On the depletion and reconstitution of both QA and metal in reaction centers of the photosynthetic bacterium Rb. sphaeroides R-26

Four possible ways to prepare QA-depleted, Fe-depleted and QA-reconstituted RCs were studied: (1) first depleting the Fe, then depleting QA and finally reconstituting QA (D-Fe, D-Q, R-Q), (2) first depleting QA, then depleting the Fe and finally reconstituting QA (D-Q, D-Fe, R-Q), (3) first depleting QA, then reconstituting QA and finally depleting Fe (D-Q, R-Q, D-Fe), (4) first depleting QA, then depleting the Fe and reconstituting QA in the same step (D-Q, D-Fe-R-Q). Our results showed that: method (1) results in the irreversible loss of photochemical activity; method (2) and (3) result in low recovery of the photochemical activity and poor yield of Fe-depleted, QA-reconstituted RCs; method (4) gives surprisingly good results. This method allows for the first time to prepare the QA-depleted, Fe-depleted, QA-reconstituted RCs with high recovery of the photochemical activity and good yield. The sample has 98% of photochemical activity (yield of P+ QA-) compared with that of the native RCs and shows strong polarization of the EPR signal of QA- under continuous illumination at 5K. The decay halftime of I- is slow (~5 ns) compared with that of the native RCs, but it is the same as that measured for the RCs from which only iron is removed. These results indicate that the depletion of iron and the reconstitution of QA have been successful. Reconstitution of the QA-depleted, Fe-depleted and QA-reconstituted RCs with Zn2+ gives also the spin-polarized QA-, and yields the same decay of I- (halftime 200 ps) as that of the native RCs.