Near-infrared in vitro measurements of photosystem I cofactors and electron-transfer partners with a recently developed spectrophotometer

A kinetic-LED-array-spectrophotometer (Klas) was recently developed for measuring in vivo redox changes of P700, plastocyanin (PCy), and ferredoxin (Fd) in the near-infrared (NIR). This spectrophotometer is used in the present work for in vitro light-induced measurements with various combinations of photosystem I (PSI) from tobacco and two different cyanobacteria, spinach plastocyanin, cyanobacterial cytochrome c₆ (cyt. c₆), and Fd. It is shown that cyt. c₆ oxidation contributes to the NIR absorption changes. The reduction of (F_AF_B), the terminal electron acceptor of PSI, was also observed and the shape of the (F_AF_B) NIR difference spectrum is similar to that of Fd. The NIR difference spectra of the electron-transfer cofactors were compared between different organisms and to those previously measured in vivo, whereas the relative absorption coefficients of all cofactors were determined by using single PSI turnover conditions. Thus, the (840 nm minus 965 nm) extinction coefficients of the light-induced species (oxidized minus reduced for PC and cyt. c₆, reduced minus oxidized for (F_AF_B), and Fd) were determined with values of 0.207?±?0.004, –?0.033?±?0.006, –?0.036?±?0.008, and –?0.021?±?0.005 for PCy, cyt. c₆, (F_AF_B) (single reduction), and Fd, respectively, by taking a reference value of +?1 for P700⁺. The fact that the NIR P700 coefficient is larger than that of PCy and much larger than that of other contributing species, combined with the observed variability in the NIR P700 spectral shape, emphasizes that deconvolution of NIR signals into different components requires a very precise determination of the P700 spectrum.