Absolute configuration of protochlorophyllide alpha and substrate specificity of NADPH-protochlorophyllide oxidoreductase

Protochlorophyllide a was isolated from dark-grown barley (Hordeum vulgare L.) seedlings. The circular dichroism spectrum was identical with that of protochlorophyll a prepared from chlorophyll a by dehydrogenation. The mirror image was obtained for the circular dichroism spectrum of protochlorophyllide a' that had been prepared from chlorophyllide a'. These results prove the 13(2)(R) configuration of natural protochlorophyllide a. Incubation of the seedlings with 5-aminolevulinate resulted in accumulation of protochlorophyllide a. This proved to be partially racemized already within the plants. Circular dichroism spectra were also obtained for 13(2)(R)-methoxy and 13(2)(S)-methoxy derivatives of zinc protopheophorbide a that cannot racemize. Using the increment of the methoxy group by comparison with the respective methoxypyro compound, the circular dichroism spectrum of zinc protopheophorbide a was calculated. Kinetics of racemization indicated a faster reaction for zinc than for magnesium complexes. The enzyme NADPH-protochlorophyllide oxidoreductase solubilized from isolated prolamellar bodies of dark-grown wheat (Triticum aestivum L.) does not accept protochlorophyllide a' or any compound with substituents at C-13(2) different from protochlorophyllide a. The substrate specificity is compared with that of chlorophyll synthase.