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Spectroscopic characterizations of modified iron-sulfur clusters F-x, F-A and F-B in cyanobacterial photosystem I
Abstract
Electron transfer through the F$\rm\sb{X}$, F$\rm\sb{A}$ and F$\rm\sb{B}$ iron-sulfur clusters in cyanobacterial Photosystem I(PS I) was investigated by chemical and genetic modification of the cysteine ligands. The major findings include: (i) The site-directed conversion of cysteine to serine at positions 565 and 556 in PsaB leads to an accumulation of PS I in Synechocystis sp. PCC 6803. F$\rm\sb{X}$ becomes a mixed-ligand (4Fe-4S) cluster, which is an inefficient electron acceptor from A$\sb1$; (ii) The replacement of the cysteine-adjacent aspartate residues in the F$\rm\sb{X}$-binding region with alanine or lysine influences the EPR spectral properties of F$\rm\sb{X}$, but it does not affect electron transfer efficiency. This implies that aspartate plays a structural rather than functional role in the F$\rm\sb{X}$-binding domain; (ii) The absence of PsaD and PsaE, but the presence of the PsaA-PsaB heterodimer in PsaC-less mutants indicate that PsaC is necessary for stable assembly of the peripheral subunits onto PS I complexes; (iv) While Escherichia coli-expressed PsaC mutant proteins, C14$\rm\sb{PsaC}$ and C51X$\rm\sb{PsaC}$ (X= S, A, G) can be rebound to isolated P$\sb{700}$-F$\rm\sb{X}$ cores, PsaC is not found in the in vivo C14A$\rm\sb{PsaC}$-PS I or C51A$\rm\sb{PsaC}$-PS I Synechocystis mutants. These studies show that only PsaC proteins incorporating two cubane clusters can bind to PS I; (v) In vitro studies show that a (4Fe-4S) cluster in the F$\rm\sb{A}$ or F$\rm\sb{B}$ site of C14X$\rm\sb{PsaC}$-PS I and C51X$\rm\sb{PsaC}$-PS I (X = A, G) can be chemically 'rescued' by an external thiolate provided by 2-mercaptoethanol or dithiothreitol. Both the 'rescued' (4Fe-4S) clusters and the mixed-ligand (oxygen-ligated) (4Fe-4S) clusters are capable of functioning as electron transfer intermediates in PS I; and (vi) Hg-treated PS I largely lacks the F$\rm\sb{B}$ cluster and is deficient in flavodoxin- or ferredoxin-mediated NADP$\sp+$ photoreduction. After reconstitution of F$\rm\sb{B}$, NADP$\sp+$ reduction is fully recovered, thus establishing a definitive role for this iron-sulfur cluster in NADP$\sp+$ photoreduction. Serial electron transfer from F$\rm\sb{X}$ to F$\rm\sb{A}$ to F$\rm\sb{B}$ is suggested.
Subject Area
Molecular biology|Biophysics|Biochemistry|Botany
Recommended Citation
Jung, Yean Sung, "Spectroscopic characterizations of modified iron-sulfur clusters F-x, F-A and F-B in cyanobacterial photosystem I" (1996). ETD collection for University of Nebraska-Lincoln. AAI9700092.
https://digitalcommons.unl.edu/dissertations/AAI9700092