Abstract
Amino acid residue D1-Asp(170) of the D1-polypeptide of photosystem II was previously shown to be implicated in the binding and oxidation of the first manganese to be assembled into the Mn(4)Ca cluster of the oxygen-evolving complex (OEC). According to recent x-ray crystallographic structures of photosystem II, D1-Glu(333) is proposed to participate with D1-Asp(170) in the coordination of Mn4 of the OEC. Other residues in the C-terminal region of the D1-polypeptide are proposed to coordinate nearby manganese of the cluster. Site-directed replacements in Synechocystis sp. PCC 6803 at D1-His(332), D1-Glu(333), D1-Asp(342), D1-Ala(344), and D1-Ser(345) were examined with regard to their ability to influence the binding and oxidation of the first manganese in manganese-depleted photosystem II core complexes. Direct and indirect measurements reveal in all mutants, but most marked in D1-Glu(333) replaced by His, an impaired ability of Mn(2+) to reduce Y(Z)., indicating a reduced ability (elevated K(m)) compared with WT to bind and oxidize the first manganese of the OEC. The effect on the K(m) of these mutations is, however, considerably weaker than some of those constructed at D1-Asp(170) (replacement by Asn, Ala, and Ser). These observations imply that the C-terminal residues ultimately involved in manganese coordination contribute to the high affinity binding at D1-Asp(170) likely through electrostatic interactions. That these residues are far from D1-Asp(170) in the primary structure of the D1-polypeptide, imply that the C terminus of the D1-polypeptide is already close to its mature conformation at the first stages of assembly of the Mn(4)Ca cluster.
Highlights
MARCH 9, 2007 VOLUME 282 NUMBER 10 supports light-driven oxidation of water to molecular oxygen
Ananyev and co-workers [24] have demonstrated using a D2-Y160F mutant (D2-Tyr160 replaced by Phe) that redox-active tyrosine YD probably plays an indirect role in accelerating electron acceptor of Photosystem II (PSII); QA, primary quinone electron acceptor of PSII; QB, secondary quinone electron acceptor of PSII; TC31 and TC35, wild-type strains of Synechocystis 6803 in which the D1-polypeptide is encoded only by psbA3, the first two copies of psbA having been deleted; WT, wild type, referring here to strain TC31 or TC35; YD, secondary electron donor tyrosine, D2-Tyr160; YZ, secondary electron donor tyrosine, D1-Tyr161
We have previously shown that D1-Asp170 is implicated in binding and oxidation of the first manganese of the oxygen-evolving complex (OEC)
Summary
The glucose-tolerant strain of the cyanobacterium Synechocystis sp. PCC 6803 [31] was used for construction of the sitedirected mutants described in this paper. Optical spectroscopy on PSII core complexes was performed using a flash detection spectrophotometer based on a design by Joliot et al [36] and described in Metz et al [32] The same instrument was used for measurements in whole cells of the amplitude of and kinetics of relaxation of the relative quantum yield of chlorophyll fluorescence [25, 38] This kinetic relaxation, following excitation with saturating actinic light flashes (EG&G FX-199), was monitored using weak detecting flashes (EG&G FX-199) rendered monochromatic by the HL300 monochromator at 422 nm. Prior to measurements of the evolution of the variable fluorescence following actinic flash illumination, Synechocystis cells at an optical density of 0.9 cmϪ1 at 730 nm were preincubated for 10 min in BG-11 plus 50 mM HEPES-NaOH (pH 7.5), 0.3 mM p-benzoquinone, and 0.3 mM K3Fe(CN). To measure the kinetics of charge recombination between QAϪ and the oxidized
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