Biochemical evidence for the role of the bound iron-sulphur centres A and B in NADP reduction by Photosystem I

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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] terredoxin and is considered to be the solubilised 2[FeS A/B centres of Photosystem I. Oxidation-reduction potential titration of the protein showed E m ≈ −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 t 1 2 = 12 ms to t 1 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. The rereduction of P700 + returned to t 1 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 + .