Metabolic Flux of Extracellular Heme Uptake in Pseudomonas aeruginosa Is Driven by the Iron-regulated Heme Oxygenase (HemO)

• Published in: The Journal of biological chemistry Vol. 287; no. 22; pp. 18342 - 18350
• Main Authors: Barker, Kylie D., Barkovits, Katalin, Wilks, Angela
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25.05.2012; American Society for Biochemistry and Molecular Biology
• Subjects: 13C-heme Metabolism; Biliverdin; Blotting, Western; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Enzyme Catalysis; Genetic Complementation Test; Heme - metabolism; Heme Chaperone; Heme Oxygenase; Heme Oxygenase (Decyclizing) - genetics; Heme Oxygenase (Decyclizing) - metabolism; Heme Uptake; Iron - metabolism; Metabolism; Metal Homeostasis; Pseudomonas aeruginosa - metabolism; Spectrometry, Mass, Electrospray Ionization; Trafficking; Biliverdin; Heme Uptake; Enzyme Catalysis; Trafficking; Heme Oxygenase; Heme Chaperone; Metabolism; 13C-heme Metabolism; Metal Homeostasis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.359265

Heme utilization by Pseudomonas aeruginosa involves several proteins required for internalization and degradation of heme. In the following report we provide the first direct in vivo evidence for the specific degradation of extracellular heme to biliverdin (BV) by the iron-regulated HemO. Moreover, through isotopic labeling (13C-heme) and electrospray ionization-MS analysis we have confirmed the regioselectivity and ratio of 13C-δ and β-BV IX (70:30) is identical in vivo to that previously observed for the purified protein. Furthermore, the 13C-BV IXδ and BV IXβ products are effluxed from the cell by an as yet unidentified transporter. Conversion of extracellular heme to BV is dependent solely on the iron-regulated HemO as evidenced by the lack of BV production in the P. aeruginosa hemO deletion strain. Complementation of P. aeruginosa ΔhemO with a plasmid expressing either the wild type HemO or α-regioselective HemO mutant restored extracellular heme uptake and degradation. In contrast deletion of the gene encoding the cytoplasmic heme-binding protein, PhuS, homologs of which have been proposed to be heme oxygenases, did not eliminate 13C-BV IXδ and IXβ production. In conclusion the metabolic flux of extracellular heme as a source of iron is driven by the catalytic action of HemO. Pseudomonas aeruginosa utilizes extracellular heme as a source of iron. Deletion of hemO results in loss of 13C-heme uptake and degradation to 13C-BV IXδ and BV IXβ. Extracellular heme uptake is dependent on the catalytic action of HemO. Determining the role of metabolic flux in heme uptake and degradation is crucial in understanding the relationship between iron homeostasis and virulence.