Publications (58) View all
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Article: Oxidation of Heme to β- and δ-Biliverdin by Pseudomonas aeruginosa Heme Oxygenase as a Consequence of an Unusual Seating of the Heme
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ABSTRACT: The origin of the unusual regioselectivity of heme oxygenation, i.e. the oxidation of heme to δ-biliverdin (70%) and β-biliverdin (30%), that is exhibited by heme oxygenase from Pseudomonas aeruginosa (pa-HO) has been studied by 1H NMR, 13C NMR, and resonance Raman spectroscopies. Whereas resonance Raman indicates that the heme−iron ligation in pa-HO is homologous to that observed in previously studied α-hydroxylating heme oxygenases, the NMR spectroscopic studies suggest that the heme in this enzyme is seated in a manner that is distinct from that observed for all other α-hydroxylating heme oxygenase enzymes for which a structure is known. In pa-HO, the heme is rotated in-plane 110°, so the δ-meso-carbon of the major orientational isomer is located within the HO-fold in the place where the α-hydroxylating enzymes typically place the α-meso-carbon. The unusual heme seating displayed by pa-HO places the heme propionates so that these groups point in the direction of the solvent-exposed heme edge and appears to originate in large part from the absence of stabilizing interactions between the polypeptide and the heme propionates, which are typically found in α-hydroxylating heme oxygenase enzymes. These interactions typically involve Lys-16 and Tyr-112, in Neisseriae meningitidis HO, and Lys-16 and Tyr-134, in human and rat HO-1. The corresponding residues in pa-HO are Asn-19 and Phe-117, respectively. In agreement with this hypothesis, we found that the Asn-19 Lys/Phe-117 Tyr double mutant of pa-HO exists as a mixture of molecules exhibiting two distinct heme seatings; one seating is identical to that exhibited by wild-type pa-HO, whereas the alternative seating is very similar to that typical of α-hydroxylating heme oxygenase enzymes and is related to the wild-type seating by 110° in-plane rotation of the heme. Furthermore, each of these heme seatings in the pa-HO double mutant gives rise to a subset of two heme isomeric orientations that are related to each other by 180° rotation about the α−γ-meso-axis. The coexistence of these molecules in solution, in the proportions suggested by the corresponding area under the peaks in the 1H NMR spectrum, explains the unusual regioselectivity of heme oxygenation observed with the double mutant, which we found produces α- (55%), δ- (35%), and β-biliverdin (10%). α-Biliverdin is obtained by oxidation of the heme seated similar to that of α-hydroxylating enzymes, whereas β- and δ-biliverdin are formed from the oxidation of heme seated as in wild-type pa-HO.11/2002; -
Article: Homologues of neisserial heme oxygenase in gram-negative bacteria: degradation of heme by the product of the pigA gene of Pseudomonas aeruginosa.
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ABSTRACT: 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.Journal of Bacteriology 12/2001; 183(21):6394-403. · 3.83 Impact Factor -
Article: Crystal structure of heme oxygenase from the gram-negative pathogen Neisseria meningitidis and a comparison with mammalian heme oxygenase-1.
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ABSTRACT: We report the crystal structure of heme oxygenase from the pathogenic bacterium Neisseria meningitidis at 1.5 A and compare and contrast it with known structures of heme oxygenase-1 from mammalian sources. Both the bacterial and mammalian enzymes share the same overall fold, with a histidine contributing a ligand to the proximal side of the heme iron and a kinked alpha-helix defining the distal pocket. The distal helix differs noticeably in both sequence and conformation, and the distal pocket of the Neisseria enzyme is substantially smaller than in the mammalian enzyme. Key glycine residues provide the flexibility for the helical kink, allow close contact of the helix backbone with the heme, and may interact directly with heme ligands.Biochemistry 10/2001; 40(38):11552-8. · 3.42 Impact Factor -
Article: The ShuS protein of Shigella dysenteriae is a heme-sequestering protein that also binds DNA.
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ABSTRACT: The ability of Shigella dysenteriae to utilize heme as an iron source is dependent on the iron-regulated expression of a number of genes including the outermembrane receptor ShuA and the cytoplasmic protein ShuS. The ShuS protein has no sequence homology with any proteins of known function and its role in heme acquisition has not been determined. In this paper we describe the purification and characterization of ShuS. The soluble oligomeric protein (650 kDa) is composed of a single type of subunit with a molecular mass of 37 kDa and binds one heme per monomer (Kd = 13 microM). In addition, the ShuS protein was shown to nonspecifically bind double-stranded DNA. It appears, therefore, that ShuS may function as both a heme storage protein, during periods of active heme transport, and as a DNA binding protein to protect the DNA from any ensuing heme mediated oxidative damage.Archives of Biochemistry and Biophysics 04/2001; 387(1):137-42. · 2.93 Impact Factor -
Article: Degradation of heme in gram-negative bacteria: the product of the hemO gene of Neisseriae is a heme oxygenase.
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ABSTRACT: 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.Journal of Bacteriology 01/2001; 182(23):6783-90. · 3.83 Impact Factor
