The biosynthesis of porphyrins by species of Athiorhodaceae

Introduction
The biosynthetic reactions leading from the initial substrates for porphyrin biosynthesis, glycine and succinyl coenzyme - A, to the monopyrrole porphobilinogen have been studied in detail in work with animals, plants and photosynthetic bacteria. Less is known about the reactions from porphobilinogen to protoporphyrin. Recent studies with enzyme systems from animals and algae have shown that colourless precursors of porphyrins, resembling the porphyrinogens produced by chemical reduction of porphyrins, are synthesised from porphobilinogen, and that chemically prepared porphyrinogens may be utilised for synthesis of other porphyrins; little is known of the properties of either the chemically prepared porphyrinogens or the biologically synthesised precursors of porphyrins.
The reactions leading from protoporphyrin to chlorophylls have been studied in detail only in mutant strains of Chlorella which excrete magnesium protoporphyrin and other intermediates containing magnesium.
The conditions of incubation necessary for the synthesis of free porphyrins, haem and bacteriochlorophyll have been studied in detail with suspensions of Rhodopseudomonas spheroides, and provided a basis for the present study.
The present investigation
A photosynthetic bacterium was isolated from mud, using enrichment cultures incubated in the light, and Identified as a strain of Rhodopseudomonas capsulatus, a member of the Athiorhodaceae, from its morphological and nutritional properties.
To provide a basis for later work on the biosynthesis of porphyrins, the nutrition of Rps. capsulatus was investigated and compared with that of Rhodopseudomonas palustris; standard growth media were devised for each organism. When ammonium was the sole source of combined nitrogen, there was a delay of 4 days before cultures of Rps. capsulatus incubated in the light in the presence of air started to grow; no such delay was observed with Rps. palustris is under similar conditions. This delay was prevented by adding 10-6 M methionine or 2 × 10-10 M cobalamin to growth media, or by incubating cultures anaerobically . When high concentrations of acid-hydrolysed casein were incorporated into growth media for Rps. capsulatus, growth yields of up to 8 mg. dry wt./ml. were obtained. The growth of Rps. palustris was sharply inhibited by concentrations of glycine greater than 4 × 10-3 M; no such inhibition was observed when glycine-leucine peptides were used in place of glycine.
Rps. capsulatus, but not Rps. palustris, excreted magnesium protoporphyrin into culture fluids. This compound, previously reported only in work with algae, was identified by its absorption spectrum and that of the protoporphyrin derived from it, and by testing for magnesium.
Both Rps. capsulatus and Rps. palustris formed coproporphyrin and bacteriochlorophyll in cultures incubated in the light; the formation of bacteriochlorophyll was reduced and that of coproporphyrin much increased when iron citrate was withheld from the medium.
The synthesis of porphyrin (mainly coproporphyrin III), magnesium protoporphyrin and bacteriochlorophyll was investigated in suspensions of Rps. capsulatus incubated in the light. Incubation mixtures were devised to support the maximum synthesis of each of these compounds, and the effect of omitting various components was recorded. The concentrations of added iron citrate supporting optimal synthesis of coproporphyrin, magnesium protoporphyrin and bacteriochlorophyll were, respectively, nil, 2 × 10-5 M and 2 × 10-4 M, suggesting that iron may be required for the conversion of magnesium protoporphyrin, or some related compound, to bacteriochlorophyll, as well as for earlier steps in the biosynthetic pathway. Added DL-methionine increased the synthesis of coproporphyin and, (at a lower concentration), of magnesium protoporphyrin, but not of bacteriochlorophyll. The anionic detergent, Tween 80, was essential in both cultures and suspensions if more than trace amounts of magnesium protoporphyrin were to be formed.
Suspensions of Rps. capsulatus, incubated anaerobically in the light in the medium designed for optimal synthesis of coproporphyrin, gave rise to almost colourless supernatant fluids after centrifuging. However, when such supernatant fluids were allowed to stand in air, the colour changed to orange and then red, and the concentration of coproporphyrin increased 20-fold. When freshly prepared supernatant fluids were acidified, the increase in the content of coproporphyrin was accompanied by the appearance of a strong absorption band at 500 mμ which increased and then decreased in intensity. These changes were accelerated by light, oxidising agents, alkali, or organic solvents, and retarded by reducing agents.
The time course of the changes in optical densities at 548 mμ (due to coproporphyrin} and at 300 mμ was followed when iodine or hydrogen peroxide was added to freshly prepared supernatant fluids from suspensions of Rps. capsulatus. The two oxidising agents produced similar sequences of changes in optical density, and in each case when the optical density at 500 mμ had begun to decrease, the ratio of decrease at 500 mμ to increase at 548 mμ was constant at 2.1; this same ratio was also observed in acidified supernatant fluids under other conditions. It was concluded that a colourless precursor of coproporphyrin III (designated compound x) was produced in these suspensions of Rps. capsulatus; compound x was spontaneously oxidised to coproporphyrin III via. an intermediate absorbing at 300 mμ in acid solution (compound y). The probable molar extinction coefficient of compound y at 500 mμ was estimated from the above data.
A method was devised for the extraction of compounds x and y from supernatant fluids into ether followed by re-extraction into aqueous alkali. The chemical and spectrometric properties of the extracted preparation were surveyed; three tests for bile pigments were applied with negative results. The spectrum in ether showed peaks at 48l mμ and 372 mμ probably due to compound y, and a further, sharp peak at 437 mμ.
The preparation of compounds x and y was compared with coproporphyrinogen, the colourless tetrapyrrole produced w&en; six hydrogen atoms are added to the coproporphyrin molecule, and prepared in the present work by reduction of coproporphyrin by sodium amalgam. The two preparations were identical in all observations made, including solubility, spectral and chemical properties. Hence compound x is probably coproporphyrinogen III; compound y may be a bilene, that is a tetrapyrrole containing one dipyrryl methane group.
The following conversions were not observed when preparations of the substrates were added to suspensions incubated in the light and synthesis of the products compared with that in controls:
coproporphyrinogen to magnesium protoporphyrin (Rps. capsulatus and Rps. spheroides)
coproporphyrinogen to bacteriochlorophyll (Rps. capsulatus and Rps. spheroides)
magnesium protoporphyrin to bacteriochlorophyll (Rps. capsulatus)
14C - labelled coproporphyrinogen and bacteriopheophytin were prepared by incubating suspensions of Rps. capsulatus with 2 - 14C glycine. The molar specific activity of the pure bacteriopheophytin, prepared by chemical removal of the magnesium atom of bacteriochlorophyll followed by column chromatography, was 7 - 8 times that of the original 2 - 14C glycine. The corresponding ratio for the coproporphyrin preparation was 10 - 11, but the purity of this preparation was uncertain.
The radioactivity of bacteriopheophytin synthesised when suspensions of Rps. capsulatus
were incubated in the presence of the 14C - labelled coproporphyrinogen was negligible. Pyrrole compounds may be unable to penetrate from the external medium to the sites of porphyrin and bacteriochlorophyll synthesis in this organism.