A Periplasmic Iron-binding Protein Contributes toward Inward Copper Supply

Abstract

Periplasmic substrate binding proteins are known for iron, zinc, manganese, nickel, and molybdenum but not copper. Synechocystis PCC 6803 requires copper for thylakoid-localized plastocyanin and cytochrome oxidase. Here we show that mutants deficient in a periplasmic substrate binding protein FutA2 have low cytochrome oxidase activity and produce cytochrome c 6 when grown under copper conditions (150 nM) in which wild-type cells use plastocyanin rather than cytochrome c 6 . Anaerobic separation of extracts by two-dimensional native liquid chromatography followed by metal analysis and peptide mass-fingerprinting establish that accumulation of copperplastocyanin is impaired, but iron-ferredoxin is unaffected in ⌬futA2 grown in 150 nM copper. However, recombinant FutA2 binds iron in preference to copper in vitro with an apparent Fe(III) affinity similar to that of its paralog FutA1, the principal substrate binding protein for iron import. FutA2 is also associated with iron and not copper in periplasm extracts, and this Fe(III)-protein complex is absent in ⌬futA2. There are differences in the soluble protein and small-molecule complexes of copper and iron, and the total amount of both elements increases in periplasm extracts of ⌬futA2 relative to wild type. Changes in periplasm protein and small-molecule complexes for other metals are also observed in ⌬futA2. It is proposed that FutA2 contributes to metal partitioning in the periplasm by sequestering Fe(III), which limits aberrant Fe(III) associations with vital binding sites for other metals, including copper.Oxygenic phototrophs have exceptional requirements for metals such as iron, copper, and manganese within the photosynthetic machinery (1, 2). There is interest in understanding how metal ions partition to the correct cellular destinations in all organisms (3), including to the approximately one-third of proteins that need metals (4). The periplasm is a key location for metal partitioning. Bacterial ATP binding cassette (ABC)-type importers include substrate binding proteins which may be free within the periplasm or anchored to the outer face of the plasma membrane. It is anticipated that these proteins assist in loading metal ions onto the correct importers. There is similarity in the metal-binding sites of several substrate binding proteins that contribute toward the import of distinct metals, such as manganese versus zinc, and subtle differences in second coordination spheres may be crucial for metal selectivity (5). It has also been noted that the nature of interactions between substrate binding proteins and their respective metal transporters may make a key contribution to metal specificity (5

Coordinated transporter activity shapes high-affinity iron acquisition in cyanobacteria

“…Upon entering the periplasm FutA2 binds Fe(III), generating the chemical gradient that facilitates the influx of Fe. This is consistent with the suggested role for FutA2 in metal partitioning in the periplasm (Waldron et al, 2007). On the basis of our findings (Figures 2 and 3), FutA2 is a major factor in determining iron reduction and subsequent transport.…”

The Structure of the Iron-binding Protein, FutA1, from Synechocystis 6803

“…The binding of ferrous iron by FutA2 in this investigation contrasts the results of the work done by Waldron et al, which concluded that FutA2 binds ferric iron in vitro (9). The data presented here represent the first test of both ferric and ferrous iron binding to FutA1 or FutA2.…”

“…FutA2 Purification-FutA2 was purified and refolded from inclusion bodies as previously described (9). Approximately 2 g of cells were thawed in 35 ml of inclusion body buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 5 mM MgCl 2 , 1% Triton X-100) with 10 mM ␤-mercaptoethanol, DNase I, and lysozyme.…”

“…These results suggested that there was at least some redundancy in the functions of FutA1 and FutA2. Recently, it was shown that FutA2 binds iron with approximately the same relative affinity as FutA1 (9). However, when futA2 is deleted, the mutants accumulate normal levels of iron-containing ferredoxin in the periplasm but fail to accumulate copper in plastocyanin.…”

Redox control of copper homeostasis in cyanobacteria

“…7 Another protein that have been implicated in copper transport is FutA2, which electrophoretic mobility changes in the presence of the Cu + -chelator, and a mutant in the corresponding gene is impaired in copper import and has reduced levels of intracellular copper proteins. 8 CtaA and PacS are thought to transport reduced copper, although copper is present in the growth medium in it oxidized state, pointing to the existence of an unidentified copper reductase in cyanobacteria.…”