Interaction of 2 ,3 -0-(2,4,6-Trinitriphenyl)adenosine Diphosphate and 2,4,6-Trinitrophenol with Thylakoid Membrane Proteins

2',3'-0-(2,4,6-trinitrophenyl)ADP (= T N P-A DP) is a competitive inhibitor o f photophos­ phorylation o f ADP. Here, the interaction o f TNP-A D P with the H +-translocating chloroplast ATPase and other thylakoid membrane proteins was studied more in detail and compared with the interactions o f the parental compounds ADP and 2,4,6-trinitrophenol (= picric acid). The following results were obtained: (1) Preilluminated thylakoids incorporate about 1 nmol [l4C]ADP per mg chlorophyll or the same amount o f TNP-[14C]ADP in the following dark. If TNP-[I4C]ADP is present in the light, however, up to 4 nmol label is bound per mg chlorophyll whereas the amount o f incorporated [14C]ADP is unchanged. [14C]ADP is exclusively bound to CF,, yielding a molar ratio o f 1 nu­ cleotide per enzyme. Likewise 1 mol o f l4C label per mol enzyme is found in CF, when thyla­ koids are illuminated in the presence o f TNP-[14C]ADP; the residual bound radioactivity is related with other components o f the thylakoid membrane. (2) TNP-A DP as well as picrate are electron acceptors o f the photosynthetic electron trans­ port chain. Picramate is one main product o f photosynthetic reduction o f picrate. Hence at least one o f the three nitro groups o f the trinitrophenyl moiety is reduced to an amino group. However, additional non-identified, reactive intermediates are formed, which can bind to membrane components. About 20% o f the bound label is covalently linked to membrane pro­ teins. (3) The patterns o f covalently labeled polypeptides are identical whether [l4C]picrate or TNP[l4C]ADP is employed, except for a and ß subunits o f CF, which are specifically labeled in the presence o f TNP-[14C]ADP. This reaction is protected by (ADP + P,) > ATP > ADP > P;. M ost likely radicalic intermediates o f picrate or T NP-A DP photoreduction are responsible for covalent binding. In case o f CF, the radicalic intermediate o f TNP-A DP reduction may be accomodated by its A D P moiety into a nucleotide binding site. Subsequent covalent linkage via the reactive phenyl ring substituent seems to occur in both, the nucleotide and the P( bind­ ing domain.