Ferrisiderophore reductases of Pseudomonas. Purification, properties and cellular location of the Pseudomonas aeruginosa ferripyoverdine reductase.
Purification of the ferripyoverdine reductase from Pseudomonas aeruginosa, strain PAO1, lead to the isolation of a soluble protein of M(r) 27,000-28,000, as determined by HPLC sieving filtration and by denaturating gel electrophoresis. In the presence of NADH as the reductant, ferripyoverdine as the iron substrate, ferrozine as an iron(II)-trapping agent and FMN, this protein displayed an iron-reductase activity which resulted in the formation of ferrozine-iron(II) complex, providing that the enzymic assay was run under strict anaerobiosis. FMN was absolutely required for the activity to occur, but the lack of a visible spectrum and the lack of fluorescence for the protein in solution suggested that ferripyoverdine reductase is not a flavin-containing protein and that covalently bound FMN is not a prerequisite for the enzymatic reaction. A search of ferripyoverdine reductase by immunological detection amongst the different cellular compartments of P. aeruginosa lead to the conclusion that the soluble enzyme, which represented more than 95% of the total cellular enzyme, is not located in the periplasm but specifically in the cytoplasm. A strongly immunoreacting material, corresponding to a protein with identical M(r) as the ferripyoverdine reductase of P. aeruginosa PAO1, was detected in all the eighteen fluorescent pseudomonad strains belonging to the P. aeruginosa, P. fluorescens, P. putida and P. chlororaphis species, as well as in P. stutzeri, a non-fluorescent species, suggesting that the enzyme acting as a ferripyoverdine reductase in P. aeruginosa PAO1 is ubiquitous among the Pseudomonas.
Hallé
F
F
Laboratoire de Microbiologie, Unité de Recherche Associée au Centre National de la Recherche Scientifique, no. 1481, Université Louis Pasteur, Strasbourg, France.
Meyer
J M
JM
D015153:Blotting, Western; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D038181:FMN Reductase; D006801:Humans; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology / Q000302:isolation & purification; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology
D006882:Hydroxyapatites; D017886:Durapatite; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; C077757:ferripyoverdine reductase