Abstract
The oxidation-reduction potential of the reaction-centre chlorophyll of Photosystem I (P700) in spinach chloroplasts was determined by using the ability of the reaction centre to photoreduce the bound ferredoxin and to photo-oxidize P700 on illumination at 20K as an indicator of the oxidation state of P700. This procedure shows that P700 is oxidized with Em (pH8.0)(mid-point redox potential at pH8.0)congruent to +375mV. Further oxidation of the chloroplast preparations by high concentrations of K3Fe(CN)6(10mM) in the presence of mediating dyes leads to the appearance of a large radical signal with an apparent Em congruent to +470mVA second, light-inducible, radical also appears over the same potential range. We propose that these signals are due to bulk chlorophyll oxidation and not, as was previously thought [Knaff & Malkin (1973) Arch. Biochem. Biophys. 159, 555-562], to reaction-centre oxidation. A number of optical techniques were used to determine Em of P700. Dual-wavelength spectroscopy (697-720nm) indicates Em congruent to +460-+480mV. The spectrum of the sample during the titration showed a large contribution to the signal by bulk chlorophyll oxidation, in agreement with the electron-paramagnetic-resonance results and those of Ke, Sugahara & Shaw [(1975) Biochim. Biophys. Acta 408, 12-25]. The light-induced absorbance change at 435 nm, usually attributed to P700, showed a potential dependence similar to that of bulk chlorophyll oxidation. Determination of Em of P700 on the basis of the appearance of the P700 signal in oxidized-versus-reduced difference spectra showed Em (pH8.0) congruent to +360mV. Measurements of the effect of potential on the irreversible photo-oxidation of P700 at 77K showed that P700 became oxidized in this potential range. We conclude that the reaction-centre chlorophyll of Photosystem I has Em (pH8.0) congruent to +375mV.