependent hemin transporter. General Significance A detailed understanding of the nutrient acquisition pathways in common pathogens such as N. meningitidis provides a foundation for new antimicrobial strategies.

Differential Contributions of the Outer Membrane Receptors PhuR and HasR to Heme Acquisition in Pseudomonas aeruginosa

“…This is in contrast to previous reports where the purified S. marcescens HasR receptor was reported to strip heme from holo-HasA (21). Additionally, the ShuA receptor of S. dysenteriae was shown to accept heme from methemoglobin in lipid bicelles but not free heme (23) and the recently characterized N. meningitidis HmbR receptor was unable to be reconstituted with heme or hemoglobin following purification (26). Therefore given the variation in the heme-binding properties of the detergent-solubilized receptors we have employed a combination of bacterial genetics and 13 C-isotopic heme labeling to assess the contributions of the OM hemophore-dependent HasR and PhuR receptors to heme acquisition in P. aeruginosa.…”

“…Interestingly, the majority of heme receptors so far characterized, both hemophore-dependent and independent, have the characteristic bis-His coordination. An exception to the bis-His coordination was the recently reported five coordinate Tyr ligation in the HmbR receptor of Neisseria meningitidis (26). Recent spectroscopic characterization of the PhuR receptor by our laboratory revealed heme is coordinated through His-124 of the N-terminal plug and Tyr-519 of the FRAP/PNPL loop.…”

Spectroscopic Determination of Distinct Heme Ligands in Outer-Membrane Receptors PhuR and HasR of Pseudomonas aeruginosa

“…This is in keeping with previous studies on the S. dysenteriae ShuA 12 and recent characterization of the N. meningitidis HmbR receptor. 15 The addition of free heme to the detergent-solubilized proteins is due, in part, to nonspecific binding to the protein surface. However, we were able to show that apo-HasR is functional to bind hemin-agarose (Figure 3A).…”

“…We cannot reconcile these results with those for the S. marcescens HasA–HasR system, although the present data is consistent with the substoichiometric heme levels and inability to extract heme from metHb reported for the heterologous expressed OM heme receptors ShuA and HmbR of S. dysenteriae and N. meningitides , respectively. 10,15 Conflicting data on the isolated receptors is further complicated by the fact that previous in vivo reports on the S. marcescens system determined that heme release from HasA to HasR required interaction with the TonB-dependent energy transducing system. 33 Our current studies are consistent with the latter observation that the TonB-dependent energy transducing system is critical for heme extraction from the hemeprotein substrate.…”

“…An exception to the bis-His coordination was the recently reported five-coordinate Tyr-ligation in the HmbR receptor of Neisseria meningitides . 15 …”