Pigment‐free NADPH:protochlorophyllide oxidoreductase from Avena sativa L

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Abstract
The enzyme NADPH:protochlorophyllide oxidoreductase (POR) is the key enzyme for light‐dependent chlorophyll biosynthesis. It accumulates in dark‐grown plants as the ternary enzyme–substrate complex POR‐protochlorophyllide a‐NADPH. Here, we describe a simple procedure for purification of pigment‐free POR from etioplasts of Avena sativa seedlings. The procedure implies differential solubilization with n‐octyl‐β‐d‐glucoside and one chromatographic step with DEAE‐cellulose. We show, using pigment and protein analysis, that etioplasts contain a one‐to‐one complex of POR and protochlorophyllide a. The preparation of 13 analogues of protochlorophyllide a is described. The analogues differ in the side chains of the macrocycle and in part contain zinc instead of the central magnesium. Six analogues with different side chains at rings A or B are active substrates, seven analogues with different side chains at rings D or E are not accepted as substrates by POR. The kinetics of the light‐dependent reaction reveals three groups of substrate analogues with a fast, medium and slow reaction. To evaluate the kinetic data, the molar extinction coefficients in the reaction buffer had to be determined. At concentrations above 2 mole substrate/mole enzyme, inhibition was found for protochlorophyllide a and for the analogues.

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