BIOCHEMICAL-EVIDENCE FOR THE ROLE OF THE BOUND IRON-SULFUR CENTER-A AND CENTER-B IN NADP REDUCTION BY PHOTOSYSTEM-I

The Photosystem I reaction centre contains three iron-sulphur centres. Although extensively characterised at low temperature, the role of these centres in room-temperature electron transport to NADP has never been demonstrated. An iron-sulphur protein was solubilised from spinach Photosystem I particles by butanol extraction. The reduced protein has the characteristic EPR spectrum of a 2[4Fe4S] ferredoxin and is considered to be the solubilised Fe-S(A/B) centres of Photosystem I. Oxidation-reduction potential titration of the protein showed E(m) almost-equal-to -510 mV. Photosystem I particles depleted of the Fe-S(A/B) centres were prepared by urea treatment. The depleted preparation had only 15% of the original EPR signals due to the Fe-S(A/B) centres, showed a rate of P700+ rereduction following flash illumination changed from t1/2 = 12 ms to t1/2 = 1 ms, and catalysed NADP+ photoreduction at only 5% of the initial rate. Reconstitution with the solubilised protein led to recovery of the EPR spectrum (80%) and low-temperature electron transfer from P700 to Fe-S(A). The rereduction of P700+ returned to t1/2 = 12 ms and NADP+ reduction was recovered to 90% of the initial rate. Oxidative destruction of the iron-sulphur centres in the solubilised protein prevented reconstitution. The results show that the Fe-S(A/B) a centres can be removed from Photosystem I and reconstituted, and that these iron-sulphur centres are essential components of the overall electron transfer to NADP+.