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The role of the cytoplasmic heme-binding protein (PhuS) of Pseudomonas aeruginosa in intracellular heme trafficking and iron homeostasis.

ABSTRACT: The cytoplasmic heme-binding protein PhuS, encoded within the Fur-regulated Pseudomonas heme utilization (phu) operon, has previously been shown to traffic heme to the iron-regulated heme oxygenase (HO). We further investigate the role of PhuS in heme trafficking to HO on disruption of the phuS and hemO genes in a Pseudomonas aeruginosa siderophore-deficient and wild-type background. Previous studies have shown that deletion of hemO prevents the cells from utilizing heme as the sole source of iron. However, disruption of phuS alone resulted in a slow growth phenotype, consistent with its role as a heme-trafficking protein to HO. Furthermore, in contrast to the hemO and hemO/phuS deletion strains, the phuS knockout prematurely produced pyocyanin in the presence of heme. Western blot analysis of PhuS protein levels in the wild-type strain showed that Fur-regulation of the phu operon could be derepressed in the presence of heme. In addition the premature onset of pyocyanin production requires both heme and a functional HO. Suppression of the phenotype on increasing the external heme concentration suggested that the decreased heme-flux through HO results in premature production of pyocyanin. The premature production of pyocyanin was not due to lower intracellular iron levels as a result of decreased heme flux through HO. However, transcriptional analysis of the phuS mutants indicates that the cells are sensing iron deprivation. The present data suggest that PhuS has a dual function in trafficking heme to HO, and in directly or indirectly sensing and maintaining iron and heme homeostasis.

SUBMITTER: Kaur AP

PROVIDER: S-EPMC2610525 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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The role of the cytoplasmic heme-binding protein (PhuS) of Pseudomonas aeruginosa in intracellular heme trafficking and iron homeostasis.

Kaur Ajinder P AP Lansky Ila B IB Wilks Angela A

The Journal of biological chemistry 20081105 1

The cytoplasmic heme-binding protein PhuS, encoded within the Fur-regulated Pseudomonas heme utilization (phu) operon, has previously been shown to traffic heme to the iron-regulated heme oxygenase (HO). We further investigate the role of PhuS in heme trafficking to HO on disruption of the phuS and hemO genes in a Pseudomonas aeruginosa siderophore-deficient and wild-type background. Previous studies have shown that deletion of hemO prevents the cells from utilizing heme as the sole source of ir ...[more]

PMID: 18990702

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Project description: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 iron-regulated heme oxygenase (pa-HO). While the heme association rate for PhuS was similar to that of myoglobin, a markedly higher rate of heme dissociation (approximately 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 (kH = 1.3 x 10(-8) microM) than that of PhuS (0.2 microM). 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. On the basis of 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 the 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 a 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.

Dataset Information

The role of the cytoplasmic heme-binding protein (PhuS) of Pseudomonas aeruginosa in intracellular heme trafficking and iron homeostasis.

Publications

The role of the cytoplasmic heme-binding protein (PhuS) of Pseudomonas aeruginosa in intracellular heme trafficking and iron homeostasis.

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