Spectroscopic characterization of photosystem I: Functional study of electron acceptors A(1), F(X), F(B) and F(A)

A Photosystem I core has been isolated which contains functional P700, A$\sb0$ and A$\sb1$. The properties of this preparation are (i) over 90% of the flash-induced absorption change at 820 nm decays with a 10 $\mu$s half-time at room temperature, and a 200 $\mu$s half-time at 77 K, (ii) the terminal acceptor is chemically reducible and (iii) the room temperature flash-induced difference spectrum from 380 to 480 nm and from 720 to 910 nm shows a P700 cation. Addition of 0.1% Triton X-100 displaces the 10 $\mu$s transient with a 25 ns halftime representing direct backreaction between A$\sb0$ and P700 and a low P700 triplet yield with a decay half-time of 3 $\mu$s. A fusion protein, PsaC1, containing an amino-terminal extension of five amino acids and PsaD can be combined with the Photosystem I P700-F$\sb{\rm x}$ core to reconstitute electron transport from P700 to F$\sb{\rm A}$/F$\sb{\rm B}$. Reconstitution is dependent on reinsertion of the iron-sulfur clusters in PsaC1 and on the presence of the PsaD protein. Added PsaD enhances magnetic interaction between F$\sb{\rm A}$ and F$\sb{\rm B}$ in the in vitro reconstituted Photosystem I complex and in a PsaD-strain of the cyanobacterium Synechocystis sp. PCC 6803, probably by restricting the conformational flexibility of PsaC. The linewidth and g-values of the F$\sb{\rm A}$ and F$\sb{\rm B}$ are independent of the presence of PsaE indicating that PsaE cannot substitute for PsaD. The site-directed conversion of cysteine 565 to serine in PsaB of Synechocystis sp. PCC 6803 leads to an accumulation of Photosystem I polypeptides and the low temperature photoreduction of the terminal electron acceptors F$\sb{\rm A}$ and F$\sb{\rm B}$. A dodecyl maltoside Photosystem I complex prepared from the C565S mutant contains a (3Fe-4S) cluster which can be photochemically reduced and reoxidized and chemically reduced with dithionite at pH 6.5. The mutant complex also contains a mixed-ligand (4Fe-4S) cluster located in the F$\sb{\rm X}$ site, which has a redox potential low enough to transfer electrons to F$\sb{\rm A}$/F$\sb{\rm B}$ and is photochemically reversible