Normal and Abnormal Heme Biosynthesis. 1. Synthesis and Metabolism of Di- and Monocarboxylic Porphyrinogens Related to Coproporphyrinogen-III and Harderoporphyrinogen:  A Model for the Active Site of Coproporphyrinogen Oxidase

Abstract

Coproporphyrinogen oxidase (copro'gen oxidase), which catalyses the conversion of coproporphyrinogen-III via a monovinylic intermediate to protoporphyrinogen-IX, is one of the least well understood enzymes in the heme biosynthetic pathway. To develop a model for the substrate recognition and binding recognition for this enzyme, a series of substrate analogues were prepared with two alkyl substituents on positions 13 and 17 in place of the usual propionate residues. Although the required substrate probes are porphyrinogens (hexahydroporphyrins), the corresponding porphyrin methyl esters were initialy synthesized via a,c-biladiene intermediates. These were hydrolyzed and reduced with 3% sodium amalgam to give the unstable porphyrinogens needed for the biochemical investigations. These modified structures were metabolized by avian preparations of copro'gen oxidase to give monovinylic products, but the second propionate residue was not further metabolized. In three cases, the metabolites were isolated and further characterized by proton NMR spectroscopy and mass spectrometry. When methyl or ethyl groups were placed at the 13 and 17 positions, the resulting porphyrinogens were very good substrates (although the ethyl version, mesoporphyrinogen-VI, gave slightly better results), but when propyl units were introduced metabolism was significantly inhibited and the butyl-substituted structure was only slightly transformed after long incubation periods. These results suggest the presence of an active-site lipophobic region near the catalytic site for copro'gen oxidase. The observation that the related 3-vinyl- and 3-ethylporphyrinogens with 13,17-diethyl substituents were not substrates for this enzyme confirmed the need for a second propionate residue to hold the substrate in place at the catalytic site.

The oxygen-independent coproporphyrinogen III oxidase HemN utilizes harderoporphyrinogen as a reaction intermediate during conversion of coproporphyrinogen III to protoporphyrinogen IX

“…Harderoporphyrin trimethyl ester 7 was synthesized by condensation of pyrromethanes 3 and 5 following a modified MacDonald approach that constructs the tetrapyrrole in a convergent approach from a northern and southern segment (Figure 3) (Arsenault et al, 1960;Carr et al, 1971;Cavaleiro et al, 1973Cavaleiro et al, , 1974Chen et al, 1999;Lash et al, 1999;Ludwig and Lehr, 2004). A slightly modified protocol was used for condensation of pyrromethanes 3 and 5 to avoid large amounts of self-condensation product 8 of the southern fragment 5.…”

“…Co-crystallization of HemN with coproporphyrinogen III would provide the necessary insight into the orientation of the substrate and its propionate side chains in the active site of the enzyme. Considering our findings that harderoporphyrinogen is a possible HemN reaction Harderoporphyrin trimethyl ester (7) was synthesized by condensation of pyrromethanes (3) and (5) following the modified MacDonald approach with some minor modifications as described in the text (Arsenault et al, 1960;Carr et al, 1971;Cavaleiro et al, 1973Cavaleiro et al, , 1974Chen et al, 1999;Lash et al, 1999;Ludwig and Lehr, 2004). intermediate, the ring A propionate side chain of coproporphyrinogen III is expected to be found in close proximity to the w4Fe-4Sx-bound S-adenosyl-L-methionine. Further biochemical and crystallographic studies are needed to understand how the reaction intermediate harderoporphyrinogen is further decarboxylated.…”

“…Pyrromethanes (1) and (4) were synthesized as previously described (Carr et al, 1971;Chen et al, 1999;Lash et al, 1999;Ludwig and Lehr, 2004).…”

Normal and abnormal heme biosynthesis. Part 7. Synthesis and metabolism of coproporphyrinogen-III analogues with acetate or butyrate side chains on rings C and D. Development of a modified model for the active site of coproporphyrinogen oxidase

“…5 Copro'gen-III is transferred into the mitochondria and undergoes two oxidative decarboxylations, promoted by coproporphyrinogen oxidase (CPO, EC 1.3.3.3) to produce proto'gen-IX (Scheme 1). 4b, 12,13 Subsequent dehydrogenation by protoporphyrinogen oxidase affords the corresponding porphyrin and iron(II) insertion by ferrochelatase then gives heme b (Scheme 1). Protoporphyrin-IX is also the precursor to most of the other hemes and chlorophylls.…”

“…29,30 Meso'gen-VI and related porphyrinogens 1b-d with methyl, propyl or butyl groups at these positions are converted by CPO to the corresponding monovinyl products, but the second oxidative decarboxylation is not observed. 13,30,32 However, the specific alkyl residues at positions 13 and 17 do have an impact, as meso'gen-VI is the best substrate, followed by 1b and 1c. Porphyrinogen 1d is a very poor substrate and only gives <10% conversion to 2d.…”