Abstract:
:The active site of water oxidation in Photosystem II (PSII) is a Mn4CaO5 cluster that is located in a cavity between the D1 and CP43 protein subunits by which it is coordinated. The remainder of this cavity is filled with water molecules, which serve as a source of substrate and participate in poorly understood hydrogen bond networks that may modulate the function of the Mn4CaO5 cluster. These water molecules interact with the first and second sphere amino acid ligands to the Mn4CaO5 cluster and some water interacts directly with the Mn4CaO5 cluster. Here, the results of mutations to the amino acids that line the walls of several predicted cavities in the immediate vicinity of the Mn4CaO5 cluster were examined in Synechocystis sp. PCC 6803. Of these, mutations of Val185 in the D1 subunit resulted in the most interesting functional alterations. The hydrophobic D1-Val185 occupies a location contacting water molecules that are positioned between the redox active tyrosine (YZ) and the putative proton gate residue, D1-Asp61, and at a position opposite the oxo bridge atom, O5, of the cluster. Mutations of the residue D1-Val185 were produced, with the intention that the substitute residue would extend into the water cavity that includes H2O molecules that interact with the Mn4CaO5 cluster, amino acid ligands of the Mn4CaO5 cluster, YZ and the chloride co-factor of PSII. Three of these mutants, D1-Val185Asn, D1-Val185Thr, and D1-Val185Phe, were able to accumulate significant levels of charge separating PSII and were characterized using polarographic and fluorescent techniques. Of the three substitutions, the phenylalanine substitution was the most severe with a complete inability to evolve oxygen, despite being able to accumulate PSII and to undergo stable charge separation. The threonine substitution had no apparent effect on oxygen evolution other than a 40% reduction in the steady state rate of O2 production compared to the case of wild-type Synechocystis , due to a reduced ability to accumulate PSII centers. The asparagine substitution produced the most complex phenotype with respect to O2 evolution. Although still able to evolve oxygen, D1-Val185Asn does so less efficiently than wild-type PSII, with a higher miss factor than that for the wild type. Most significantly, asparagine substitution dramatically retards the rate of O2 release and results in an extension of the kinetic lag phase prior to O2 release that is highly reminiscent of the effects of mutations produced at D1-Asp61. The observed effects of the D1-Val185Phe and D1-Val185Asn mutations may be due to alterations in the environment of nearby chloride co-factor of PSII and/or alterations in the hydrogen bond network, perhaps impeding the movement of water to a binding site on the metal cluster.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Dilbeck PL,Bao H,Neveu CL,Burnap RLdoi
10.1021/bi400930gsubject
Has Abstractpub_date
2013-10-01 00:00:00pages
6824-33issue
39eissn
0006-2960issn
1520-4995journal_volume
52pub_type
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