Angela Wilks

Crystal structure of human heme oxygenase-1

Heme oxygenase catalyzes the first step in the oxidative degradation of heme. The crystal structure of heme oxygenase-1 (HO-1) reported here reveals a novel helical fold with the heme sandwiched between two helices. The proximal helix provides a heme iron ligand, His 25. Conserved glycines in the distal helix near the oxygen binding site allow close contact between the helix backbone and heme in addition to providing flexibility for substrate binding and product release. Regioselective oxygenation of the α-meso heme carbon is due primarily to steric influence of the distal helix.

Homologues of Neisserial Heme Oxygenase in Gram-Negative Bacteria: Degradation of Heme by the Product of the pigA Gene of Pseudomonas aeruginosa

The oxidative cleavage of heme to release iron is a mechanism by which some bacterial pathogens can utilize heme as an iron source. The pigA gene of Pseudomonas aeruginosa is shown to encode a heme oxygenase protein, which was identified in the genome sequence by its significant homology (37%) with HemO of Neisseria meningitidis. When the gene encoding the neisserial heme oxygenase, hemO, was replaced with pigA, we demonstrated that pigA could functionally replace hemO and allow for heme utilization by neisseriae. Furthermore, when pigA was disrupted by cassette mutagenesis in P. aeruginosa, heme utilization was defective in iron-poor media supplemented with heme. This defect could be restored both by the addition of exogenous FeSO4, indicating that the mutant did not have a defect in iron metabolism, and by in trans complementation with pigA from a plasmid with an inducible promoter. The PigA protein was purified by ion-exchange chromotography. The UV-visible spectrum of PigA reconstituted with heme showed characteristics previously reported for other bacterial and mammalian heme oxygenases. The heme-PigA complex could be converted to ferric biliverdin in the presence of ascorbate, demonstrating the need for an exogenous reductant. Acidification and high-performance liquid chromatography analysis of the ascorbate reduction products identified a major product of biliverdin IX-beta. This differs from the previously characterized heme oxygenases in which biliverdin IX-alpha is the typical product. We conclude that PigA is a heme oxygenase and may represent a class of these enzymes with novel regiospecificity.

Degradation of Heme in Gram-Negative Bacteria: the Product of the hemO Gene of Neisseriae Is a Heme Oxygenase

A full-length heme oxygenase gene from the gram-negative pathogen Neisseria meningitidis was cloned and expressed in Escherichia coli. Expression of the enzyme yielded soluble catalytically active protein and caused accumulation of biliverdin within the E. coli cells. The purified HemO forms a 1:1 complex with heme and has a heme protein spectrum similar to that previously reported for the purified heme oxygenase (HmuO) from the gram-positive pathogen Corynebacterium diphtheriae and for eukaryotic heme oxygenases. The overall sequence identity between HemO and these heme oxygenases is, however, low. In the presence of ascorbate or the human NADPH cytochrome P450 reductase system, the heme-HemO complex is converted to ferric-biliverdin IXalpha and carbon monoxide as the final products. Homologs of the hemO gene were identified and characterized in six commensal Neisseria isolates, Neisseria lactamica, Neisseria subflava, Neisseria flava, Neisseria polysacchareae, Neisseria kochii, and Neisseria cinerea. All HemO orthologs shared between 95 and 98% identity in amino acid sequences with functionally important residues being completely conserved. This is the first heme oxygenase identified in a gram-negative pathogen. The identification of HemO as a heme oxygenase provides further evidence that oxidative cleavage of the heme is the mechanism by which some bacteria acquire iron for further use.

Heme oxygenase structure and mechanism

ACKNOWLEDGMENTS : The work at the University of California in San Francisco and the preparation of this review were supported by National Institutes of Health grant DK30297.

HutZ Is Required for Efficient Heme Utilization in Vibrio cholerae

Vibrio cholerae, the causative agent of cholera, requires iron for growth. One mechanism by which it acquires iron is the uptake of heme, and several heme utilization genes have been identified in V. cholerae. These include three distinct outer membrane receptors, two TonB systems, and an apparent ABC transporter to transfer heme across the inner membrane. However, little is known about the fate of the heme after it enters the cell. In this report we show that a novel heme utilization protein, HutZ, is required for optimal heme utilization. hutZ (open reading frame [ORF] VCA0907) is encoded with two other genes, hutW (ORF VCA0909) and hutX (ORF VCA0908), in an operon divergently transcribed from the tonB1 operon. A hutZ mutant grew poorly when heme was provided as the sole source of iron, and the poor growth was likely due to the failure to use heme efficiently as a source of iron, rather than to heme toxicity. Heme oxygenase mutants of both Corynebacterium diphtheriae and C. ulcerans fail to use heme as an iron source. When the hutWXZ genes were expressed in the heme oxygenase mutants, growth on heme was restored, and hutZ was required for this effect. Biochemical characterization indicated that HutZ binds heme with high efficiency; however, no heme oxygenase activity was detected for this protein. HutZ may act as a heme storage protein, and it may also function as a shuttle protein that increases the efficiency of heme trafficking from the membrane to heme-containing proteins.

