USE OF A NOVEL HISTIDYL MODIFIER TO PROBE FOR RESIDUES ON TRIS-TREATED PHOTOSYSTEM-II MEMBRANE-FRAGMENTS THAT MAY BIND FUNCTIONAL MANGANESE

In this paper, we investigate the effects of histidyl amino acid modif ication on high-affinity Mn binding to photosystem II (PSII) using met hods similar to those used in the preceding paper [Ghirardi et al. (19 98) Biochemistry 37, 0000] for carboxyl amino acid modification. Given the rather low specificity of diethyl pyrocarbonate (DEPC) for histid ine modification, we modified Tris-washed PSII membranes with a novel and more specific histidyl modifier, platinum(II) (2,2':6',2 ''-terpyr idine) chloride (Pt-TP). Both the ''diphenylcarbazide (DPC)-inhibition assay'' and single-turnover flash approaches were used. The concentra tion dependence of Pt-TP modification on steady-state measurements sho ws two types of interactions, each accounting for about half of the fu ll effect. At concentrations <50 mu M, Pt-TP modifies mostly histidyls and abolishes half of the observed Mn inhibition of DPC-mediated 2,6- dichlorophenol-indophenol (DCIP) photoreduction (equivalent to two hig h-affinity, Mn-binding ligands). This effect can be blocked by additio n of Mn2+ during Pt-TP modification. Double-modification experiments w ith DEPC and Pt-TP demonstrate that both modifiers affect the same obs ervable histidyl residues in PSII. Above 50 mu M, Pt-TP modifies mostl y cysteines (or histidines in a more hydrophobic environment) and has an additional effect on the reducing side of PSII that (a) does not in volve Mn binding and (b) results in the apparent abolishment of all fo ur of the Mn-binding ligands detected by the DPC-inhibition assay, Sin gle-flash experiments show that histidyl modification does not elimina te the binding of the high-affinity, photooxidizable Mn2+ to Asp170 on D1 (nor does it significantly affect high-affinity DPC photooxidation ), but it does decrease the binding affinity (K-d) of that Mn from 0.6 to 1.5 mu M, particularly at lower (<50 mu M Pt-TP) concentrations. D ouble-modification experiments also demonstrate that the lower affinit y, photooxidizable Mn-binding site, uncovered when the high-affinity s ite is modified with 1-ethyl-3-[3-(dimethylamino)propyl]carbo hydrochl oride (EDC) [see Ghirardi et al. (1998)], is not associated with a his tidyl ligand. Three nonphotooxidizable, high-affinity Mn2+ ions bind t o a second carboxyl and two histidyl ligands, and these Mn are not pho tooxidized by a flash even when the ligand to the photooxidizable Mn i s modified by EDC. Proteolytic enzyme studies indicate that the two hi stidyl ligands identified by the DPC-inhibition assay are probably His 337 on D1 and His 339 on D2, but His 332 on D1 is not eliminated.