THE INFLUENCE OF CHELATING-AGENTS UPON THE DISSIMILATORY REDUCTION OFFE(III) BY SHEWANELLA-PUTREFACIENS .2. OXO-BRIDGED AND HYDROXO-BRIDGED POLYNUCLEAR FE(III) COMPLEXES

The susceptibility to dissimilatory reduction of polynuclear oxo- and hydroxo-bridged Fe(III) complexes by Shewanella putrefaciens intact ce lls and membranes has been investigated. These complexes were ligated by the potential tetradentates heidi (H(3)heidi = N-(2-hydroxyethyl)im inodiacetic acid) or nta (H(3)nta = nitrilotriacetic acid), or the pot ential tridentate ida (H(2)ida = iminodiacetic acid). A number of defi ned small complexes with varied nuclearity and solubility properties w ere employed, as well as undefined species prepared by mixing differen t molar ratios of ida or heidi:Fe(III) in solution. The rates of Fe(II I) reduction determined by an assay for Fe(II) formation with ferrozin e were validated by monitoring c-type cytochrome oxidation and re-redu ction associated with electron transport. For the undefined Fe(III) po lymeric species, reduction rates in whole cells and membranes were con siderably faster in the presence of heidi compared to ida. This is bel ieved to result from generally smaller and more reactive clusters form ing with heidi as a consequence of the alkoxo function of this ligand being able to bridge between Fe(III) nuclei, with access to an Fe(III) reductase located at the cytoplasmic membrane being of some importanc e. The increases in reduction rates of the undefined ida species with Fe(III)) using membranes relative to whole cells reinforce such a view . Using soluble synthetic Fe(III) clusters, slow reduction was noted f or an ore-bridged dimer coordinatively saturated with ida and featurin g unligated carboxylates. This suggests that sterically hindering the cation can influence enzyme action. A heidi dimer and a heidi multimer (17 or 19 Fe(III) nuclei), which are both of poor solubility, were fo und to be reduced by whole cells, but dissimilation rates increased ma rkedly using membranes. These data suggest that Fe(III) reductase acti vity may be located at both the outer membrane and the cytoplasmic mem brane of S. putrefaciens. Slower reduction of the heidi multimer relat ive to the heidi dimer reflects the presence of a central hydroxo(oxo) -bridged core containing nine Fe(III) nuclei within the former cluster . This unit is a poor substrate for dissimilation, owing to the fact t hat the Fe(III) is not ligated by aminocarboxylate. The faster reducti on noted for the heidi dimer in membranes than for a soluble ida monom er suggests that the presence of ligating water molecules may relieve steric hindrance to enzyme attack. Furthermore, reduction of an insolu ble oxo-bridged nta dimer featuring ligating water molecules in intact cells was faster than that of a soluble monomer coordinatively satura ted by nta and possessing an unligated carboxylate. This suggests that steric factors may override solubility considerations with respect to the susceptibility to reduction of certain Fe(III) complexes by the b acterium.