TY - JOUR
T1 - Chemical rescue of a site-modified ligand to a [4Fe-4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to Photosystem I
AU - Antonkine, Mikhail L.
AU - Maes, Estelle M.
AU - Czernuszewicz, Roman S.
AU - Breitenstein, Christoph
AU - Bill, Eckhard
AU - Falzone, Christopher J.
AU - Balasubramanian, Ramakrishnan
AU - Lubner, Carolyn
AU - Bryant, Donald A.
AU - Golbeck, John H.
N1 - Funding Information:
We thank Alan Benesi (director of NMR facility at Pennsylvania State University) for his help and cooperation. Technical help from Andrew Hansen, Bernt Minert and Irina Michin is gratefully acknowledged. We thank Veronica L. Stirewalt for technical assistance in the construction of the site-specific mutations in psaC . We are grateful to Keiichi Fukuyama from Department of Biology of the Osaka University for discussions of the structure of mono cluster ferredoxins. We sincerely thank Anton Savitsky for fruitful discussion of magnetic interaction between two reduced iron–sulfur clusters in dicluster ferredoxins. We also thank Dietmar Stehlik for stimulating discussions. We acknowledge Wolfgang Lubitz and Wolfgang Gärtner for use of the biological laboratory at the Max-Planck Institute for Bioinorganic Chemistry, which allowed preparation of the Mössbauer samples of the C13G/C33S PsaC variant. The Max-Planck Society, specifically Max-Planck Institute for Bioinorganic Chemistry, is gratefully acknowledged for funding of the Mössbauer studies. Welch Foundation grant E-1184 to R.S.C. is acknowledged for funding of resonance Raman work. A major portion of the overall work was supported by the Department of Energy (DE-FG-02-98-ER20314) and by the National Science Foundation (MCB-0117079) to J.H.G. the National Science Foundation (MCB-0077586) to D.A.B and Sfb 498 (project A3) to M.L.A. are acknowledged for additional funding.
PY - 2007/6
Y1 - 2007/6
N2 - Chemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure-function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron-sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe-4S]1+,2+ cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe-4S]1+,2+ clusters termed FA and FB. In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron-sulfur cluster FB. Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and Mössbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe-4S]1+,2+ clusters, despite the absence of one of the biological ligands. 19F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the FB cluster. The finding that site-modified [4Fe-4S]1+,2+ clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand.
AB - Chemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure-function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron-sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe-4S]1+,2+ cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe-4S]1+,2+ clusters termed FA and FB. In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron-sulfur cluster FB. Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and Mössbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe-4S]1+,2+ clusters, despite the absence of one of the biological ligands. 19F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the FB cluster. The finding that site-modified [4Fe-4S]1+,2+ clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand.
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U2 - 10.1016/j.bbabio.2007.02.003
DO - 10.1016/j.bbabio.2007.02.003
M3 - Article
C2 - 17434441
AN - SCOPUS:34249804791
SN - 0005-2728
VL - 1767
SP - 712
EP - 724
JO - Biochimica et Biophysica Acta - Bioenergetics
JF - Biochimica et Biophysica Acta - Bioenergetics
IS - 6
ER -