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A carboxylic residue at the high-affinity, Mn-binding site participates in the binding of iron cations that block the site
Authors:Boris K Semin  Michael Seibert
Institution:a Basic Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401, USA
b Department of Biophysics, Faculty of Biology, Moscow State University, Moscow 119992, Russian Federation
Abstract:The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by YZradical dot. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII−Mn]) membranes. The rate of Mn(II) oxidation saturates at ≥10 μM in PSII(−Mn) membranes and ≥500 μM in EDC-treated PSII (−Mn) samples. Intact PSII(−Mn) membranes have only one site for Mn(II) oxidation via YZ (dissociation constant, Kd = 0.64 μM), while EDC-treated PSII(−Mn) samples have two sites (Kd = 1.52 and 22 μM; the latter is the low-affinity site). When PSII(−Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (Kd = 2.3 μM) for exogenous Mn(II) oxidation by YZradical dot radical. In this case, the rate of electron donation via YZ saturated at a Mn(II) concentration ≥15 μM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HAZ site is protected from the action of the modifier by the iron cations blocking the HAZ site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HAZ site (Mn4 in the tetranuclear manganese cluster Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site.
Keywords:Chl  chlorophyll  DCIP  2  6-dichlorophenolindophenol  DCMU  3-(3  4-dichlorophenyl)-1  1-dimethylurea  DPC  1  5-diphenylcarbazide  EDC  1-ethyl-3-[(3-dimethylamino) propyl]carbodiimide  F0  fluorescence emitted by a sample at low light levels prior to flash excitation  (F   &minus     F0)/F0  fluorescence yield  Ffinal  final fluorescence yield detected after decay of the flash-induced Fmax  Fmax  maximum fluorescence yield following actinic-flash excitation  HAZ  high-affinity electron donation site to YZradical dotels-cdn   by Mn(II)" target="_blank">com/sd/entities/rad" class="glyphImg"> by Mn(II)  Kd  dissociation constant of a substrate-enzyme complex  Ki  dissociation of an inhibitor-enzyme complex  Kst  stability constant  LAZ  low-affinity electron donation site to YZradical dotels-cdn   by Mn(II)" target="_blank">com/sd/entities/rad" class="glyphImg"> by Mn(II)  MES  2-(N-morpholino) ethanesulfonic acid  Mn4  Mn coordinated at the HAZ site  (Mn)4/Ca cluster  tetrameric Mn/Ca cluster of the OEC  OEC  O2-evolving complex  pKapp  apparent pK  PSII  photosystem II  PSII(-Mn)  Mn-depleted PSII membranes  PSII(&minus  Mn  +Fe)  Fe-blocked PSII(&minus  Mn)  RC  reaction center  YZ  redox-active tyrosine D1-Tyr161  the first electron donor to P680+ in PSII
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