Abstract
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 ((13)C-heme) and electrospray ionization-MS analysis we have confirmed the regioselectivity and ratio of (13)C-δ and β-BV IX (70:30) is identical in vivo to that previously observed for the purified protein. Furthermore, the (13)C-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 (13)C-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.
Highlights
Pseudomonas aeruginosa utilizes extracellular heme as a source of iron
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
In summary we have shown that the catalytic action of HemO drives the metabolic flux of extracellular heme uptake
Summary
Pseudomonas aeruginosa utilizes extracellular heme as a source of iron. Results: Deletion of hemO results in loss of 13C-heme uptake and degradation to 13C-BV IX␦ and BV IX. 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. 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 IX␦ and IX production. In conclusion the metabolic flux of extracellular heme as a source of iron is driven by the catalytic action of HemO. In early studies a homolog of the cytoplasmic heme-binding protein PhuS, HemS from Yersinia entercolitica, had been proposed to be a heme oxygenase or hemin degrading factor, based solely on the observation of an inability to efficiently utilize heme on deletion of the hemS gene [12]. This study heme uptake we undertook a metabolic analysis of the P. aeruginosa wild type and phuS and hemO deletion strains. Lack of BV in the media on deletion of hemO is consistent with PhuS being a heme chaperone and not as previously suggested a heme-degrading enzyme
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