The Mechanism of Heme Transfer from the Cytoplasmic Heme Binding Protein PhuS to the δ-Regioselective Heme Oxygenase of Pseudomonas aeruginosa

Abstract

The opportunistic pathogen Pseudomonas aeruginosa has evolved two outer membrane receptor mediated uptake systems (encoded by the phu and has operons) by which it can utilize the hosts heme and hemeproteins as a source of iron. PhuS is a cytoplasmic heme binding protein encoded within the phu operon, and has previously been shown to function in the trafficking of heme to the ironregulated heme oxygenase (pa-HO). While the heme association rate for PhuS was similar to that of myoglobin, a markedly higher rate of heme dissociation (∼10 5 s −1 ) was observed, in keeping with a function in heme-trafficking. Additionally, the transfer of heme from PhuS to pa-HO was shown to be specific and unidirectional when compared to transfer to the non-iron regulated heme oxygenase (BphO), in which heme distribution between the two proteins merely reflects their relative intrinsic affinities for heme. Furthermore, the rate of transfer of heme from holo-PhuS to pa-HO of 0.11 ± 0.01 s −1 is 30-fold faster than that to apo-myoglobin, despite the significant higher binding affinity of apo-myoglobin for heme (k H =1.3 × 10 −8 μM) than that of PhuS (0.2 μM). This data suggests that heme transfer to pa-HO is independent of heme affinity and is consistent with temperature dependence studies which indicate the reaction is driven by a negative entropic contribution, typical of an ordered transition state, and supports the notion that heme transfer from PhuS to pa-HO is mediated via a specific protein-protein interaction. In addition, pH studies, and reactions conducted in the presence of cyanide, suggest the involvement of spin transition during the heme transfer process, whereby the heme undergoes spin change from 6-c LS to 6-c HS either in PhuS or pa-HO. Based on the magnitudes of the activation parameters obtained in the presence of cyanide, whereby both complexes are maintained in a 6-c LS state, and the biphasic kinetics of heme transfer from holo-PhuS to pa-HO-wt, supports the notion that the spin-state crossover occur within holo-PhuS prior to heme transfer step. Alternatively, the lack of the biphasic kinetic with pa-HO-G125V, 6-c LS, and with comparable rate of heme transfer as pa-HO-is supportive of mechanism in which the spin-change could occur within pa-HO. The present data suggests either or both of the two pathways proposed for heme transfer may occur under the present experimental conditions. The dissection of which pathway is physiologically relevant is the focus of ongoing studies.Heme, a cofactor of proteins involved in a variety of biological processes such as oxygen transport and storage, oxygenation reactions, electron transfer and transcriptional regulation is also a redox-reactive, hydrophobic iron chelate that readily associates with membranes, and is toxic to cells due to its ability to generate reactive oxygen species. Therefore, aerobic organisms have developed strategies to protect themselves from the harmful effects of "free" heme by sequestering it within specific proteins (1,2). While hemeproteins se...

The Role of the Cytoplasmic Heme-binding Protein (PhuS) of Pseudomonas aeruginosa in Intracellular Heme Trafficking and Iron Homeostasis

“…This is consistent with previous in vitro reports that PhuS functions to traffic heme to the iron regulated HO (10,11,47). The observation of the premature production of pyocyanin in the phuS mutants and the confirmation that heme can override suppression of the phu operon by Fur, indicates that there is a separate and distinct heme-dependent regulation of the phu operon.…”

“…1A). These data taken together are consistent with our previous in vitro studies, which have characterized PhuS as a heme-trafficking protein to the iron-regulated HO for degradation and release of iron (10,11,47). Interestingly, the phuS mutant in contrast to the parent strain or the mutants carrying the hemO gene deletion began to produce a blue-green pigment (supplemental Fig.…”

“…The phu locus consists of six open reading frames encoding the outer membrane heme receptor PhuR, the periplasmic ABC (ATP binding cassette) transport system PhuTUV, PhuW whose function has not been determined, and the cytoplasmic heme-binding protein PhuS, which is essential for optimal heme utilization (8). The cytoplasmic protein PhuS has been shown in vitro to transfer heme to the iron-regulated heme oxygenase, HO 2 encoded by the hemO (also referred to as PigA in the literature) (10,11). The expression of the phu and has operons is regulated by the ferric uptake regulator (Fur) protein, which has both negative and positive regulatory effects on the expression of iron-regulated genes (8,12,13).…”

Adaptation of Iron Homeostasis Pathways by a Pseudomonas aeruginosa Pyoverdine Mutant in the Cystic Fibrosis Lung

“…P. aeruginosa is unique in that many strains encode a second, non-iron-regulated heme oxygenase, BphO (42). However, in vitro studies show that PhuS can deliver heme to HemO but not to BphO (43). Further, P. aeruginosa strain PAO1 almost exclusively degrades extracellularly provided heme with HemO, while BphO mediates degradation of endogenously produced heme (44).…”

The Extracellular Heme-binding Protein HbpS from the Soil Bacterium Streptomyces reticuli Is an Aquo-cobalamin Binder

“…The rate constants of heme dissociation from heme-binding proteins can be determined using apomyoglobin as a heme scavenger (37,38). Timedependent heme transfer was followed by UV-visible spectroscopy.…”