• The Cytoplasmic Heme Binding Protein PhuS of P. aeruginosa: A Heme Oxygenase (HemO) Titratable Regulator of Extracellular Heme Uptake

      O'Neill, Maura Jean; Wilks, Angela (2013)
      Iron acquisition is critical for pathogenic bacteria and as such they have evolved sophisticated mechanisms to utilize the hosts heme containing proteins as an iron source. The Pseudomonas aeruginosa cytoplasmic heme binding protein (PhuS) has been shown to interact specifically with and deliver heme to the iron regulated heme oxygenase (HemO). HemO then oxidatively cleaves heme to release iron with biliverdin (BV) IX delta and IX beta and CO as by-products of the reaction. A combination of site directed mutagenesis and spectroscopic studies of holo-PhuS reveal a dynamic heme with overlapping but distinct binding sites through alternate heme ligands, His-209 or His-212. We have further investigated the role of the histidine triad (His-209, His-210 and His-212) in complex formation and heme transfer. A series of biophysical studies has shown that a heme induced conformational change drives interaction of holo-PhuS with HemO. We further show that in addition to the proximal ligand His-209 both His-210 and His-212 are required for complex formation and heme transfer. Based on these studies we propose a mechanism that couples the heme-dependent conformational switch in PhuS to protein-protein interaction, the subsequent free energy of which drives heme transfer via a His-ligand switch from His-209 to His-212, and subsequent release of heme to HemO. The in vitro characterization of PhuS as a heme trafficking protein was further confirmed in vivo utilizing a combination of isotopic labeling (13C-heme) and qRT-PCR. Under conditions of active heme uptake wild type P. aeruginosa produced exclusively 13C- BVIX delta and IX beta. In contrast the -phuS knockout strain led to loss of the heme-dependent regulation of the heme uptake proteins and an uncoupling of heme trafficking to HemO. The resulting elevated expression of the heme uptake proteins leads to increased heme uptake and degradation of heme via both HemO (13C-BVIX delta and IX beta) and the alternate non-iron-regulated BphO (13C-BVIX alpha). We propose a testable model whereby PhuS acts as a HemO titratable regulator of extracellular heme uptake that couples the metabolic flux of heme through PhuS-HemO to the regulatory RNA network.