Abstract
The NfuA protein has been postulated to act as a scaffolding protein in the biogenesis of photosystem (PS) I and other iron-sulfur (Fe/S) proteins in cyanobacteria and chloroplasts. To determine the properties of NfuA, recombinant NfuA from Synechococcus sp. PCC 7002 was overproduced and purified. In vitro reconstituted NfuA contained oxygen- and EDTA-labile Fe/S cluster(s), which had EPR properties consistent with [4Fe-4S] clusters. After reconstitution with 57Fe2+, Mössbauer studies of NfuA showed a broad quadrupole doublet that confirmed the presence of [4Fe-4S]2+ clusters. Native gel electrophoresis under anoxic conditions and chemical cross-linking showed that holo-NfuA forms dimers and tetramers harboring Fe/S cluster(s). Combined with iron and sulfide analyses, the results indicated that one [4Fe-4S] cluster was bound per NfuA dimer. Fe/S cluster transfer from holo-NfuA to apo-PsaC of PS I was studied by reconstitution of PS I complexes using P700-F(X) core complexes, PsaD, apo-PsaC, and holo-NfuA. Electron transfer measurements by time-resolved optical spectroscopy showed that holo-NfuA rapidly and efficiently transferred [4Fe-4S] clusters to PsaC in a reaction that required contact between the two proteins. The NfuA-reconstituted PS I complexes had typical charge recombination kinetics from [F(A)/F(B)](-) to P700+ and light-induced low-temperature EPR spectra. These results establish that cyanobacterial NfuA can act as a scaffolding protein for the insertion of [4Fe-4S] clusters into PsaC of PS I in vitro.
Highlights
The NfuA protein has been postulated to act as a scaffolding protein in the biogenesis of photosystem (PS) I and other iron-sulfur (Fe/S) proteins in cyanobacteria and chloroplasts
In the photosynthetic electron transport chains of cyanobacteria and chloroplasts of higher plants, iron-sulfur (Fe/S)4 clusters serve as the terminal electron-accepting cofactors in photosystem (PS) I but they act as electron transfer cofactors in the cytochrome b6f complex and in soluble ferredoxin
PCC 7002 have shown that cyanobacteria primarily rely on the SUF system rather than the ISC system for the biogenesis and maintenance of Fe/S clusters [9]
Summary
The NfuA protein has been postulated to act as a scaffolding protein in the biogenesis of photosystem (PS) I and other iron-sulfur (Fe/S) proteins in cyanobacteria and chloroplasts. The NfuA-reconstituted PS I complexes had typical charge recombination kinetics from [FA/FB]؊ to P700؉ and lightinduced low-temperature EPR spectra These results establish that cyanobacterial NfuA can act as a scaffolding protein for the insertion of [4Fe-4S] clusters into PsaC of PS I in vitro. In cyanobacteria and in the chloroplasts of algae and higher plants, oxygen evolution accompanying water oxidation by PS II is one of the major processes associated with the light reactions Perhaps for this reason, an oxygen-resistant system is essential for Fe/S cluster biogenesis in such organisms (see Ref. 9 for a review). These pseudorevertants were able to suppress the inability of certain PsaC variants to assemble Fe/S clusters when non-Cys ligands to FA and FB were present in vivo Characterization of these intergenic suppressors identified the sufR gene, whose product was subsequently shown to encode a negative transcriptional regulator of the suf regulon [11, 12]. NifU-dependent activation of the Fe protein of nitrogenase (NifH) is lost if any of the Cys residues in the N- and C-terminal domains is replaced by alanine [16]
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