The oxidation-reduction potential of the reaction-centre chlorophyll (P700) in Photosystem I. Evidence for multiple components in electron-paramagnetic-resonance signal 1 at low temperature

Abstract

The oxidation-reduction potential of the reaction-center chlorophyll of Photosystem I (P700) in spinach [Spinacia oleracea] chloroplasts was determined by using the ability of the reaction center to photoreduce the bound ferredoxin and to photo-oxidize P700 on illumination at 20.degree. K as an indicator of the oxidation state of P700. This procedure shows that P700 is oxidized with Em [membrane potential] (pH 8.0) (mid-point redox potential at pH 8.0) .simeq. +375 mV. Further oxidation of the chloroplast preparations by high concentrations of K3Fe(CN)6 (10 mM) in the presence of mediating dyes leads to the appearance of a large radical signal with an apparent Em .simeq. +470 mV. A 2nd, light-inducible, radical appears over the same potential range. Possibly these signals are due to bulk chlorophyll oxidation and not, as was previously thought, to reaction-center oxidation. A number of optical techniques were used to determine Em of P700. Dual-wavelength spectroscopy (697-720 nm) indicates Em .simeq. +460 to +480 mV. The spectrum of the sample during the titration showed a large contribution to the signal by bulk chlorophyll oxidation, in agreement with the EPR results and those of Ke, Sugahara and Shaw. 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-vs.-reduced difference spectra showed Em (pH 8.0) .simeq. +360 mV. Measurements of the effect of potential on the irreversible photo-oxidation of P700 at 77.degree. K showed that P700 became oxidized in this potential range. It is concluded that the reaction-center chlorophyll of Photosystem I has Em (pH 8.0) .simeq. +375 mV.