Identification of N-Terminal Regions of Wheat Leaf Ferredoxin NADP+ Oxidoreductase Important for Interactions with Ferredoxin

摘要

Wheat leaves contain two isoproteins of the photosynthetic ferredoxin:NADPþ reductase (pFNRInand pFNRII). Truncated forms of both enzymes have been detected in vivo, but only pFNRII displaysnN-terminal length-dependent changes in activity. To investigate the impact of N-terminal truncation onninteraction with ferredoxin (Fd), recombinant pFNRII proteins, differing by deletions of up to 25 aminonacids, were generated.During purification of the isoproteins found in vivo, the longer forms of pFNRII boundnmore strongly to a Fd affinity column than did the shorter forms, pFNRIIISKK and pFNRII[N-2]KKQD.nFurther truncation of theN-termini resulted in a pFNRII protein which failed to bind to a Fd column. Similarnkcat values (104-140 sn-1n) for cytochrome c reduction were measured for all but the most truncatednpFNRII[N-5]DEGV, which had a kcat of 38 sn-1n. Stopped-flow kinetic studies, examining the impact ofntruncation on electron flow between mutant pFNRII proteins and Fd, showed there was a variation in kobsnfrom 76 to 265 sn-1ndependent on the pFNRII partner. To analyze the sites which contribute to Fd binding atnthe pFNRIIN-terminal, threemutants were generated, in which a single or double lysine residue was changednto glutaminewithin the in vivoN-terminal truncation region. Themutations affected binding of pFNRII to thenFd column. Based on activity measurements, the double lysine residue change resulted in a pFNRII enzymenwith decreased Fd affinity. The results highlight the importance of this flexible N-terminal region of thenpFNRII protein in binding the Fd partner