[14C2]Coproporphyrin III, 14C-labelled in the carboxyl carbon atoms of the 2- and 4-propionate substituents, was prepared by stepwise modification of the vinyl groups of protoporphyrin IX. The corresponding porphyrinogen was used as substrate in a specific sensitive assay for coproporphyrinogen oxidase (EC 1.3.3.3) in which the rate of production of 14CO2 is measured. With this method, the Km of the enzyme from rat liver for coproporphyrinogen III is 1.2 micron. Coproporphyrin III is a competitive inhibitor of the enzyme (Ki 7.6 micron). Apparent Km values for other substrates were measured by a mixed-substrate method: that for coproporphyrinogen IV is 0.9 micron and that for harderoporphyrinogen 1.6 micron. Rat liver mitochondria convert pentacarboxylate porphyrinogen III into dehydroisocoproporphyrinogen at a rate similar to that for the formation of protoporphyrinogen IX from coproporphyrinogen III. Mixed-substrate experiments indicate that this reaction is catalysed by coproporphyrinogen oxidase and that the Km for this substrate is 29 micron. It is suggested that the ratio of the concentration of pentacarboxylate porphyrinogen III to coproporphyrinogen III in the hepatocyte determines the relative rates of formation of dehydroisocoproporphyrinogen and protoporphyrinogen IX.

Preferential rupture of the outer membrane of mitochondria from rat liver releases coproporphyrinogen oxidase in parallel with components of the intermembrane space. Coproporphyrinogen III enters the mitochondrion through the freely-permeable outer membrane. Either protoporphyrinogen IX or protoporphyrin IX must then cross the inner membrane before haem synthesis can be completed.

1. A new method for the measurement of uroporphyrinogen decarboxylase (EC 4. 1.1.37) in rat liver homogenates, with 5- carboxyl porphyrinogen as substrate, is described. 2. The administration of a diet containing 0-3% (w/w) hexachlorobenzene produces porphyria in female Wistar rats after a delay of at least 4 weeks. The development of porphyria is accompanied by a progressive fall in hepatic uroporphyrinogen decarboxylase activity to 18% of control values after 11 weeks. The features of hexachlorobenzene prophyria are consequences of this enzyme defect. 3. Feeding with hexachlorobenzene did not lead to the accumulation of iron in the liver. It is suggested that hexachlorobenzene or a metabolite acts directly to decrease the activity of the enzyme.

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 [(14)C]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 (14)CO(2) from the substrate labelled in the 2-propionate group therefore measures the formation of harderoporphyrinogen, whereas (14)CO(2) 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.

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