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UBC Theses and Dissertations

Iron binding and oxidation by Pseudo-nitzschia multiseries ferritin Pfaffen, Stephanie


A novel ferritin was identified in marine pennate diatoms, unicellular photosynthetic organisms that play a major role in global primary production and carbon sequestration in the deep ocean. The expression of the iron storage and detoxifying protein ferritin is thought to facilitate the blooming of pennate diatoms after iron fertilization in the open ocean. X-ray structures of Pseudo-nitzschia multiseries ferritin (PmFTN) from crystals soaked for various durations in ferrous iron and zinc sulfate revealed three distinct metal binding sites; sites A and B comprise the catalytic ferroxidase centre, and site C forms a pathway leading toward the central cavity where iron storage occurs. In contrast, crystal structures derived from anaerobically grown and ferrous iron soaked crystals revealed only one ferrous ion occupying site A. Together with kinetic analysis, these studies suggest a model of stepwise iron binding to the ferroxidase centre of PmFTN followed by a very fast iron oxidation phase and partial mobilization of iron from the ferroxidase centre. Using a combination of rapid reaction kinetics and high resolution crystallography, the function of site C was investigated with site C and site B/C variants. Glu130, a site B/C ligand, functions in stabilizing Fe(III) bound at the ferroxidase centre and as a consequence reducing the rate of mineralization. Furthermore, Glu44, a site C ligand, is shown to be important for limiting the rate of post-oxidation reorganisation of iron coordination. Iron was observed within the B-channels, first identified in prokaryotic ferritins and BFRs, of the E44Q variant of PmFTN and provides the first evidence that these channels are possible routes for Fe(II) entry into the cavity. The anaerobic crystal structure of the bacterioferritin from E. coli (EcBFR) revealed two Fe(II) at the ferroxidase centre sites A and B. In comparison with PmFTN, differences in ferrous iron binding and reaction rates are further evidence that in EcBFR a distinct mechanism is in operation. Clearly, PmFTN shows some characteristics of bacterial ferritins. Moreover, retention of iron at the ferroxidase centre at the expense of mineralization points to a role for this diatom ferritin in facilitating short term rather than long term iron storage.

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