Unequivocal Proof of Chlorophyll Formation by the C5 Pathway and Tentative Evidence for Shemin Pathway Activity in Greening Leaves. Is mitochondrial Haem Formed by the Shemin Pathway in Leaf Cells

The tetrapyrrole precursor 5-aminolaevulinate (ALA) may be formed by Shemin-pathway enzyme ALA synthase from glycine and succinate or by the C5 pathway (Beale, Castelfranco 1973) from the intact C5 skeleton of 2-oxoglutarate or glutamate which are interconvertible. The evidence for the C5 pathway depends on the derivation of the C-5 of ALA, accumulated by greening plant or algal cells in the presence of laevulinate (LA), from the C-1 of 2-oxoglutarate or glutamate and not from the C-2 of glycine as in the Shemin pathway. This evidence, however, does not indicate the type of tetrapyrroles formed by pathways involving the C5 pathway. To prove whether the C5 or Shemin pathways are involved in chlorophyll (Chl) formation, Porra et al. (1982;1983) illuminated excised etiolated maize leaves in the presence of [1-13C]glutamate or [2-13C]-glycine, respectively. The 13C-NMR spectra of the Chl a formed (Table 1) show that the eight Chl carbons labelled by the glutamate are those derived from the C-5 of ALA (Fig. lA) proving C5-pathway involvement in Chl biosynthesis. Absence of enhancement of the same eight peaks by [2-13C]glycine proved Shemin-pathway inactivity in Chl formation in greening leaves; however, the enhancement of the 51.4 ppm peak indicated that the C-2 of glycine forms the methyl ester carbon of Chl (Fig. 1A), an esterification which occurs in the chloroplast after completion of the tetrapyrrole ring (cf Porra et al. 1983).