OVEREXPRESSION IN ESCHERICHIA-COLI OF THE COMPLETE PETH GENE-PRODUCT FROM ANABAENA - PURIFICATION AND PROPERTIES OF A 49 KDA FERREDOXIN-NADP(+) REDUCTASE

The complete petH gene product from Anabaena PCC 7119 bar been overexp ressed in E. coli and purified in order to determine the influence of the N-terminal extension on the interaction of ferredoxin-NADP(+) redu ctase with its substrates. The intact 49 kDa FNR can be easily purifie d in a two-step procedure using batch extraction with DEAE-cellulose f ollowed by Cibacron blue-Sepharose chromatography, of the proteins unb ound to DEAE. Isoelectric focusing of FNR shows several forms, with th e major band at pH 6.26. The presence of the N-terminal extension incr eases the K-m, of FNR for NADPH by 4-fold and by 16.4-fold in the redu ction reactions of DCPIP and cytochrome c, However, the K-m, for ferre doxin is 12-fold lower in the reaction catalyzed by the 49 kDa FNR tha n with the 36 kDa protein. This indicates that the presence of the thi rd domain favours the interaction of FNR with ferredoxin, possibly due to the more positive net charge of the N-terminal extension. Comparab le rate constants for both enzymes, were obtained for the photoreducti on of NADP(+) using photosynthetic membranes and also using rapid kine tic techniques. Slightly different ionic strength dependences of the r ate constants were obtained, nevertheless. for both forms of the enzym e. These are a consequence of the structural differences that the prot eins show at the N-terminal and of their effect on the interaction wit h ferredoxin.