The lumenal loop connecting transmembrane helices I and II of the D1 polypeptide is important for assembly of the photosystem two complex

Current structural models indicate that the D1 and D2 polypeptides of the Photosystem two reaction center complex (PS II RC) each span the thylakoid membrane five times. In order to assess the importance of the lumenal extrinsic loop that connects transmembrane helices I and II of D1 we have constructed five deletion mutants and two double mutants in the cyanobaterium Synechocystic sp. PCC 6803. Four of the deletion mutants (59–65, 69–74, 79–86 and 109–110) are obligate photoheterotrophs unable to accumulate D1 in the membrane as assayed by immunoblotting experiments or pulse-labelling experiments using [³⁵S]-methionine. In contrast deletion mutant 100 which lacks A100 behaved very similarly to the WT control strain in terms of photoautotrophic growth rate, saturated rates of oxygen evolution, flash-induced oxygen evolution, fluorescence induction and decay, and thermoluminescence. 100 is the first example of an internal deletion on the lumenal side of the D1 polypeptide that is benign to photosystem two function. Double mutant D103G/E104A also behaves similarly to the WT control strain leading to the conclusion that residues D103 and E104 are unlikely to be involved in ligating the metal ions Mn or Ca²⁺, which are needed for photosynthetic oxygen evolution. Double mutant, G109A/G110A, was constructed to assess the significance of this GlyGly motif which is also conserved in the L subunit of purple bacterial reaction centres. The G109A/G110A mutant is able to evolve oxygen at approximately 50–70% of WT rates but is unable to grow phatoautotrophically apparently because of an enhanced sensitivity to photoinactivation than the WT control strain. A photoautotropic revertant was isolated from this strain and shown to result from a mutation that restored the WT codon at position 109. Pulse-chase experiments in cells using [³⁵S]-methionine showed that resistance to photoinhibition in the revertant correlated with an enhanced rate of incorporation of D1 into the membrane compared to mutant G109A/G110A. The sensitivity to photoinhibition shown by the G109A/G110A mutant is therefore consistent with a perturbation to the D1 repair cycle possibly at the level of D1 synthesis or incorporation of D1 into the PS II complex.Abbreviations DCMU- 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Hepes- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes- 4-morpholineethanesulfonic acid - PCR- polymerase chain reaction - PS II- Photosystem II - TL- thermoluminescence - PQ- plastoquinone - PS II^–- absence of PS II activity - PS^–- incapable of photoautotrophic growth - Q_A- primary plastoquinone electron acceptor - Q_B- secondary plastoquinone electron acceptor - SDS- sodium dodecyl sulphate