The Functionality of the three-sited Ferroxidase center of E. coli Bacterial Ferritin (EcFtnA)

At least three ferritins are found in the bacterium Escherichia coli, the heme-containing bacterioferritin (EcBFR) and two non-heme bacterial ferritins (EcFtnA and EcFtnB). In addition to the conserved A- and B-sites of the diiron ferroxidase center, EcFtnA has a third iron-binding site (the C-site) of unknown function that is nearby the diiron site. In the present work, the complex chemistry of iron oxidation and deposition in EcFtnA has been further defined through a combination of oximetry, pH stat, stopped-flow and conventional kinetics, UV-visible, fluorescence and EPR spectroscopic measurements on the wildtype protein and site-directed variants of the A-, B- and C-sites. The data reveal that, while H2O2 is a product of dioxygen reduction in EcFtnA and oxidation occurs with a stoichiometry of Fe(II)/O2 ~ 3:1, most of the H2O2 produced is consumed in subsequent reactions with a 2:1 Fe(II)/H2O2 stoichiometry, thus suppressing hydroxyl radical formation. While the A- and B-sites are essential for rapid iron oxidation, the C-site slows oxidation and suppresses iron turnover at the ferroxidase center. A tyrosyl radical, assigned to Tyr24 near the ferroxidase center, is formed during iron oxidation and its possible significance to the function of the protein is discussed. Taken as a whole, the data indicate that there are multiple iron-oxidation pathways in EcFtnA with O2 and H2O2 as oxidants. Furthermore, the data are inconsistent with the C-site being a transit site, providing iron to the A- and B-sites, and does not support a universal mechanism for iron oxidation in all ferritins as recently proposed

Culmination in the slime mould <i>Dictyostelium discoideum</i> studied with a scanning electron microscope

ABSTRACT Myxamoebae of Dictyostelium discoideum were allowed to develop on cellulose acetate filters, and specimens taken at various stages of fruiting body formation were prepared for study by scanning electron microscopy. In the immature fruiting body where the mass of pre-spore cells has just been lifted off the substratum by the developing stalk, the pre-spore cells are irregular in shape and are similar in appearance to cells in aggregates at earlier stages of development. As the stalk lengthens, the pre-spore cells gradually separate from one another and become rounded and elongate, but mature spores are not visible until the fruiting body reaches its maximum height. It is concluded that, contrary to previous reports, spore maturation is a slow process and is not completed until the sorus becomes pigmented. The mature stalk is surrounded by a smooth cellulose sheath but this does not envelop the cells of the basal disc, which remain discrete. The fruiting body is enclosed in a slime sheath and this may be important in holding together the mass of spores.</jats:p

Studies of early stages of differentiation of the cellular slime mould <i>Dictyostelium discoideum</i>

ABSTRACT Countercurrent distribution in a polymer, two-phase system has been used to study changes in the cell surface properties of amoebae of Dictyostelium discoideum. Amoebae harvested during exponential growth in axenic culture and during the subsequent first six hours of development on Millipore filters were distributed as a single peak. However, the position of the peak changed during the period of early development which showed that changes in cell surface properties were occurring. At aggregation (8 h), the peak markedly broadened, indicating considerable increase in cell surface heterogeneity amongst the amoebae, and heterogeneity was so great by 9 –10 h that the amoebae distributed as two peaks. Amoebae from one peak were shown to be precursors of spores while amoebae from the other peak appeared to be precursors of stalk cells. Similarly, amoebae from the trailing and leading edges of the broad peak, formed from amoebae beginning to aggregate (8 h), were found to have different fates. Thus cell differentiation had been found at times of development prior to formation of aggregates having apical tips or anterior-posterior polarity and neither of these features of aggregates can be essential for initiation of cell differentiation. It is therefore concluded that differentiation is not initiated in D. discoideum in response to ‘positional information’.</jats:p