Recent genetic characterization of bacterial heme uptake and transport systems in Pseudomonas aerugenosa and Shigella dysenteriae have raised interesting questions regarding the functional details of heme binding, translocation, and release by the protein players, the heme-binding proteins (HBPs) and the heme-transport receptors (HTRs), in these processes. Many gram negative bacterial pathogens use the most abundant source of soluble iron, heme from their host, as a source of the iron essential to establish infection. However, heme is cytotoxic and must be highly sequestered throughout the course of uptake and transport. This requirement for protection against the toxicity of heme imposes two design criteria on the proteins involved in the process. The first is that, in order to protect the organism from the toxic effects of free heme, all heme:HBP and heme:HTR complexes must exhibit intrinsically high thermodynamic stabilities. Yet, facile transfer of heme between these proteins having high heme affinities must be possible. Thus the second design criterion is that it must be possible to modulate the high stabilities of the heme:HBPs and heme:HTRs through specific interactions with the partners to which they pass their cargo. The free energy of interaction between heme-loaded proteins and their targets must be transduced to impose driving force and kinetic lability on the transfer of heme from the donor to its target, i.e. effective direction of heme to HO for liberation of its iron can be viewed as being mechanistically driven. Work on bacterial assimilation of the iron from heme along with our recent biophysical characterization of some of the proteins involved suggests that the HBPs can be classified in three groups;those having anionic tyrosinate axial ligands, those having axial His ligands, and those having axial His and Met ligands. In this study, the HBPs and HTRs from S. dysenteriae and P. aerugenosa will be examined. Based on genetic studies, the proteins involved in heme uptake and transport have been identified. The general hypothesis to be addressed by this proposed study is that the aforementioned axial ligand environments correlate with distinct mechanisms for heme binding, transport, and release in the aforementioned organisms. We will identify contribution(s) from heme coordination chemistry to the stabilities of the heme:HBP and heme:HTR complexes. We will further investigate which of those contributions could be modulated by complexation of heme:HBP or heme:HTR with its target protein for heme transfer. The long-term goal of our work with heme uptake and transport proteins is to elucidate the mechanistic parameters that govern the specificity and efficacy of heme transfer. The relevance of this work to human health lies in the potential of the results to ultimately provide new inroads into treatment of bacterial infections. In this project, the means by which bacterial pathogens acquire heme from their hosts and assimilate the iron necessary to establish infection will be investigated at the level of molecular mechanism. The relevance of this work to human health lies in the potential for knowledge of heme uptake by bacterial pathogens to ultimately provide new inroads into treatment of bacterial infections.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Academic Research Enhancement Awards (AREA) (R15)
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Macromolecular Structure and Function A Study Section (MSFA)
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Mills, Melody
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North Dakota State University
Schools of Arts and Sciences
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