N-5-METHYLASPARAGINE AND ENERGY-TRANSFER EFFICIENCY IN C-PHYCOCYANIN

A posttranslationally methylated asparagine residue, N-5-methylasparag ine, is found at the beta-72 site in many phycobiliproteins. Two mutat ions (Asp and Gin) in the beta-72 position of Agmenellum quadruplicatu m C-phycocyanin were investigated to clarify the role of the wild-type N-5-methylasparagine near the beta-84 ''fluorescing'' bilin tetrapyrr ole chromophore. Chemical analysis for amide modification revealed tha t the beta-72Q protein was partially methylated with a stoichiometry o f 0.27, suggesting that either the asparagine methyltransferase is non specific or a glutamine methyltransferase exists. Urea denaturation st udies could detect no difference in protein stability for any of the C -phycocyanin species. Steady-state spectroscopic measurements demonstr ate that Asp and Gin substitution for the C-phycocyanin beta-72 NMA af fects both the ground to excited state transition and the excited-stat e characteristics of the beta-84 chromophore, while the rate of radiat ive energy transfer is unaffected. Energy-transfer efficiency within p hycobilisomes (represented by steady-state fluorescence quantum yields ) was also negatively impacted by the beta-72 substitutions. Time-reso lved fluorescence emission spectroscopic studies with C-phycocyanin re veal three distinguishable fluorescence lifetimes. The longest fluores cence lifetime is diminished 7-10% by the Asp and Gin mutations in com parison to a control sample where beta-72 is NMA. Molecular dynamics c alculations implicate a change in the bilin tetrapyrrole chromophore r ing geometry as a Likely source of the altered photophysics induced by the mutations. We conclude that N-5-methylasparagine plays a special role in establishing the environment surrounding the beta-84 chromopho re which minimizes the rates of nonradiative energy losses that would otherwise defeat the high quantum yield for energy transfer within the phycobilisomes.