Role of positively charged residues in Chlamydomonas reinhardtii ferredoxin-NADP+-reductase

We have previously reported that ferredoxin-NADP-reductase (FNR) from the photosynthetic alga, Chlamydomonas reinhardtii was fully N-trimethylated in vivo on three specific lysines (K83, K89 and K135) located in two sequence insertions comparative to photosynthetic FNRs. This paper deals with the comparison of the biochemical properties of the “native” algal FNR (thus methylated) and the homologous recombinant protein expressed in E. coli, which did not undergo this post-translational modification. Few differences could be observed between the two forms except that their apparent molecular mass was significantly different. This could be indicative that methylation induced changes in the tertiary structure of FNR, making the protein more compact. Another difference was the fivefold higher Km value for NADPH exhibited by the recombinant protein. However, the binding of ferredoxin (Fd) to its reductase seems to be independent of the methylation status. We have also studied four mutated FNRs, in which each Lys was substituted with a hydrophobic amino acid. The results indicate that a cluster of positive charges including K83 and K89 is required for catalytic activity, like in photosynthetic FNRs although the fine interactions are probably different in the algal protein.