Spectroscopic characterization of quinone acceptors and modified iron-sulfur clusters F(A) and F(B) in type I photosynthetic reaction centers

Electron transfer cofactors in Photosystem I (PS I) of cyanobacteria and the reaction center of green sulfur bacteria have been studied by a combination of biochemical and biophysical techniques. The major findings include: (1) electron transfer from P700 to F$\rm\sb{x}$ is unidirectional. Removal of the C$\sb3$-distal subunits, including PsaE/PsaF/PsaJ, in Triton X-100 isolated PS I complexes results in the loss of the EPR signal from the photoaccumulated phylloquinone anion radical. Only one of the two phylloquinones is photoactive and it is located at the intersection of the m and n helices associated with the PsaA polypeptide; (2) menaquinone-7 in the reaction center of green sulfur bacteria functions analogously to phylloquinone in cyanobacterial PS I. Two molecules of menaquinone-7 were found per P840. EPR spectroscopy showed a photoaccumulated signal with a g tensor similar to that of A$\sb1\sp-$ in PS I. This signal disappeared after a short period of illumination, reflecting a difference in the quinone binding sites between green sulfur bacteria and PS I; (3) the EPR signal of the $\rm F\sb{A}/F\sb{B}$ clusters from the unbound PscB polypeptide in Chlorobium tepidum exhibited an interaction spectrum from two iron-sulfur clusters similar to that of PsaC in PS I. This demonstrates the equality of these two reaction centers in terms of the terminal electron acceptors; (4) aryl thiolates were proven to be functional in the reconstitution of iron-sulfur clusters in the unbound mutant C14G-PsaC. The EPR spectrum of F$\rm\sb{A}$-deleted PsaC demonstrated that the signal from the site-modified PsaC in $g=2.0$ region was exclusively from the unmodified cluster; (5) the finding of a (4Fe-4S) cluster EPR signal in a three-cysteine ligand containing synthetic protein, a 4$\alpha$-helical bundle, supports a chemical rescue hypothesis where $\beta$-mercaptoethanol serves as the fourth ligand. Differences in the lineshapes and the g values of (4Fe-4Se) clusters from single- and di-cluster proteins reflect differences in the protein environment near the cluster binding sites