Absolute Configuration of Protochlorophyllideaand Substrate Specificity of NADPH−Protochlorophyllide Oxidoreductase

Abstract

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.

Substrate Recognition of Nitrogenase-like Dark Operative Protochlorophyllide Oxidoreductase from Prochlorococcus marinus

“…Taken together, these findings clearly demonstrate that substrate recognition of DPOR differs considerably from the LPOR system; the latter does not accept compounds 12 and 13 as substrates (47).…”

Pre-Loading of Chlorophyll Synthase with Tetraprenyl Diphosphate Is an Obligatory Step in Chlorophyll Biosynthesis

“…When this group in Chlide a and Zn Pheide a is replaced by a methoxy group (compound 9) or by a proton (compound 6), reactivity does not significantly change. It is not self-evident that replacement of a large residue by a smaller group in a natural substrate must be tolerated by its enzyme since replacement of the 13 2 -methoxycarbonyl group by a proton in protochlorophyllide leads to complete loss of reactivity with NADPH:protochlorophyllide oxidoreductase (Helfrich et al, 1996). Thus the methoxycarbonyl group must play a specific role in NADPH:protochlorophyllide oxidoreductase while the results obtained with chlorophyll synthase can be explained by steric constraint around the binding site at C-13 2 of the substrate.…”

Chlorophylls, Symmetry, Chirality, and Photosynthesis

“…The new hydrogen atom on C17 is derived from the pro-S hydride of NADPH and the one on C18 from a tyrosine residue whose pKa is lowered by a neighboring lysine [140,141]. Intriguingly, the wheat enzyme requires a substrate with 13 2 R configuration and did not tolerate any modifications of the 13 2 position, possibly indicating an involvement of this part in the photo-reduction [142]. In anoxygenic bacteria, and other organisms, a light-independent, dark-operative pathway is used for the formation of the chlorophyllides.…”