Studies on the ferrochelatase activity of isolated rat liver mitochondria with special reference to the effect of oxidizable substrates and oxygen concentration

The mitochondrial ferrochelatase activity has been studied in coupled rat liver mitochondria using deuteroporphyrin IX (incorporated into liposomes of lecithin) and Fe(III) or Co(II) as the substrates.

1. 1. It was found that respiring mitochondria catalyze the insertion of Fe(II) and Co(II) into deuteroporphyrin. When Fe(III) was used as the metal donor, the reaction revealed an absolute requirement for a supply of reducing equivalents supported by the respiratory chain.

2. 2. A close correlation was found between the disappearance of porphyrin and the formation of heme which allows an accurate estimate of the extinction coefficient for the porphyrin to heme conversion. The value Δ (mM^?1 · cm^?1) = 3.5 for the wavelength pair 498 509 nm, is considerably lower than previously reported.

3. 3. The maximal rate of deuteroheme synthesis was found to be approx. 1 nM · min^?1 · mg^?1 of protein at 37 °C, pH 7.4 and optimal substrate concentrations, i.e. 75 μM Fe(III) and 50 μM deuteroporphyrin.

4. 4. Provided the mitochondria are supplemented with an oxidizable substrate, the presence of oxygen has no effect on the rate of deuteroheme synthesis.

Abbreviations: EPPS, (4-(2-hydroxyethyl)-1-piperazine propane sulphonic acid); HEPES, N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid; PIPES, piperazine-N,N′-2-bis(2-ethanesulphonic acid)