The derivation of the formyl‐group oxygen of chlorophyll b in higher plants from molecular oxygen

Abstract

The mechanism of formation of the formyl group of chlorophyll b has long been obscure but, in this paper, the origin of the 7‐formyl‐group oxygen of chlorophyll b in higher plants was determined by greening etiolated maize leaves, excised from dark‐grown plants, by illumination under white light in the presence of either H 2 18 O or 18 O 2 and examining the newly synthesized chlorophylls by mass spectroscopy. To minimize the possible loss of 18 O label from the 7‐formyl substituent by reversible formation of chlorophyll b ‐7 1 ‐ gem ‐diol (hydrate) with unlabelled water in the cell, the formyl group was reduced to a hydroxymethyl group during extraction with methanol containing NaBH 4 : chlorophyll a remained unchanged during this rapid reductive extraction process. Mass spectra of chlorophyll a and [7‐hydroxymethyl]‐chlorophyll b extracted from leaves greened in the presence of either H 2 18 O or 18 O 2 revealed that 18 O was incorporated only from molecular oxygen but into both chlorophylls: the mass spectra were consistent with molecular oxygen providing an oxygen atom not only for incorporation into the 7‐formyl group of chlorophyll b but also for the well‐documented incorporation into the 13 1 ‐oxo group of both chlorophylls a and b [see Walker, C. J., Mansfield, K. E., Smith, K. M. & Castelfranco, P. A. (1989) Biochem. J. 257 , 599–602]. The incorporation of isotope led to as much as 77% enrichment of the 13 1 ‐oxo group of chlorophyll a : assuming identical incorporation into the 13 1 oxygen of chlorophyll b , then enrichment of the 7‐formyl oxygen was as much as 93%. Isotope dilution by re‐incorporation of photosynthetically produced oxygen from unlabelled water was negligible as shown by a greening experiment in the presence of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea. The high enrichment using 18 O 2 , and the absence of labelling by H 2 18 O, unequivocally demonstrates that molecular oxygen is the sole precursor of the 7‐formyl oxygen of chlorophyll b in higher plants and strongly suggests a single pathway for the formation of the chlorophyll b formyl group involving the participation of an oxygenase‐type enzyme.