Identification of Two Heme-Binding Sites in the Cytoplasmic Heme-Trafficking Protein PhuS from <i>Pseudomonas aeruginosa</i> and Their Relevance to Function

Block, Darci R.; Lukat-Rodgers, Gudrun S.; Rodgers, Kenton R.; Wilks, Angela; Bhakta, Mehul N.; Lansky, Ila B.

doi:10.1021/bi701509n

Cited by 28 publications

(46 citation statements)

References 32 publications

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“…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.…”

Section: Discussionsupporting

confidence: 92%

“…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.…”

Section: Phus Is Required For Efficient Heme Utilization In Psupporting

confidence: 91%

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

Kaur

Lansky

Wilks

2009

Journal of Biological Chemistry

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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 Furregulation 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.Iron is essential for the growth, survival, and virulence of most bacterial pathogens, with only a few exceptions (1-3). However, within the human body, bacteria encounter an extremely low iron milieu, because the majority of iron is sequestered in iron and heme proteins, such as transferrin and hemoglobin, respectively (4). Bacterial pathogens have therefore evolved multiple mechanisms to obtain iron from the ironand heme-containing proteins of the host. The opportunistic pathogen Pseudomonas aeruginosa, which is responsible for severe nosocomial infections in immunocompromised patients (5, 6), secretes an array of high affinity iron binding siderophores (pyoverdin and pyochelin) and in addition can directly utilize the host iron-and heme-containing proteins (7,8).The P. aeruginosa genome encodes two heme acquisition systems: the has (heme assimilation system) and the phu (Pseudomonas heme utilization) operons. The has locus encodes the hemophore HasA, the hemophore receptor HasR, and a potential ABC transporter HasDEF for export of HasA (8, 9). 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-re...

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Section: Discussionsupporting

confidence: 92%

Section: Phus Is Required For Efficient Heme Utilization In Psupporting

confidence: 91%

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

Kaur

Lansky

Wilks

2009

Journal of Biological Chemistry

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“…The heme trafficking protein CcmE from E. coli, which trafficks heme to periplasmic cytochrome enzymes, is covalently linked to heme at His130 (30). Heme is coordinated to the PhuS protein at the conserved His209 residue or the nonconserved His212 residue (31). Therefore, given the absence of a crystal structure or a clear hemin-binding motif, the conserved tyrosine (Y57), cysteine (C89), and histidine (H46) residues in Cj1386 were mutated to identify residues important for hemin ligand binding in Cj1386.…”

Section: Discussionmentioning

confidence: 99%

“…It is possible that additional amino acids compensate for hemin binding in the absence of Y57 to produce the 6-coordinate peak. Indeed, heme coordination to PhuS in P. aeruginosa still can be achieved in a His209 mutant by compensation by an alternative heme coordinating residue, His212 (31). From the multiple-sequence alignment of Cj1386, a less conserved tyrosine residue (Y61), which is in close proximity to Y57, could play a role as an alternative hemin-coordinating residue.…”

Section: Discussionmentioning

confidence: 99%

Cj1386, an Atypical Hemin-Binding Protein, Mediates Hemin Trafficking to KatA in Campylobacter jejuni

Flint

Stintzi

2015

J Bacteriol

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. While the structure and biochemical properties of catalase enzymes have been well characterized over many decades of research, it remained unclear how catalases acquire hemin. We have previously reported that Cj1386 is essential for ensuring proper hemin content in Campylobacter jejuni catalase (KatA) (A. Flint, Y. Q. Sun, and A. Stintzi, J Bacteriol 194:334 -345, 2012). In this report, an in-depth molecular characterization of Cj1386 was performed to elucidate the mechanistic details of this association. Coimmunoprecipitation assays revealed that KatA-Cj1386 transiently interact in vivo, and UV-visible spectroscopy demonstrated that purified Cj1386 protein binds hemin. Furthermore, hemin titration experiments determined that hemin binds to Cj1386 in a 1:1 ratio with hexacoordinate hemin binding. Mutagenesis of potential hemin-coordinating residues in Cj1386 showed that tyrosine 57 was essential for hemin coordination when Cj1386 was overexpressed in Escherichia coli. The importance of tyrosine 57 in hemin trafficking in vivo was confirmed by introducing the cj1386 Y57A allele into a C. jejuni ⌬cj1386 mutant background. The cj1386 Y57A mutation resulted in increased sensitivity toward H 2 O 2 relative to the wild type, suggesting that KatA was not functional in this strain. In support of this finding, KatA immunoprecipitated from the ⌬cj1386؉cj1386 Y57A mutant had significantly reduced hemin content compared to that of the cj1386 WT background. Overall, these findings indicate that Cj1386 is involved in directly trafficking hemin to KatA and that tyrosine 57 plays a key role in this function.

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“…Once internalized heme is sequestered by the cytoplasmic heme-binding protein PhuS. In a series of in vitro studies we have shown that the cytoplasmic heme-binding protein PhuS forms a specific protein complex with the ironregulated heme oxygenase, HemO (7)(8)(9). Heme is transferred to HemO for further degradation with the release of iron, CO, and biliverdin IX (BVIX) ␦-and ␤-isomers (10,11).…”

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confidence: 99%

Differential Contributions of the Outer Membrane Receptors PhuR and HasR to Heme Acquisition in Pseudomonas aeruginosa

Smith

Wilks

2015

Journal of Biological Chemistry

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Background: Pseudomonas aeruginosa utilizes extracellular heme as a source of iron on infection of the host. Results: Both PhuR and HasR are required for efficient utilization of heme by P. aeruginosa. Conclusion: Non-redundant outer membrane receptors allow for heme acquisition across a range of physiological conditions. Significance: PhuR and HasR have complimentary roles in heme acquisition and utilization.

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Identification of Two Heme-Binding Sites in the Cytoplasmic Heme-Trafficking Protein PhuS from Pseudomonas aeruginosa and Their Relevance to Function

Cited by 28 publications

References 32 publications

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

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

Cj1386, an Atypical Hemin-Binding Protein, Mediates Hemin Trafficking to KatA in Campylobacter jejuni

Differential Contributions of the Outer Membrane Receptors PhuR and HasR to Heme Acquisition in Pseudomonas aeruginosa

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