Chloroplast HCF101 is a scaffold protein for [4Fe-4S] cluster assembly.
Oxygen-evolving chloroplasts possess their own iron-sulfur cluster assembly proteins including members of the SUF (sulfur mobilization) and the NFU family. Recently, the chloroplast protein HCF101 (high chlorophyll fluorescence 101) has been shown to be essential for the accumulation of the membrane complex Photosystem I and the soluble ferredoxin-thioredoxin reductases, both containing [4Fe-4S] clusters. The protein belongs to the FSC-NTPase ([4Fe-4S]-cluster-containing P-loop NTPase) superfamily, several members of which play a crucial role in Fe/S cluster biosynthesis. Although the C-terminal ISC-binding site, conserved in other members of the FSC-NTPase family, is not present in chloroplast HCF101 homologues using Mössbauer and EPR spectroscopy, we provide evidence that HCF101 binds a [4Fe-4S] cluster. 55Fe incorporation studies of mitochondrially targeted HCF101 in Saccharomyces cerevisiae confirmed the assembly of an Fe/S cluster in HCF101 in an Nfs1-dependent manner. Site-directed mutagenesis identified three HCF101-specific cysteine residues required for assembly and/or stability of the cluster. We further demonstrate that the reconstituted cluster is transiently bound and can be transferred from HCF101 to a [4Fe-4S] apoprotein. Together, our findings suggest that HCF101 may serve as a chloroplast scaffold protein that specifically assembles [4Fe-4S] clusters and transfers them to the chloroplast membrane and soluble target proteins.
Schwenkert
Serena
S
Biozentrum der Ludwig-Maximilians-Universität München, Department Biologie I, Lehrstuhl für Botanik, Grosshaderner Strasse 2-4, 82152 Planegg-Martinsried, Germany.
Netz
Daili J A
DJ
Frazzon
Jeverson
J
Pierik
Antonio J
AJ
Bill
Eckhard
E
Gross
Jeferson
J
Lill
Roland
R
Meurer
Jörg
J
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002736:Chloroplasts / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods
D029681:Arabidopsis Proteins; C484275:HCF101 protein, Arabidopsis; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; C021588:ferredoxin-thioredoxin reductase; D003545:Cysteine