KINETICS OF IRON ACQUISITION FROM FERRIC SIDEROPHORES BY PARACOCCUS-DENITRIFICANS

The kinetics of iron accumulation by iron-starved Paracoccus denitrificans during the first 2 min of exposure to 55Fe-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 μM < [Fe L-parabactin] < 10 μM fit a regression line which suggests a low-affinity system (K(m) = 3.9 ± 1.2 μM, V(max) = 494 pg-atoms of 55Fe min-1 mg of protein-1), whereas the data for 0.1 μM ≤ [Fe L-parabactin] ≤ 1 μM fit another line consistent with a high-affinity system (K(m) = 0.24 ± 0.06 μM, V(max) = 108 pg-atoms of 55Fe min-1 of protein-1). The K(m) 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 Michaelis-Menten process with comparatively low affinity (K(m) = 3.1 ± 0.9 μM, V(max) = 125 pg-atoms of 55Fe min-1 mg of protein-1). Other catecholamide siderophores with an intact oxazoline ring derived from L-threonine (L-monoparabactin, 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 Λ 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 K(m) (1.4 ± 0.3 μM) and high V(max) (324 pg-atoms of 55Fe min-1 mg of protein-1). 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.