Holo- and Apo-bound Structures of Bacterial Periplasmic Heme-binding Proteins

An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an “in” position where it can coordinate the heme iron to an “out” orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg228 in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg228, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B12-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B12, compared with ligands for FhuD, a peptide siderophore.

The Cytoplasmic Heme-binding Protein (PhuS) from the Heme Uptake System of Pseudomonas aeruginosa Is an Intracellular Heme-trafficking Protein to the δ-Regioselective Heme Oxygenase

The uptake and utilization of heme as an iron source is a receptor-mediated process in bacterial pathogens and involves a number of proteins required for internalization and degradation of heme. In the following report we provide the first in-depth spectroscopic and functional characterization of a cytoplasmic heme-binding protein PhuS from the opportunistic pathogen Pseudomonas aeruginosa. Spectroscopic characterization of the heme-PhuS complex at neutral pH indicates that the heme is predominantly six-coordinate low spin. However, the resonance Raman spectra and global fit analysis of the UV-visible spectra show that at all pH values between 6 and 10 three distinct species are present to varying degrees. The distribution of the heme across multiple spin states and coordination number highlights the flexibility of the heme environment. We provide further evidence that the cytoplasmic heme-binding proteins, contrary to previous reports, are not heme oxygenases. The degradation of the heme-PhuS complex in the presence of a reducing agent is a result of H2O2 formed by direct reduction of molecular oxygen and does not yield biliverdin. In contrast, the heme-PhuS complex is an intracellular heme trafficking protein that specifically transfers heme to the previously characterized iron-regulated heme oxygenase pa-HO. Surface plasmon resonance experiments confirm that the transfer of heme is driven by a specific protein-protein interaction. This data taken together with the spectroscopic characterization is consistent with a protein that functions to shuttle heme within the cell.

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

Cystic fibrosis (CF) patients suffer from chronic bacterial lung infections, most notably by Pseudomonas aeruginosa, which persists for decades in the lungs and undergoes extensive evolution. P. aeruginosa requires iron for virulence and uses the fluorescent siderophore pyoverdine to scavenge and solubilize ferric iron during acute infections. Pyoverdine mutants accumulate in the lungs of some CF patients, however, suggesting that the heme and ferrous iron acquisition pathways of P. aeruginosa are more important in this environment. Here, we sought to determine how evolution of P. aeruginosa in the CF lung affects iron acquisition and regulatory pathways through the use of longitudinal CF isolates. These analyses demonstrated a significant reduction of siderophore production during the course of CF lung infection in nearly all strains tested. Mass spectrometry analysis of one of these strains showed that the later CF isolate has streamlined the metabolic flux of extracellular heme through the HemO heme oxygenase, resulting in more-efficient heme utilization. Moreover, gene expression analysis shows that iron regulation via the PrrF small RNAs (sRNAs) is enhanced in the later CF isolate. Finally, analysis of P. aeruginosa gene expression in the lungs of various CF patients demonstrates that both PrrF and HemO are consistently expressed in the CF lung environment. Combined, these results suggest that heme is a critical source of iron during prolonged infection of the CF lung and that changes in iron and heme regulatory pathways play a crucial role in adaptation of P. aeruginosa to this ever-changing host environment.

Characterization of the outer membrane receptor ShuA from the heme uptake system of Shigella dysenteriae. Substrate specificity and identification of the heme protein ligands.

Shigella dysenteriae, like many bacterial pathogens, has evolved outer membrane receptor-mediated pathways for the uptake and utilization of heme as an iron source. As a first step toward understanding the mechanism of heme uptake we have undertaken a site-directed mutagenesis, spectroscopic, and kinetic analysis of the outer membrane receptor ShuA of S. dysenteriae. Purification of the outer membrane receptor gave a single band of molecular mass 73 kDa on SDS-PAGE. Initial spectroscopic analysis of the protein in either detergent micelles or lipid bicelles revealed residual heme bound to the receptor, with a Soret maximum at 413 nm. Titration of the protein with exogenous heme gave a Soret peak at 437 nm in detergent micelles, and 402 nm in lipid bicelles. However, transfer of heme from hemoglobin yields a Soret maximum at 413 nm identical to that of the isolated protein. Further spectroscopic and kinetic analysis revealed that hemoglobin in the oxidized state is the most likely physiological substrate for ShuA. In addition, mutation of the conserved histidines, H86A or H420A, resulted in a loss of the ability of the receptor to efficiently extract heme from hemoglobin. In contrast the double mutant H86A/H420A was unable to extract heme from hemoglobin. These findings taken together confirm that both His-86 and His-420 are essential for substrate recognition, heme coordination, and transfer. Furthermore, the full-length TonB was shown to form a 1:1 complex with either apo-ShuA H86A/H420A or the wild-type ShuA. These observations provide a basis for future studies on the coordination and transport of heme by the TonB-dependent outer membrane receptors.

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

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.