The role of the membrane-bound iron-sulphur centres A and B in the Photosystem I reaction centre of spinach chloroplasts

Photosystem I particles prepared from spinach chloroplast using Triton X-100 were frozen in the dark with the bound iron-sulphur Centre A reduced. Illumination at cryogenic temperatures of such samples demonstrated the photoreduction of the second bound iron-sulphur Centre B. Due to electron spin-electron spin interaction between these two bound iron-sulphur centres, it was not possible to quantify amounts of Centre B relative to the other components of the Photosystem I reaction centre by simulating the line-shape of its EPR spectrum. However, by deleting the free radical signal I from the EPR spectra of reduced Centre A alone or both Centres A plus B reduced, it was possible to double integrate these spectra to demonstrate that Centre B is present in the Photosystem I reaction centre in amounts comparable to those of Centre A and thus also signal I ($\text{P-700}$) and X.Oxidation-reduction potential titrations confirmed that Centre A had $\text{E}{}_{\mathrm{<mtext>m</mtext>}}\text{? ?550}\text{mV}$, Centre B had $\text{E}{}_{\mathrm{<mtext>m</mtext>}}\text{? ?585}\text{mV}$. These results, and those presented for the photoreduction of Centre B, place Centre B before Centre A in the sequence of electron transport in Photosystem I particles at cryogenic temperatures. When both A and B are reduced, $\text{P-700}$ photooxidation is reversible at low temperature and coupled to the reduction of the component X. The change from irreversible to reversible $\text{P-700}$ photooxidation and the photoreduction of X showed the same potential dependence as the reduction of Centre B with $\text{E}{}_{\mathrm{<mtext>m</mtext>}}\text{? ?585}$ mV, substantiating the identification of X as the primary electron acceptor of Photosystem I.