The in vitro effects of ATP and protein phosphorylation on the activity of ferredoxin: NADP + oxidoreductase from spinach chloroplasts

Abstract

When ferredoxin:NADP + oxidoreductase (FNR) was preincubated with [ γ- 32P]ATP-Mg and separated by native PAGE it was found to be radioactive. This was not seen on SDS-PAGE, unless FNR was preincubated with a crude protein kinase extract with FNR kinase activity under phosphorylating conditions. These observations suggest that FNR contains an ATP-binding domain. It was found that ATP caused an inhibition of FNR diaphorase activity, which when analysed by Lineweaver-Burk plots indicated that ATP was a non-competitive inhibitor with respect to NADPH. This shows that the ATP site is distinct from the NADP(H) active site. The in vitro phosphorylation of FNR on a serine residue(s) by the crude FNR kinase extract led to a modification of ferredoxin (Fd)-dependent FNR activity. An analysis of the data showed that after phosphorylation the apparent K m for Fd and V max both increased. This observation suggests that the Fd-FNR interaction is modified after FNR phosphorylation in vitro. Limited proteolysis of phosphorylated FNR followed by SDS-PAGE infers that the phosphorylated amino acid(s) is located near the N-terminus.