Study of the Mn-binding sites in photosystem II using antibodies raised against lumenal regions of the D1 and D2 reaction center proteins

Abstract

The experiments discussed in this tl_esi.sfocus on identifying the protein segments or specific amino acids which provide ligands to the Mn cluster of photosystcm H (PS 11).This Mn cluster plays a central role in the oxygen-evolving complex (OEC) of PS H. The Mn cluster is thought to be bound by lumenal regions of the PS II reaction center proteins known as D 1 and D2. First, several peptides were synthesized which correspond to • specific lumenal segments of the D1 and D2 proteins. Next, polyclonal antibodies were successfufly elicited using three of these peptides. The peptides recognized by these antibodies correspond to protein segments of the spinach reaction center proteins: I1e-321 to l_ Ala-344 of D1 (DI-a), Asp-319 to Arg-334 of D1 (Dl-b), and Val-300 to Asn-319 of D2 (D2-a). These antibodies were then used in assays which were developed to structurally or°f unctionally probe the potential Mn-binding regions of the D I and D2 proteins. -The two assays yielding structural information were western blotting and solution binding experiments. Western blotting identified the denatm_ed proteins of spinach PS II membrane preparations to which the antibodies bound. The solution antibody binding assay was used to confirm antibody binding to native PS II membranes. Three assays which examined functional aspects of PS II were measurement of oxygen evolution, yield of photoactivation, and MnCI2 inhibition of diphenylcarbazide (DPC) to 2,6-dichlorophenol indophenol (DCIP)photoreduction. Assaysofoxygenevolution tested the ability ofantibodies toaffect thenaturally boundMn ofPS II. The yield ofphotoa"°ivation assays investigated theability ofantibody toblock thebinding andsubsequent photoligation ofMn toPS IImembraneslacking theendogenous Mn cluster. Finally, experiments whichassayed theMnCl2 inl_bition ofDPC toDCIP photoreduction addressed whethcr boundantibody couldblock anyoftheibur components ofthehighaffinity Mn-binding site that becomeavailable inthepresence ofDPC. The results ofthese experiments include thefollowing: Allthree antibodies reacted withtheir corresponding denatured protein (DIorD2) on western blots. In_lution binding assays, thebinding ofthethree antibodies toseveral PS IIpreparations was demonstrated andcharacterized. The first antibodies available, those against DI-a,were, usedininitial experiments toexamineeffects onoxygcnevolution. UsingPS IImembrane preparations ofvarying cxtriP._ic protein composition, no adverse effects ofthese antibodies wereobserved onoxygenevolution. Alldlrce antibodies werethenusc_totest for possible effects on theability tophotoactivate theMn cluster oftheOEC. Theseassays showedno effect for theantibodies recogifizing theD l-borD2-a peptide and some decrease inyield fortheantibodies recognizing theD 1-apeptide. Finally, incubation of Tris-treated PS II membrane preparations with antibodies to the D 1-a peptide resulted in an inhibition of one component of the high-aff'mity Mn-binding site as measured by the MnC12 inhibition of DPC to DCIP photoreduction. This did not occu...