Studies On The Structure And Function Of Extrinsic Proteins Of Higher Plant Photosystem II

Photosystem II (PSII) of higher plants catalyzes the light-driven reduction ofplastoquinone, and the oxidation of water to molecular oxygen and protons at its electrondonor side. On the luminal side of PSII membranes, three extrinsic proteins, named by of 33,23 and 18 kDa protein, play important roles in oxygen evolution. Among these extrinsicproteins, the 33 kDa protein is particularly important for stabilizing the manganese cluster inthe photosynthetic oxygen evolving center under physiological conditions. 23 and 18 kDaproteins are intimately related to the unique requirement of Ca2+ and Cl–, respectively, foroxygen evolution. This research work is mainly focused on the structure and function of threeextrinsic proteins, the main results could be concluded in following four parts.1. Structural and functional differentiation of three groups of tyrosine residues byacetylation of N-acetylimidozole in the 33 kDa proteinIn order to study its contribution to the assembly of the 33 kDa protein into photosystemII (PSII), tyrosine residues were specifically acetylated using N-acetylimidozole (NAI). Insoluble 33 kDa protein, three groups of Tyr residues could be differentiated by NAIacetylation: approximately 5 (actually ~5.2) Tyr residues could be easily acetylated(superficial), 1-2 Tyr residues could be acetylated when the NAI concentration wassufficiently high (superficially buried) and 1-2 Tyr residues could only be acetylated in thepresence of the denaturant, urea (deeply buried). Acetylation of the 5.2 Tyr residues did notaffect the reconstitution or oxygen-evolving activities of the 33 kDa protein, and far-UVcircular dichroism (CD) analysis showed that the altered 33 kDa protein, retained most of itsnative secondary structure. These results suggested that 5.2 Tyr residues are not absolutelyessential to the function of 33 kDa protein, However, further modification of the 1-2superficially buried Tyr residues (for a total acetylation of ~6.4 Tyr residues) completelyabrogated rebinding and oxygen evolution activities of the 33 kDa protien. Finally, at leastone tyrosine residue was inaccessible to NAI until the 33 kDa protein was completelyunfolded by 8 M urea. A prominent red shift in fluorescence spectra of the 33 kDa protein,(excited at 280 or 295 nm) was observed after modification of 6.4 Tyr residues. and a furtherred shift could be found after all 8 Tyr residues were modified, indicating a great loss ofnative secondary structure. Far-UV CD revealed that the 33 kDa protein was mostly unfoldedwhen 6.4 Tyr residues were modified and completely unfolded when all 8 Tyr residues weremodified. Fluorescence and far-UV CD studies revealed that loss of 33 kDa protein functionfollowing NAI modification correlated well with conformational changes in 33 kDa protein,.Together, these results indicate that different tyrosine residues have different contributions tothe binding and assembly of 33 kDa protein, into PSII..2. Lysine residues are involved in maintaining the structure and function of the 33 kDaproteinTo clarify structure-function relationships of lysine residues in the 33 kDa protein, lysineresidues were specifically modified with N-succinimidyl propionate (NSP). When the 33 kDaprotien was treated with 0.5 mM NSP, its reconstitution with PSII membrane and oxygenevolving activities after the reconstitution were little affected. Analysis with far UV circulardichroism (CD) or intrinsic fluorescence spectra showed that 0.5 mM NSP-modified 33 kDaprotein retained most of its native secondary structure. Increasing the NSP concentration to4mM, the modified protein cannot rebind to PSII membranes and completely eliminated thephotosynthetic oxygen evolution. The results from both far UV-CD and intrinsic fluorescencespectra analysis showed a clearly conformational change for 4 mM NSP-modified 33 kDaprotein, suggesting part of Lys residues are involved in maintaining the structure and functionof 33 kDa protein. Mapping of the sites of NSP modification was performed bystaphylococcus V8 protease digestion of the modified protein and analysis by MALDI-TOFMS. Studies indicated that six lysine residues including Lys20, Lys101, Lys196, Lys207,Lys130 (or 137) and Lys66 (or 76) were modified only with 4mM NSP. These candidatelysine residues seem to play a crucial role in maintaining structure and function of the33 kDaprotein.3. Influences of NAI modification on the structure and reconstitution ability of the 23kDa protein of spinach PSII membranes1, 4 and 8 (real number 0.78, 3.66, 8.02) tyrosine (Tyr) residues on the extrinsic 23 kDaprotein were modified with 5, 10 and 40 mM NAI) respectively, the amount of the rebound ofNAI-modified extrinsic 23 kDa protein was 98%, 80%, 5% of that in the control. The resultsindicated that 1 Tyr residue is not absolutely essential to reconstitution ability of the extrinsic23 kDa protein. Other 3 Tyr residues are absolutely essential to keep the reconstitution ability.More 4 Tyr residues are absolutely essential to reconstitution ability. Furthermore, thefluorescence and circular dichroism spectra of native and NAI-modified 23 kDa extrinsicproteins were identical, suggesting that the NAI modification did not influence the structureof the 23 kDa protein. Thus, it is conclude that all Tyr residues in the protein are superficialburied and important for interaction with PSII. In addition, effects of pH and temperature onthe structure of the extrinsic 23 kDa protein were studied, suggesting that the structure of theextrinsic 23 kDa protein behaves as a stable in suspension ( pH 4-9 or Tm 25-55 °C).4. Lysines of the extrinsic 18 kDa protein are important to its interaction withphotosystem II membrans.To determine the contribution of charged amino acids to binding with the photosystem II(PSII), the amino or carboxyl groups of the extrinsic 18 kDa protein were modified withN-succinimidyl propionate (NSP) or glycine methyl ester (GME) in the presence of awater-soluble carbodiimide, respectively. Based on pI shift, 4-10 and 10-14 amino groupswere modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxylgroups were modified by reaction with 100 mM GME. Neutralization of negatively-chargedcarboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein.However, NSP-modified 18 kDa protein, in which the positively charged amino groups hadbeen modified to uncharged methyl esters, failed to bind PSII in the presence of extrinsic 23kDa protein. These defects cannot be attributed to structural or conformational alterationsimposed by chemical modification, since the fluorescence and circular dichroism spectraamong native, GME-and NSP-modified extrinsic 18 kDa proteins were identical. Thus, itwas concluded that the positive charges of Lys residues in the extrinsic 18 kDa protein arethese superficial or superficial buried and important for interaction with PSII in the presenceof the extrinsic 23 kDa protein. Furthermore, it was concluded that the negative charges ofcarboxyl groups of this protein do not participate.