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

C. G. Bowsher, L. M. Eyres, J. O. Gummadova, P. Hothi, K. J. McLean, A. W. Munro, N. S. Scrutton, G. T. Hanke, Y. Sakakibara, T. Hase

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

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 kcat values (104-140 s-1) for cytochrome c reduction were measured for all but the most truncated pFNRII[N-5] DEGV, which had a kcat 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 kobs 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.

Original languageEnglish
Pages (from-to)1778-1787
Number of pages10
JournalBiochemistry
Volume50
Issue number11
Early online date23 Feb 2011
DOIs
Publication statusPublished - 22 Mar 2011
Externally publishedYes

Fingerprint Dive into the research topics of 'Identification of N-Terminal Regions of Wheat Leaf Ferredoxin NADP<sup>+</sup>  Oxidoreductase Important for Interactions with Ferredoxin'. Together they form a unique fingerprint.

  • Cite this

    Bowsher, C. G., Eyres, L. M., Gummadova, J. O., Hothi, P., McLean, K. J., Munro, A. W., Scrutton, N. S., Hanke, G. T., Sakakibara, Y., & Hase, T. (2011). Identification of N-Terminal Regions of Wheat Leaf Ferredoxin NADP+  Oxidoreductase Important for Interactions with Ferredoxin. Biochemistry, 50(11), 1778-1787. https://doi.org/10.1021/bi1014562