IDENTIFICATION OF SER-543 AS THE MAJOR REGULATORY PHOSPHORYLATION SITE IN SPINACH LEAF NITRATE REDUCTASE

Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signal s and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major re gulatory phosphorylation site as Ser-543, which is located in the hing e 1 region connecting the cytochrome b domain with the molybdenum-pter in cofactor binding domain of NR, using recombinant NR fragments conta ining or lacking the phosphorylation site sequence, Studies with NR pa rtial reactions indicated that the block in electron flow caused by ph osphorylation also could be localized to the hinge 1 region. A synthet ic peptide (NR6) based on the phosphorylation site sequence was phosph orylated readily by NR kinase (NRk) in vitro. NR6 kinase activity trac ked the ATP-dependent inactivation of NR during several chromatographi c steps and completely inhibited inactivation/phosphorylation of nativ e NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a ba sic residue at P-3 (i.e., three amino acids N-terminal to the phosphor ylated serine) and a hydrophobic residue at P-5, Both forms are strict ly calcium dependent but belong to distinct families of protein kinase s because they are distinct immunochemically.