Abstract
For many pathogenic bacteria like Pseudomonas aeruginosa heme is an essential source of iron. After uptake, the heme molecule is degraded by heme oxygenases to yield iron, carbon monoxide, and biliverdin. The heme oxygenase PigA is only induced under iron-limiting conditions and produces the unusual biliverdin isomers IXbeta and IXdelta. The gene for a second putative heme oxygenase in P. aeruginosa, bphO, occurs in an operon with the gene bphP encoding a bacterial phytochrome. Here we provide biochemical evidence that bphO encodes for a second heme oxygenase in P. aeruginosa. HPLC, (1)H, and (13)C NMR studies indicate that BphO is a "classic" heme oxygenase in that it produces biliverdin IXalpha. The data also suggest that the overall fold of BphO is likely to be the same as that reported for other alpha-hydroxylating heme oxygenases. Recombinant BphO was shown to prefer ferredoxins or ascorbate as a source of reducing equivalents in vitro and the rate-limiting step for the oxidation of heme to biliverdin is the release of product. In eukaryotes, the release of biliverdin is driven by biliverdin reductase, the subsequent enzyme in heme catabolism. Because P. aeruginosa lacks a biliverdin reductase homologue, data are presented indicating an involvement of the bacterial phytochrome BphP in biliverdin release from BphO and possibly from PigA.
Highlights
In the presence of a suitable electron donor the enzyme heme oxygenase (HO,1 EC 1.14.99.3) catalyzes the oxidative degradation of heme to yield equimolar amounts of biliverdin (BV), iron, and CO
Because P. aeruginosa lacks a biliverdin reductase homologue, data are presented indicating an involvement of the bacterial phytochrome BphP in biliverdin release from BphO and possibly from PigA
The production of BV was further confirmed by its conversion to bilirubin (BR) upon addition of rat biliverdin reductase (BVR)
Summary
All chemicals, including glutathione-agarose, were purchased from Sigma (Munich, Germany) and were American Chemical Society grade or better. Restriction enzymes were from Invitrogen (Cleveland, OH). MasterTaqTM was purchased from Eppendorf Scientific (Westbury, NY). HPLC-grade acetone and 80% formic acid were purchased from Fisher Scientific (Pittsburgh, PA). The expression vector pGEX-6P-1 and PreScissionTM protease were obtained from Amersham Biosciences. Centricon-10 concentrator devices were purchased from Amicon (Beverly, MA). Cytochrome P450 reductase was from Merck Biosciences (Bad Soden, Germany), all ferredoxins, ferredoxin-NADPϩ oxidoreductase, and catalase were from Sigma
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have