Near infrared in vitro measurements of photosystem I cofactors and electron- transfer partners with a recently-developed spectrophotometer
Résumé
A kinetic-LED-array-spectrophotometer (Klas) was recently developed for measuring in vivoredox changes of P700, plastocyanin (PCy)and ferredoxin (Fd)in the near-infrared (NIR). This spectrophotomer is used in the present work for in vitrolight-induced measurements withvariouscombinations of photosystem I(PSI)fromtobacco and2 different cyanobacteria, spinach plastocyanin,cyanobacterial cytochrome c6(cyt. c6) and Fd. It is shownthat cyt. c6oxidation contributesto the NIR absorption changes.The reduction of (FAFB), the terminalelectron acceptor of PSI, was also observed and the shape of the (FAFB) NIR difference spectrum is similar to that of Fd.The NIR difference spectra of the electron-transfer cofactorswere compared betweendifferentorganisms and to those previously measured in vivowhereasthe relative absorption coefficients of all cofactorswere determined by using single PSI turnover conditions.Thus, the (840 nm minus965 nm) extinction coefficients of the light-induced species (oxidized minusreduced for PC and cyt. c6, reduced minusoxidized for (FAFB) and Fd) were determined with valuesof 0.207±0.004, -0.033±0.006, -0.036±0.008 and -0.021±0.005 for PCy, cyt. c6, (FAFB) (single reduction) and Fd, respectively, by taking a reference value of +1 for P700+.The fact that the NIR P700 coefficient is largerthan that of PCy and much larger than that of other contributing species, combined withthe observed variability in the NIR P700spectral shape, emphasizes that deconvolution of NIR signals into different components requires a very precise determination of the P700 spectrum.
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