Introduction of a [4Fe‐4S (S‐cys)4]^+1,+2 iron‐sulfur center into a four‐α helix protein using design parameters from the domain of the F_x cluster in the Photosystem I reaction center

Abstract

We describe the insertion of an iron‐sulfur center into a designed four α‐helix model protein. The model protein was re‐engineered by introducing four cysteine ligands required for the coordination of the mulinucleate cluster into positions in the main‐chain directly analogous to the domain predicted to ligand the interpeptide [4Fe‐4S (S‐cys)4] cluster, Fx, from PsaA and PsaB of the Photosystem I reaction center. This was achieved by inserting the sequence, CDGPGRGGTC, which is conserved in PsaA and PsaB, into interhelical loops 1 and 3 of the four α‐helix model. The holoprotein was characterized spectroscopically after insertion of the iron‐sulfur center in vitro. EPR spectra confirmed the cluster is a [4Fe‐4S] type, indicating that the cysteine thiolate ligands were positioned as designed. The midpoint potential of the iron‐sulfur center in the model holoprotein was determined via redox titration and shown to be −422 mV (pH 8.3, n = 1). The results support proposals advanced for the structure of the domain of the [4Fe‐4S] Fx cluster in Photosystem I based upon sequence predictions and molecular modeling. We suggest that the lower potential of the Fx cluster is most likely due to factors in the protein environment of Fx rather than the identity of the residues proximal to the coordinating ligands.

Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii

“…The shape of the EPR signals of the two samples were similar; the better resolution of the signals in the H 6 -PS1 sample is likely due to their higher purity, since equal amounts of Chl were present in each tube. Both samples therefore contain photoreduced [4Fe-4S] + clusters (Scott and Biggins 1997). The EPR spectra exhibited no other resonances in the low-field region (g [ 3), indicating the absence of any high-spin iron (data not shown).…”

“…The EPR spectra exhibited no other resonances in the low-field region (g [ 3), indicating the absence of any high-spin iron (data not shown). Thus, the EPR-active species can be characterized as low-spin (S = 1/2) FeS centers with unaltered cysteine ligands (Scott and Biggins 1997). In the H 6 -PS1 sample, the EPR resonances observed at g = 1.84, *1.93, and *2.04 are characteristic of F A - (Fromme et al 2002;Jung et al 1996).…”

Protein Design: Toward Functional Metalloenzymes

“…A [4Fe–4S] center incorporated in a de novo designed α -helix bundle was previously shown to have a midpoint redox potential of −442 mV (at pH 8.2, vs NHE). 912 They attributed the decreased redox potential as compared to the other mimetic system to the H-bond interactions of sulfur atoms in the cluster with the backbone amide protons, and the easy solvent accessibility of their models revealed by ESEEM and 2 H ENDOR. Furthermore, they showed that these models can bind to the native P700-F x core and participate in the light-induced electron transfer.…”

Artificial photoactive proteins

“…The yield of the 4Fe-4S cluster was very low, when all intervening amino acids were exchanged for glycine, GGCGGGCGGCGGCGGW, while holding the cysteines in their consensus. A similar approach was applied to form a single 4Fe-4S cluster resembling the F X cluster of PSI by inserting the 10-residue CDGPGRGGTC sequence into the interhelical loops 1 and 3 of the a4 protein which was originally designed to form a synthetic four-helix bundle (Regan and DeGrado 1988;Scott and Biggins 1997). A miniferredoxin protein was also created from a 31-residue peptide carrying a 4Fe-4S cluster with a redox potential of -370 mV (Sow et al 1996).…”