- Antonkine, Mikhail L.
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Doctor of Philosophy
- Document Type:
- Date of Defense:
- March 14, 2001
- Committee Members:
- John H Golbeck, Committee Chair/Co-Chair
Donald Ashley Bryant, Committee Member
Christain J Falzone, Committee Member
B Tracy Nixon, Committee Member
James Gregory Ferry, Committee Member
- The PsaC subunit of Photosystem I (PS I) is a small (9.3 kDa) protein that harbors binding sites for two [4Fe-4S] clusters usually referred to as FA and FB. PsaC is presumed to have evolved from a soluble bacterial ferredoxin. It has a high sequence similarity to dicluster ferredoxins in the vicinity of two iron-sulfur cluster consensus binding sites CxxCxxCxxxCP. PsaC has an additional sequence insertion between the binding sites for the two clusters as well as a C-terminal extension. The g-tensor orientation of FA- and FB- is believed to be correlated to the preferential localization of the mixed-valence and equal-valence (ferrous) iron pairs in each [4Fe-4S]1+ cluster. Previous studies of ferrodoxins show that the preferential position of the mixed-valence and equal-valence pairs could be inferred from the study of the temperature dependence of contact-shifted resonances, belonging to bCH2 and Ha protons of cysteines ligating iron-sulfur cluster, by 1H NMR spectroscopy. For this, a sequence-specific assignment of these signals is required. Despite its relative instability, 1H NMR spectra of fully reduced, unbound PsaC could be recorded and a self-consistent, sequence-specific and stereospecific assignment of all 18 observable hyperfine-shifted signals through a model-based 1D NOE analysis was obtained. This enabled me to assign sequence-specifically the signals of three cysteines from each of the two reduced [4Fe-4S] clusters and to obtain the preferential localization of mixed- and equal-valence pairs in FA- and FB-. In FA- the mixed-valence pair is ligated by cysteines 47 and 53; the equal valence pair is ligated by cysteines 20 and 50. In FB- the mixed-valence pair is ligated by cysteines 10 and 16; the equal valence pair is ligated by cysteines 13 and 57. From the position of the equal- and mixed-valance pairs, the orientation of the g-tensor in [4Fe-4S] centers FA and FB was inferred. The magnetic properties of the two [4Fe-4S] clusters in unbound, reduced PsaC are essentially indistinguishable, though they are quite different in PsaC bound to PS I. From the changes in the g-tensor of FA- and FB- that occur upon binding of PsaC to the PS I core, it is likely that the protein undergoes significant structural changes upon binding to PS I. To study these changes, it is necessary to know the three-dimensional structure of both bound and unbound PsaC. The Clostridium acidi urici ferredoxin structure has served as a model for the refinement of a portion of the electron density map in a 4 Å resolution X-ray structure of PS I (Klukas, O., Schubert, W.D., Jordan, P., Krauss, N., Fromme, P., Witt, H.T. and Saenger, W. (1999) J. Biol. Chem., 274, 7351-60). Thus a model for PsaC bound to PS I exists. However, no X-ray structure of unbound PsaC was available. In this work the three-dimensional NMR solution structure of recombinant, oxidized, unbound PsaC from Synechococcus sp. PCC 7002 was determined. Constraints are derived from homo- and heteronuclear, one-, two- and three-dimensional NMR data. The overall quality of the NMR solution structure is equivalent to approx. 3 Å resolution by X-ray crystallography. PsaC is only the third two [4Fe-4S] cluster-containing, low-potential, iron-sulfur ferredoxin-like protein whose structure has been determined in solution. Moreover, PsaC is the rare case of a protein tightly bound to a membrane protein complex, whose structure is known in both the bound and unbound states. It is the only dicluster ferredoxin-like protein studied in such a context. Significant differences are observed between the unbound PsaC structure presented here and the available X-ray structure of bound PsaC as an integral part of the whole cyanobacterial PS I complex. These differences mainly concern the arrangement of the N- and C-termini with respect to the [4Fe-4S] core domains. In the NMR solution structure of PsaC, the C-terminal region assumes a disordered helical conformation. This is clearly different from the extended coil conformation, which is one of the structural elements that anchors PsaC to the PS I core polypeptides. The N-terminal end of PsaC bends and slides in between the coiled-up C-terminus and the iron-sulfur core of the protein. These structural changes result in a concerted movement of the N- and C-termini of PsaC away from the FA binding site. The same regions are positioned much closer to FA in a near parallel arrangement to each other in PsaC bound to PS I. The observed structural features in solution may be relevant for the proposed stepwise assembly of the stromal PsaC, PsaD and PsaE subunits to the PS I core heterodimer. Cysteine 13 and cysteine 50, which ligate FB and FA respectively, were each changed to a serine, aspartate, alanine, or glycine in unbound PsaC in the previous work. It was proposed that 2-mercaptoethanol, a reagent used for iron-sulfur cluster reconstitution, serves as an external rescue ligand in the absence of a biological ligand to the fourth iron (Yu, L.A., Vassiliev, I.R., Jung, Y.S., Bryant, D.A., Golbeck, J.H. (1995) J. Biol. Chem., 270, 28118-28125). Instead of the expected [3Fe-4S] cluster, chemical rescue by an external thiolate ligand allows for formation of a [4Fe-4S] cluster in the alanine and glycine mutants. To support this hypothesis, C13G/C33S PsaC was reconstituted using 2-mercaptoethanol, 4-fluorothiophenol and 2,2,2-trifluoro-ethanethiol as the rescue ligands to FB. Resonance Raman and EPR data provide strong evidence that only the [4Fe-4S] clusters are assembled in C13G/C33S PsaC in the absence of a suitable biological ligand. Upon reconstitution of the mutant PsaC with 4-fluorothiophenol or 2,2,2-trifluoroethanethiol, it was possible to detect hyperfine contact-shifted 19F resonances by NMR spectroscopy showing that a thiolate ligand is bound to FB in C13G/C33S mutant PsaC, when Cysteine II is changed to amino acid that can not ligate iron-sulfur cluster. This proves the chemical rescue hypothesis. The results presented here show that both aryl and acyl thiolates can serve as external thiolate ligands to protein-bound [4Fe-4S] clusters.