Kinetics of iron acquisition from ferric siderophores by Paracoccus denitrificans

Abstract

The kinetics of iron accumulation by iron-starved Paracoccus denitrificans during the first 2 min of exposure to "5Fe-labeled ferric siderophore chelates is described. Iron is acquired from the ferric chelate of the natural siderophore L-parabactin in a process exhibiting biphastic kinetics by Lineweaver-Burk analysis. The kinetic data for 1 ,uM < [Fe L-parabactin] < 10 ,uM fit a regression line which suggests a low-affinity system (Km = 3.9 ± 1.2 ,uM, Vma,,j = 494 pg-atoms of 55Fe min-' mg of protein-'), whereas the data for 0.1 5AM < [Fe L-parabactin] c 1 1AM fit another line consistent with a high-affinity system (Km = 0.24 ± 0.06 ,uM, Vmax = 108 pg-atoms of 55Fe min'-mg of protein-l). The Km of the high-affinity uptake is comparable to the binding affinity we had previously reported for the purified ferric L-parabactin receptor protein in the outer membrane. In marked contrast, ferric D-parabactin data fit a single regression line corresponding to a simple MichaelisMenten process with comparatively low affinity (Km = 3.1 ± 0.9 ,uM, Vmax = 125 pg-atoms of 55Fe min-' mg of protein-'). Other catecholamide siderophores with an intact oxazoline ring derived from L-threonine (L-homoparabactin, L-agrobactin, and L-vibriobactin) also exhibit biphasic kinetics with a high-affinity component similar to ferric L-parabactin. Circular dichroism confirmed that these ferric chelates, like ferric L-parabactin, exist as the A enantiomers. The A forms of ferric parabactin (ferric D-and L-parabactin A), in which the oxazoline ring is hydrolyzed to the open-chain threonyl structure, exhibit linear kinetics with a comparatively high Km (1.4 ± 0.3 1AM) and high Vmax (324 pg-atoms of 55Fe min-' mg of protein-').Furthermore, the marked stereospecificity seen between ferric D-and L-parabactins is absent; i.e., iron acquisition from ferric parabactin A is nonstereospecific. The mechanistic implications of these findings in relation to a stereospecific high-affinity binding followed by a nonstereospecific postreceptor processing is discussed.The gram-negative soil bacterium Paracoccus denitrificans excretes a hexacoordinate catecholamide iron chelator, a siderophore, parabactin ( Fig. 1) (36, 42). The five aromatic hydroxyls and the oxazoline ring nitrogen of this ligand have been implicated as the donor centers coordinated to iron in the ferric L-parabactin-chelate complex (34). Previously we demonstrated that the kinetics of iron incorporation by P. denitrificans grown in iron-deficient media is consistent with an "iron taxi" mechanism (4). In the mechanism envisaged, the ferric siderophore binds to a specific receptor protein in the outer membrane, with subsequent release of iron from the siderophore complex while still on the extracellular side of the inner cytoplasmic membrane. The released iron is then transported into the cell, while the deferrated ligand remains extracellular where it may be reused for iron transport. We have identified, isolated, and partially purified a stereospecific high-affinity receptor for ferric L-para...

Increase in spermine content coordinated with siderophore production in Paracoccus denitrificans

“…Catecholamide siderophore production was estimated by addition of 20 [lI of 50 mM ferric nitrilotriacetate to 1.00 ml of culture sample, filtration through a 0.2-jm membrane, and spectrophotometric assay of the filtrate. Ferric catecholamide content was calculated by using the extinction coefficient for ferric parabactin (E516nm = 3.1 X 103 M-1 cm-1 [6]). units, suspension of the pellet in 500 ,ul of fresh minimal salts medium, and then addition of 500 pLI of 1.2 M perchloric acid with vigorous mixing.…”

Ferric iron uptake in Erwinia chrysanthemi mediated by chrysobactin and related catechol-type compounds

“…vations were also made in bioassays, in which growth of At the concentrations, M/L ratios, and pH used here, iron-deprived Fct-PMV 4082 cells could be stimulated by ferric bis-Cb would be the almost exclusively present species supernatants from low-iron cultures of E. chrysanthemi 3937 (39). The high efficiency of Cb in mediating Fe(III) transport (38) (6,33,45,49). enterobactin in E. chrysanthemi 3937; it was recently deterAlthough the Cb analogs used in this study formed optically mined that E. coli could transport both dihydroxybenzoyl active ferric Tris complexes, whose conformation around serine and DHBA by three receptors, albeit with varying the iron center depended on the chirality of the amino acid to efficiency (24).…”

“…Furthermore, other reports have described which DHBA was attached, the bis complexes were optisiderophore transport systems which consisted of more than cally inactive (39). No conclusion can therefore be drawn one receptor, or mechanism, with different affinities for one regarding the sensitivity of the ferric Cb receptor to the ferric siderophore species (6,31,41 (3,24,27,40,47). Dihydroxybenzoyl serine was also shown to efficiently remove iron from human transferrin (20).…”

Isolation and biochemical characterization of two soluble iron(III) reductases from Paracoccus denitrificans

“…Evidence for a high‐affinity iron‐scavenging system involving low molecular weight, Fe(III)‐specific siderophores was first presented by Tait [21] who isolated three iron‐binding catechol compounds from the culture supernatant of P. denitrificans and provided initial data on their structure, metal‐binding properties and enzymes needed for their biosynthesis. Since then, several thorough investigations into the mechanism, stereospecificity and kinetics of siderophore‐mediated iron transport in P. denitrificans have been carried out [22–24], but information on the enzymology of Fe(III) reduction still remains scarce and incomplete. Tait [21] described formation of Fe(II) from the Fe(III)–siderophore complex upon incubation with NADH, succinate and an ultracentrifuged and dialyzed cell‐free extract while Dailey and Lascelles [25] noticed reduction of Fe(III) citrate by NADH or succinate as electron donors in the presence of crude cell extracts containing membranes.…”