| Summary: | Database searches revealed that some species of cyanobacteria, such as Synechocystis sp. PCC 6803 and Nostoc sp. PCC 7120, contain homologues of the Fet3p-Ftrlp high-affinity ferrous-iron uptake system, defined originally in the yeast Saccharomyces cerevisiae. These homologues include the ftr (ferric permease homologue, encoding a protein similar to Ftr 1 p) gene in both Synechocystis and Nostoc, and mco (a1l3942 a multicopper oxidase homologue- encoding a protein somewhat similar to Fet3p) found only in Nostoc where it is close to ftr. RT-PCR analysis indicated that ftr and mco in Nostoc are induced upon iron restriction as is fir in Synechocystis, thus supporting a role for these genes in iron uptake. Phenotypic studies in E. coli JC28 (a strain deficient in iron uptake) revealed that Ftr from Synechocystis is not functional in compensating for lack of iron-uptake capacity in E. coli. However, phenotypic studies revealed that the mco-all3941-bfr-ftr fragment of Nostoc causes a major enhancement of growth under aerobic iron restriction in E. coli JC28. However, the mco gene was not required for this growth promotion effect although inclusion of bfr (encoding the iron-storage protein, bacterioferritin) upstream of fir enhanced the effect of fir, and the inclusion of all3941-bfr upstream of fir raised growth still further. Thus, the Ftr of Nostoc does not require a Mco protein for its activity, but may instead use a bacterioferritin. Ftr-mediated growth enhancement was promoted by low pH and by the reductant ascobate, suggesting ferrous iron is the preferred Ftr substrate. In addition, the Ftr system had a weaker iron- restriction growth benefit anaerobically than that seen aerobically. Bioinformatics analysis indicated that All3942 (Mco) is a Tat exported periplasmic protein containing two cupredoxin domains with the blue copper and tri-nuclear inter-domain copper binding sites characteristic of multi copper oxidases. Overproduced and purified MalE-Mco was able to bind 5.8, 1.2 and 3.1 atoms of Cu, Zn and Fe, respectively, per subunit which is consistent with its Mco designation. UV-visible and EPR spectroscopy revealed the likely presence of Tl, T2, and T3 copper centres. Upon addition of ferrous iron to the copper-containing Mal-Mco, Tl signals were lost, presumably due to reduction of the centre, suggesting ferroxidase activity. Copper- soaked MalE-Mco showed phenoloxidase activity, as found for other Mco proteins, as well as limited ferroxidase activity indicating that it may, like Fet3p, oxidise ferrous iron in vivo. However, no requirement for the All3942/Mco protein in Ftr-mediated iron uptake was found in E. coli JC28 which suggests that the Nostoc Ftr protein may transport ferrous iron using a mechanism distinct from that of the yeast Ftr1p.
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