Vermass 9316857 The D2 protein is one of the two reaction center proteins of photosystem II, a pigment protein complex in the thylakoid membrane of plants and cyanobacteria. This photosystem utilizes light energy to oxidize water (resulting in oxygen) and to produce reducing equivalents to be used in subsequent steps of photosynthesis. The D2 protein binds a number of cofactors and prosthetic groups, which are involved in photosynthetic electron transport. In addition, it contains a redox-active tryosyl residue, which is stable in its oxidized (radical) form for up to several hours. The protein environment provides cofactors and prosthetic groups with the appropriate orientation, localization, and redox properties to make light-induced electron-transfer reactions fast and efficient. Targeted mutagenesis of the D2 protein is applied to identify the domains and residues in D2 that interact with cofactors and affect their functional characteristics. Targeted mutations are introduced into the transformable (photo)heterotrophic cyanobacterium Synechocystis sp. PCC 6803, which incorporates foreign DNA into its genome by homologous recombination. This allows a deletion of the wild-type genes coding for the D2 protein, followed by an introduction of a gene copy (at its native position in the genome) carrying a mutation. To avoid complications in the functional analysis stemming from the presence of photosystem I (a chlorophyll-binding protein complex, which also shows light-induced electron transport), a light-tolerant photosystem I-less strain of Synechocystis 6803 has been constructed, which can be genetically tai lored as a convenient receptor strain to introduce D2 mutations. On the basis of existing data, a number of highly interesting D2 residues have been identified, and mutations will be introduced in these residues in a photosystem I-less background. The resulting mutants will be analyzed, either in vivo or in thylakoid preparations, by a number of methods, including fluorescence induction and emission, electron paramagnetic resonance spectroscopy, and flash-induced oxygen evolution. This experimental system provides an excellent opportunity for detailed analysis of cofactor properties as modulated by the protein environment. The photosystem II complex from cyanobacteria has proven to be a suitable model system to study protein/cofactor interactions, and the development of a light-tolerant photosystem I-less strain now allows such studies to be carried out in relatively intact systems. In addition, the light-tolerant photosystem I-less Synechocystis strain provides a very good system for random mutagenesis of the D2 protein, because it allows for positive selection of mutants with impaired photosystem II activity. Photosystem I-less mutants retaining the peripheral antenna system propagate well at 10% of the light intensity used for wild type, but not a full light intensity; this appears to be caused by an inability of the cell to cope with too many photosystem II-generated electrons in the absence of photosystem I. This property can be take advantage of to select randomly generated D2 mutants with impaired electron transport. After introduction of random mutations in a plasmid carrying the D2 gene in Escherichia coli, this plasmid can be used for transformation of a photosystem I-less and D2-less strain of Synechocystis. After selection for transformants, cells with impaired electron flow (resulting from a D2 mutation) easily can be picked out by a light-resistant phenotype. Subsequently, the site of the mutation and its structural and functional effects can be determined. The propo sed project on the D2 protein studied in a photosystem I-less background will result in detailed insight in the role various residues and domains play in facilitating efficient and rapid electron transfer. The research not only will enhance the understanding of photosystem II, but also will be applicable in a broader perspective of cofactor/protein interactions in protein folding and stability, and will be informative regarding rather unusual protein redox chemistry, such as tyrosyl oxidation leading to a stable radical, as is found in the D2 protein. this work involves molecular biology, microbiology, biochemistry, and certain biophysical aspects, and is very well suited for a broad and interdisciplinary graduate and postdoctoral training. %%% Photosynthesis is the process in which light is utilized to fuel carbon fixation and oxygen evolution. Essentially all organic material and oxygen on earth originates from photosynthesis. The question addressed in this project is how one of the fundamental steps in photosynthesis works. Such understanding is important from both a biochemical and biophysical point of view , and has general implications for structure, function, and assembly of protein complexes in biological membranes. The step that will be addressed is made possible by a pigment-binding protein complex, and involves a very efficient conversion of light energy to chemical energy that can be used by living organisms, with a concomitant conversion of water to oxygen. We will use genetic manipulation in a cyanobacterium (blue-green alga) to specifically change pigment-binding proteins involved in this process. Genetic manipulation also will be utilized to delete selected other pigment-binding proteins from the organism, so that functional analysis of the steps being studied is greatly facilitated. This project will result in (a) enhanced understanding of the interplay between pigments and proteins that is crucial for photosynthesis, and (b) improved insight in the role specific r esidues of proteins play in catalysis (enhancement) of particular biochemical reactions. *** u ~ R b C E S s ? $ - / 1 3 C E ? $ $ $ $ $ ! $ $ $ F E e S E 2 Times New Roman Symbol & Arial & France p- " h
