Factors determining the sequence of oxidative decarboxylation of the 2- and 4-propionate substituents of coproporphyrinogen III by coproporphyrinogen oxidase in rat liver

Coproporphyrinogen oxidase (EC 1.3.3.3) catalyses the oxidative decarboxylation of the 2- and 4-propionate substituents of coproporphyrinogen III to form protoporphyrinogen IX. A 4-propionate-substituted porphyrinogen, harderoporphyrinogen, which is also a substrate for coproporphyrinogen oxidase, is formed during the reaction. Synthetic [¹⁴C]coproporphyrinogens III, specifically labelled in the carboxyl carbon atoms of either the 2- or 4-propionate substituents, were used to measure the rate of decarboxylation of each substituent by rat liver coproporphyrinogen oxidase. The experimental results, together with the recognition that in all known substrates of coproporphyrinogen oxidase only those propionate groups flanked by a specific arrangement of substituents are decarboxylated, indicate that the 4-propionate group of coproporphyrinogen III cannot be attacked until the 2-propionate group has been decarboxylated. Production of ¹⁴CO₂ from the substrate labelled in the 2-propionate group therefore measures the formation of harderoporphyrinogen, whereas ¹⁴CO₂ from the 4-propionate-labelled substrate measures protoporphyrinogen IX formation. The rate of harderoporphyrinogen formation is about twice that of protoporphyrinogen, and this ratio is unchanged by varying the concentration of coproporphyrinogen III or by competitive inhibition of the enzyme. When coproporphyrinogen III is present in an excess, two fractions of harderoporphyrinogen can be distinguished. One accumulates during the reaction, and the other, which is destined to become protoporphyrinogen IX, does not equilibrate with added harderoporphyrinogen. It is suggested that both decarboxylations take place at the same active centre, which becomes temporarily inaccessible to coproporphyrinogen III and added harderoporphyrinogen, and that the molecule rotates after the first decarboxylation to allow the second to take place.