No abstract

Release of iron from various ferrisiderophores (ferripyoverdines, ferrioxamines B and E, ferricrocin, ferrichrome A, ferrienterobactin and its analog ferric N,N',N"-tri(l,3,5-'Tris) 2,3-dihydroxybenzoylaminomethylbenzene) was obtained through an enzymic reduction of iron, involving NADH, FMN and the ferripyoverdine reductase of Pseudomonas aeruginosa PA01 . The iron released from the same complexes was also obtained through chemical reduction of iron involving FMNH,. Evidence is given that the enzymic process acts through a FMNHz reduction; the P. aeruginosu enzyme, purified according to its ferripyoverdine-reductase activity [Halle, F. & Meyer, J. M., Eur.J . Biochem. 209, 613 -6201, functions as a NADH : FMN oxidoreductase, the FMNH2 produced being able lo chemically reduce the iron complexed by siderophores. The general occurence of such a multi-step mechanism, which denies the existence of specific ferrisiderophore reductases, is discussed.One of the major questions which remain to be answered in microbial siderophore-mediated iron metabolism concerns the intracellular release of iron from ferrisiderophores. These iron chelates are formed through complexing of environmental iron(II1) with the siderophores produced by iron-limited growing microorganisms [ 11. Ferrisiderophores are transported from the outside to the inside of the cells via several ironregulated membrane proteins, the best known being the outermembrane receptors of Gram-negative bacteria [2]. Characterized by a very high and specific affinity for iron(II1) and not iron(II), siderophores are thought to be released intracellularly from the transported ion mainly by an iron-reductive mechanism. A ligand hydrolysis has been implicated, in some cases involving trimeric siderophores such as enterobactin (enterochelin) in Escherichia coli [3] or fusarinines in various species of fungi [4, 51. Studies on ferrienterobactin iron-release mechanisms illustrate the poor understanding that we have of this problem. It is particularly difficult to resolve in E. coli, since enterobactin is known to have the highest affinity constant for iron(II1) (10") [6]. It also has the lowest redox potential (En' = -750 mV) [7] among siderophores, far below the values of the common physiological reductants like NADH or FMNHz (E" = -320 mV and -216 mV, respectively) [S]. The ligand-hydrolysis hypothesis is supported by the physiological and genetic characterization of an enterobactin esterase [9]. Another mechanism which may also release iron Correspondence to J. M.

Purification of the ferripyoverdine reductase from Pseudomonas aeruginosa, strain PAOI, lead to the isolation of a soluble protein of M , 27000-28000, 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(I1)-trapping agent and FMN, this protein displayed an ironreductase activity which resulted in the formation of ferrozine-iron(I1) 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 prcrequisite 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 950/0 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 , as the ferripyoverdine reductase of P. aeruginosa PA01 , was detected in all the eighteen fluorescent pseudomonad strains belonging to the P . aeruginosa, P. jluorescens, P. putidu 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 PA01 is ubiquitous among the Pseudomonas.Siderophore-mediated iron metabolism in microorganisms involves scveral stcps, the first one occurring outside the cells and involving the complexing of iron(II1) by the siderophore(s) excreted by the microorganisms under iron stress. This reaction results in the solubilization of iron, otherwise insoluble in aerated growth media at physiological pH values, through the formation of very tightly iron-bound forms characterized by stability constants as high as [l], with an average value for most ferrisiderophores of lo3*-lo3' [2]. Following translocation through the cell membranes, iron must be released from its carrier in order to reach its different biological sites. Such a mechanism implicates a cytoplasmic localization of the process, although other locations of release could be postulated, i. e. directly at the cell membrane or inside the periplasm for Gram-negative bacteria. In Escherichia coli, which produces enterobactin as a siderophore, the trimer of ] that this enzyme is in fact a NADHiFMN oxidoreductase, displaying a ferripyoverdine-reductase activity due to the chemical reduction of iron in ferrisiderophores by FMNH2. dihydroxybenzoylserine [3], a cytoplasmic esterase, is thought to be responsible for the release of iron, occurring through the hydrolysis of the chelator [4]. A similar activity, involving an ornithine-esterase, has been found in various fusarinineproducing or fusarinine-like-producing fungi [5]...