THE STABILITY OF THE MOLYBDENUM-AZOTOCHELIN COMPLEX AND ITS EFFECT ONSIDEROPHORE PRODUCTION IN AZOTOBACTER-VINELANDII

Azotobacter vinelandii produces five siderophores with different metal binding properties, depending on the concentrations of Fe(III) and mo lybdate in the growth medium. The three lower protonation constants of the unusual bis(catecholamide) siderophore azotochelin (L) were deter mined by a simultaneous spectrophotometric and potentiometric titratio n as log K-5 = 3.65(5), log K-4 = 7.41(3) and log K-3 = 8.54(4). The m etal-ligand equilibrium constant for [MoO2(L)](3-) was obtained from a nalysis of the absorbance concentration data: at 20 degrees C and pH 6 .6, log K-eq = 4(1). Based on an average log K-a value of 12.1 for the two basic phenolic oxygens of azotochelin, the equilibrium formation constant was converted into the conventional formation constant K-f(Mo L) = [MoO2L3-]/[MoO22+][L5-] = 10(35) M-1. To assess the influence of molybdenum-siderophore interactions on metal uptake in A. vinelandii, the dose-response effect of molybdate in the growth medium on sideroph ore biosynthesis was followed by UV-vis spectroscopy and HPLC. It coul d be shown that the formation of molybdenum siderophore complexes clea rly reduces the concentration of free siderophores available for iron solubilization. Furthermore, in media with initial molybdate concentra tions up to 100 mu M, the molybdenum azotochelin complex is the predom inant molybdenum species, suggesting that azotochelin might also posse ss sequestering activity towards molybdenum. Even higher molybdate lev els result in a complete repression of the synthesis of the tetradenta te siderophore azotochelin, while they initiate the alternative releas e of the more efficient iron chelator, the hexadentate siderophore pro tochelin.