A respiring culture of Rhodobacter sphneroides, grown in the dark under defined aerobic conditions, produced cells capable of immediately commencing adaptation to photosynthetic growth on exposure to light and further reduction of oxygen tension. Adaptation was complete after 12 h and the bacteriochlorophyll a content increased 10-20-fold. This adaptation was performed in the presence of either H,"0 or "0,. The extracted bacteriochlorophyll a was examined by mass spectrometry to determine the origin of both the 3-acetyl and 13'-oxo oxygen atoms: both were derived from water.The derivation of the 13'-0xo group from water in R. sphaeroides indicates that the formation of isocyclic ring E from the 13-propionic acid methylester side chain of Mgz'-protoporphyrin IX monomethylester is an anaerobic process involving a hydratase. This is very different to the situation in higher plants and green algae where the formation of isocyclic ring E is an aerobic process in which the 13'-0x0 group is derived from molecular oxygen via an oxygenase.In contrast to adapting R. sphaeroides cells, the 13'-oxo group of bacteriochlorophyll a i n growing cells of the obligate aerobic chemotrophic bacterium Roseobacter denitrificans, was labelled by ''02 and is, therefore, derived from molecular oxygen like in higher plants and green algae; however, the 3-acetyl group was not labelled by "0,. Thus, while the 13'-oxo group has different origins in R. sp4aeroide.r and R . denitrificans, the 3-acetyl group arises in both bacteria by enzymic hydration of the vinyl group of a chlorophyll a derivative.

Using mass spectrometry, we have demonstrated ~sO-labelling of both the 13Loxo and 3-acetyl groups of newly-formed bacteriochlorophyll a synthesized by Rhodobacter sphaeroides cells during adaptation from respiratory to photosynthetic conditions in the presence of H21so. This derivation of the 131-oxo group of bacteriochlorophyll a from water provides a stark contrast with that of chlorophylls in higher plants where ring E formation is an aerobic process in which the 13Loxo group arises from molecular oxygen via an oxygenase activity. The formation of the 3-acetyl group of bacteriochlorophyll a, however, is consistent with the enzymic hydration of the 3-vinyl group of a derivative of chlorophyll a.

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,"0 or I8O2 and examining the newly synthesized chlorophylls by mass spectroscopy. To minimize the possible loss of label from the 7-formyl substituent by reversible formation of chlorophyll b-7l-gem-diol (hydrate) with unlabelled water in the cell, the formyl group was reduced to a hydroxymethyl group during extraction with methanol containing NaBH, : 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,' *O or 1802 revealed that "0 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 131-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, 599-6021. The incorporation of isotope led to as much as 77% enrichment of the 131-oxo group of chlorophyll a : assuming identical incorporation into the 13' 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)-l ,l-dimethylurea.The high enrichment using '*02, and the absence of labelling by H,"0, 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.The biosynthesis of chlorophylls (Chls) a (I) and b (111), the two major chlorophylls of higher plants, green algae and some prochlorophytes, is now largely understood [l-31, but the mechanism of the formation of the 7-formyl group of Chl b has been an outstanding gap in our current knowledge.

Using mass spectroscopy, we demonstrate as much as 93% enrichment of the 7-formyl group oxygen of chlorophyll b when dark-grown, etiolated maize leaves are greened under white light in the presence of "OZ. This suggests that a mono-oxygenase is mvolved in the oxidation of tts methyl group precursor.The concomitant enrichment of about 75% of the 13'-oxygen confirms the well-documented finding that this 0x0 group, in both chlorophyll (I and b, also arises from 0,. High "0 enrichment into the 7-formyl oxygen relative to the substrate "0, was achieved by optimization of the greenmg conditions in combination with a reductive extraction procedure. It indicates not only a single pathway for Chl b formyl group formation, but also unequivocally demonstrates that molecular oxygen is the sole precursor of the 'I-formyl oxygen.

Triplet state electron paramagnetic resonance (EPR) experiments have been carried out at X-band on Rb. sphaeroides R-26 reaction centers that have been reconstituted with the carotenoid, spheroidene, and exchanged with 132-OH-Zn-bacteriochlorophyll a and [3-vinyl]-132-OH-bacteriochlorophyll a at the monomeric, 'accessory' bacteriochlorophyll sites BA. B or with pheophytin a at the bacteriopheophytin sites HA B. The primary donor and carotenoid triplet state EPR signals in the temperature range 95 -150 K are coi~pared and contrasted with those from native Rb. sphaeroides wild type and Rb. sphaeroides R-26 reaction centers reconstituted with spheroidene. The temperature dependencies of the EPR signals are strikingly different for the various samples. The data prove that triplet energy transfer from the primary donor to the carotenoid is mediated by the monomeric, BChl B molecule. Furthermore, the data show that triplet energy transfer from the primary donor to the carotenoid is an activated process, the efficiency of which correlates with the estimated triplet state energies of the modified pigments.