The role of protein surface charge in catalytic activity and chloroplast membrane association of the pea NADPH: protochlorophyllide oxidoreductase (POR) as revealed by alanine scanning mutagenesis

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependen t reduction of protochlorophyllide (pchlide) to chlorophyllide (chlide) in the biosynthesis of chlorophyll. FOR is a peripheral membrane protein that accumulates to high levels in the prolamellar bodies of vascular plant etio plasts and is present at low levels in the thylakoid membranes of developin g and mature plastids. Clustered charged-to-alanine scanning mutagenesis of the pea (Pisum sativum L.) FOR was carried out and the resulting mutant en zymes analyzed for their ability to catalyze pchlide photoconversion in viv o and to associate properly with thylakoid membrane preparations in vitro. Of 37 mutant enzymes examined, 5 retained wild-type levels of activity, 14 were catalytically inactive, and the remaining 18 exhibited altered levels of function. Several of the mutant enzymes showed temperature-dependent enz ymatic activity, being inactive at 32 degrees C, but partially active at 24 degrees C. Mutations in predicted cr-helical regions of the protein showed the least effect on enzyme activity, whereas mutations in predicted B-shee t regions of the protein showed a consistent adverse affect on enzyme funct ion. In the absence of added NADPH, neither wildtype FOR nor any of the mut ant PORs resisted proteolysis by thermolysin following assembly onto the th ylakoid membranes. In contrast, when NADPH was present in the assay mixture , 13 of the 37 mutant PORs examined were found to be resistant to thermolys in upon treatment, suggesting that the mutations did not affect their abili ty to be properly attached to the thylakoid membrane. In general, the repla cement of charged amino acids by alanine in the most N- and C-terminal regi ons of the mature protein did not significantly affect FOR assembly, wherea s mutations within the central core of the protein (between residues 86 and 342) were incapable of proper attachment to the thylakoid. Failure to prop erly associate with the thylakoid membrane in a protease resistant manner w as only weakly correlated to loss of catalytic function. These studies are a first step towards defining structural determinants crucial to FOR functi on and intraorganellar localization.