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The P. aeruginosa heme binding protein PhuS is a heme oxygenase titratable regulator of heme uptake.

ABSTRACT: The Pseudomonas aeruginosa heme utilization (Phu) system encodes several proteins involved in the acquisition of heme as an iron source. Once internalized, heme is degraded by the iron-regulated heme oxygenase, HemO to biliverdin (BV) IX? and ?. In vitro studies have shown holo-PhuS transfers heme to the iron-regulated HemO. This protein-protein interaction is specific for HemO as PhuS does not interact with the ?-regioselective heme oxygenase, BphO. Bacterial genetics and isotopic labeling ((13)C-heme) studies confirmed extracellular heme is converted to (13)C-BVIX ? and ? through the catalytic action of HemO. In an effort to further understand the role of PhuS, similar studies were performed on the P. aeruginosa PAO1 ?phuS and ?phuS/?hemO strains. In contrast to wild-type strain, the absence of PhuS results in extracellular heme uptake and degradation via the catalytic action of HemO and BphO. At low heme concentrations, loss of PhuS leads to inefficient extracellular heme uptake supported by the fact the mRNA levels of PhuR, HemO, and BphO remain elevated when compared to the wild-type PAO1. On increasing extracellular heme concentrations, the elevated levels of PhuR, HemO, and BphO allow "leaky uptake" and degradation of heme via HemO and BphO. Similarly, in the ?phuS/?hemO strain, the higher heme concentrations combined with elevated levels of PhuR and BphO leads to nonspecific heme uptake and degradation by BphO. Thus we propose heme flux into the cell is driven by the catalytic action of HemO with PhuS acting as a "control valve" to regulate extracellular heme flux.

SUBMITTER: O'Neill MJ

PROVIDER: S-EPMC3748626 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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The P. aeruginosa heme binding protein PhuS is a heme oxygenase titratable regulator of heme uptake.

O'Neill Maura J MJ Wilks Angela A

ACS chemical biology 20130606 8

The Pseudomonas aeruginosa heme utilization (Phu) system encodes several proteins involved in the acquisition of heme as an iron source. Once internalized, heme is degraded by the iron-regulated heme oxygenase, HemO to biliverdin (BV) IXδ and β. In vitro studies have shown holo-PhuS transfers heme to the iron-regulated HemO. This protein-protein interaction is specific for HemO as PhuS does not interact with the α-regioselective heme oxygenase, BphO. Bacterial genetics and isotopic labeling ((13 ...[more]

PMID: 23947366

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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.

Project description:The secreted Mycobacterium tuberculosis (Mtb) heme binding protein Rv0203 has been shown to play a role in Mtb heme uptake. In this work, we use spectroscopic (absorption, electron paramagnetic resonance, and magnetic circular dichrosim) methods to further characterize the heme coordination environments of His-tagged and native protein forms, Rv0203-His and Rv0203-notag, respectively. Rv0203-His binds the heme molecule through bis-His coordination and is low-spin in both ferric and ferrous oxidation states. Rv0203-notag is high-spin in both oxidation states and shares spectroscopic similarity with pentacoordinate oxygen-ligated heme proteins. Mutagenesis experiments determined that residues Tyr59, His63, and His89 are required for Rv0203-notag to efficiently bind heme, reinforcing the hypothesis based on our previous structural and mutagenesis studies of Rv0203-His. While Tyr59, His63, and His89 are required for the binding of heme to Rv0203-notag, comparison of the absorption spectra of the Rv0203-notag mutants suggests the heme ligand may be the hydroxyl group of Tyr59, although an exogenous hydroxide cannot be ruled out. Additionally, we measured the heme affinities of Rv0203-His and Rv0203-notag using stopped flow techniques. The rates for binding of heme to Rv0203-His and Rv0203-notag are similar, 115 and 133 ?M(-1) s(-1), respectively. However, the heme off rates differ quite dramatically, whereby Rv0203-His gives biphasic dissociation kinetics with fast and slow rates of 0.0019 and 0.0002 s(-1), respectively, and Rv0203-notag has a single off rate of 0.082 s(-1). The spectral and heme binding affinity differences between Rv0203-His and Rv0203-notag suggest that the His tag interferes with heme binding. Furthermore, these results imply that the His tag has the ability to stabilize heme binding as well as alter heme ligand coordination of Rv0203 by providing an unnatural histidine ligand. Moreover, the heme affinity of Rv0203-notag is comparable to that of other heme transport proteins, implying that Rv0203 may act as an extracellular heme transporter.

Dataset Information

The P. aeruginosa heme binding protein PhuS is a heme oxygenase titratable regulator of heme uptake.

Publications

The P. aeruginosa heme binding protein PhuS is a heme oxygenase titratable regulator of heme uptake.

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