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

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 FeSO 4 , 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-␤. This differs from the previously characterized heme oxygenases in which biliverdin IX-␣ is the typical product. We conclude that PigA is a heme oxygenase and may represent a class of these enzymes with novel regiospecificity.

1H NMR Study of the Magnetic Properties and Electronic Structure of the Hydroxide Complex of Substrate-bound Heme Oxygenase from Neisseria Meningitidis: Influence of the Axial Water Deprotonation on the Distal H-bond Network

“…The relative values for the pKs for the two complexes are consistent with, but not proof for, stronger H-bond stabilization of the ligated OH -by proton donation by a non-ligated water in PaHO than NmHO (see Figure 1C). At this time, the 13 Inspection of Table 1 shows that, while δ DSS (obs) differs by as much as 1.6 ppm between the two complexes, correction for δ dip (calc) for each complex reduces the difference in δ DSS (dia*) to well under 0.2 ppm for all but a few labile protons. Hence the strength of the majority of the H-bonds is strongly conserved upon deprotonating the axial water.…”

“…Integration of a high-spin and low-spin resolved methyl peak indicate that 1:1 population occurs at pH 9. 13 C contact shift pattern of the PH substrate. The significant difference in the pKs for the acid-alkaline transition in PaHO and NmHO complexes would allow for significant differences in the effective axial field strength of the OH -ligand, such that different orbital ground states could, in principle, be populated for the two complexes.…”

Crystal Structures of the NO- and CO-bound Heme Oxygenase from Neisseriae meningitidis

“…HO also is present in some pathogenic bacteria such as Corynebacterium diphtheriae, Neisseriae meningitidis, and Pseudomonas aeruginosa (11)(12)(13)(14)(15). In bacteria, heme oxygenase is a soluble cytoplasmic protein, which takes part in the mining of iron from host heme.…”

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

“…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). P. aeruginosa encodes a second non-iron-regulated heme oxygenase, BphO directly upstream of the phytochrome two-component sensor kinase, BphP (12,13).…”