Use of a novel histidyl modifier to probe for residues on tris-treated photosystem II membrane fragments that may bind functional manganese

被引:29
作者
Ghirardi, ML
Preston, C
Seibert, M [1 ]
机构
[1] Natl Renewable Energy Lab, Basic Sci Ctr, Golden, CO 80401 USA
[2] Univ Adelaide, Waite Agr Res Inst, Dept Plant Protect, Glen Osmond, SA 5064, Australia
关键词
D O I
10.1021/bi980359o
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
In this paper, we investigate the effects of histidyl amino acid modification on high-affinity Mn binding to photosystem II (PSII) using methods similar to those used in the preceding paper [Ghirardi et al. (1998) Biochemistry 37, 0000] for carboxyl amino acid modification. Given the rather low specificity of diethyl pyrocarbonate (DEPC) for histidine modification, we modified Tris-washed PSII membranes with a novel and more specific histidyl modifier, platinum(II) (2,2':6',2 "-terpyridine) chloride (Pt-TP). Both the "diphenylcarbazide (DPC)-inhibition assay" and single-turnover flash approaches were used. The concentration dependence of Pt-TP modification on steady-state measurements shows two types of interactions, each accounting for about half of the full 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 high-affinity, Mn-binding ligands). This effect can be blocked by addition of Mn2+ during Pt-TP modification. Double-modification experiments with DEPC and Pt-TP demonstrate that both modifiers affect the same observable histidyl residues in PSII. Above 50 mu M, Pt-TP modifies mostly cysteines (or histidines in a more hydrophobic environment) and has an additional effect on the reducing side of PSII that (a) does not involve Mn binding and (b) results in the apparent abolishment of all four of the Mn-binding ligands detected by the DPC-inhibition assay, Single-flash experiments show that histidyl modification does not eliminate 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. Double-modification experiments also demonstrate that the lower affinity, photooxidizable Mn-binding site, uncovered when the high-affinity site is modified with 1-ethyl-3-[3-(dimethylamino)propyl]carbo hydrochloride (EDC) [see Ghirardi et al. (1998)], is not associated with a histidyl ligand. Three nonphotooxidizable, high-affinity Mn2+ ions bind to a second carboxyl and two histidyl ligands, and these Mn are not photooxidized by a flash even when the ligand to the photooxidizable Mn is modified by EDC. Proteolytic enzyme studies indicate that the two histidyl ligands identified by the DPC-inhibition assay are probably His337 on D1 and His 339 on D2, but His 332 on D1 is not eliminated.
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页码:13567 / 13574
页数:8
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