Biogenesis of iron-sulfur clusters in photosystem I: holo-NfuA from the cyanobacterium Synechococcus sp. PCC 7002 rapidly and efficiently transfers [4Fe-4S] clusters to apo-PsaC in vitro.
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.
Jin
Zhao
Z
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Heinnickel
Mark
M
Krebs
Carsten
C
Shen
Gaozhong
G
Golbeck
John H
JH
Bryant
Donald A
DA
D000458:Cyanobacteria / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D010100:Oxygen / Q000737:chemistry; D045331:Photosystem I Protein Complex / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D013053:Spectrophotometry / Q000379:methods; D015204:Spectroscopy, Mossbauer / Q000379:methods; D046940:Synechococcus / Q000378:metabolism*; D013696:Temperature
D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; C529800:NfuA protein, E coli; D045331:Photosystem I Protein Complex; C089909:photosystem I, psaB subunit; D010100:Oxygen