Iron is an essential nutrient for most organisms, including pathogenic organisms. Pathogenic bacteria attempting to colonize (infect) an organism are confronted with extremely low concentrations of free iron. Consequently, many pathogens have evolved sophisticated mechanisms for iron acquisition, including the utilization of heme iron. Thus, it is possible that targeting paths used by pathogenic bacteria to assimilate iron and heme-iron from their host is a viable approach to the development of new antibiotics. Many of the proteins involved in heme uptake and heme utilization in the opportunistic pathogen Pseudomonas aeruginosa have designated functions. However, their structure, dynamics and inter-protein interactions needed to facilitate host-heme capture, internalization and degradation in the cytosol, are largely unknown. We aim at contributing to fill this gap by studying the structure, function, dynamics and association of the soluble proteins that aid in the capture of heme from hemoglobin and help degrade it in the cytosol of P. aeruginosa. Current efforts entail recombinant DNA methodology, NMR spectroscopy and bioelectrochemistry in this laboratory and X-ray crystallography, EPR spectroscopy and resonance Raman spectroscopy via collaborations. This approach is aimed at:
(1) Understanding how polypeptide dynamics controls and contributes to the complicated catalytic cycle of heme degradation whereby heme oxygenase releases iron from heme for subsequent metabolic needs. These studies build from our previous investigations aimed at determining structure-function relationships in heme oxygenase from P. aeruginosa (pa-HO). A recent finding demonstrates that disrupting the network of hydrogen bonding waters in the distal site of pa-HO, accomplished by replacing Arg 80 (see figure) for Leu, leads to chaotic global μs-ms motions of the polypeptide and significant loss of heme oxidation activity.
(2) Biochemically and structurally characterizing two previously unknown electron transport proteins (pa-Bfd and pa-Fpr), which we hypothesize function to deliver the 7 electrons needed by heme oxygenase to cleave the heme and release its iron in the cytosol of P. aeruginosa. It has been recently demonstrated that the novel ferredoxin reductase (pa-FPR) efficiently delivers the 7 electrons needed by pa-HO to oxidize heme and release iron, without the need of a mediating ferredoxin. The structure of pa-FPR is now characterized and this information is being used in the study of protein-protein interactions that facilitate electron transfer to heme oxygenase.
(3) Structurally and biochemically characterizing HasAp, a secreted heme binding protein capable of capturing heme from hemoglobin and delivering it to the outer membrane receptor for internalization. The structural information is being used to define its interactions with hemoglobin using NMR spectroscopic methods, in an attempt to gain molecular understanding of the mechanism whereby HasAp “steals” the heme from hemoglobin for subsequent delivery to a receptor, internalization to the cytosol and degradation by pa-HO to release the iron.
(4) Characterizing structure-function relationships in the two iron storage bacterioferritins of P. aeruginosa, which are likely involved in facilitating its survival under low-iron and oxidative stress conditions. These investigations also encompass the study of pa-Bfd, a ferredoxin likely involved in the mobilization of iron from bacterioferritin.