RESUMO
Direct EPR evidence of the photo-generation of superoxide radicals (O(2) (-.)) was obtained by using a novel spin trapping probe in spinach Photosystem II (PS II) membrane fragments. The production of O(2) (-.) was detected by following the formation of 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) superoxide adducts (DEPMPO-OOH). The inhibition of O(2) (-.) formation by 3-(3,4-dichlorophenyl) -1,1-dimethylurea (DCMU) and the 77 K fluorescence spectrum indicated that O(2) (-.) were generated from PS II, not from PS I. The inhibition of O(2) (-.) formation by DCMU also suggested that O(2) (-.) were generated from the Q(B)binding site, not at a site prior to DCMU blockage. The extrinsic proteins and Mn are very important to eliminate O(2) (-.), showing that the oxygen-evolving system is involved in O(2) (-.) removal rather than production.
RESUMO
ESR and laser flash photolysis studies have determined a reasonable order of reactivity of nucleotides with triplet riboflavin (3Rb*) for the first time. ESR detection of triplet state reactivity of Rb with nucleoside, polynucleotide and DNA has been obtained simultaneously. In addition, ESR spin elimination measurement of the reactivity of 3Rb* with nucleotides in good accord with laser flash photolysis determination of the corresponding rate constants offers a simple and reliable method to detect the reactivities of nucleic acids and its components with photoexcited flavins. Kinetic, ESR and thermodynamic studies have demonstrated that Rb should be a strong endogenous photosensitizer capable of oxidizing all nucleic acid bases, and preferentially two purine nucleotides with high rate constants.