Identification of N-terminal regions of wheat leaf ferredoxin NADP+ oxidoreductase important for interactions with ferredoxin.
Wheat leaves contain two isoproteins of the photosynthetic ferredoxin:NADP(+) reductase (pFNRI and pFNRII). Truncated forms of both enzymes have been detected in vivo, but only pFNRII displays N-terminal length-dependent changes in activity. To investigate the impact of N-terminal truncation on interaction with ferredoxin (Fd), recombinant pFNRII proteins, differing by deletions of up to 25 amino acids, were generated. During purification of the isoproteins found in vivo, the longer forms of pFNRII bound more strongly to a Fd affinity column than did the shorter forms, pFNRII(ISKK) and pFNRII[N-2](KKQD). Further truncation of the N-termini resulted in a pFNRII protein which failed to bind to a Fd column. Similar k(cat) values (104-140 s(-1)) for cytochrome c reduction were measured for all but the most truncated pFNRII[N-5](DEGV), which had a k(cat) of 38 s(-1). Stopped-flow kinetic studies, examining the impact of truncation on electron flow between mutant pFNRII proteins and Fd, showed there was a variation in k(obs) from 76 to 265 s(-1) dependent on the pFNRII partner. To analyze the sites which contribute to Fd binding at the pFNRII N-terminal, three mutants were generated, in which a single or double lysine residue was changed to glutamine within the in vivo N-terminal truncation region. The mutations affected binding of pFNRII to the Fd column. Based on activity measurements, the double lysine residue change resulted in a pFNRII enzyme with decreased Fd affinity. The results highlight the importance of this flexible N-terminal region of the pFNRII protein in binding the Fd partner.
Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Manchester M13 9PT, UK. caroline.bowsher@manchester.ac.uk
C G
CG
Bowsher
L M
LM
Eyres
J O
JO
Gummadova
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P
Hothi
K J
KJ
McLean
A W
AW
Munro
N S
NS
Scrutton
G T
GT
Hanke
Y
Y
Sakakibara
T
T
Hase
D001665:Binding Sites; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005288:Ferredoxins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D018515:Plant Leaves / Q000201:enzymology* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014908:Triticum / Q000201:enzymology* / Q000378:metabolism
D005288:Ferredoxins; D010940:Plant Proteins; D005287:Ferredoxin-NADP Reductase