Ferrochelatase of spinach chloroplasts

Abstract

Spinach chloroplasts catalyse the incorporation of Fe²⁺ into protoporphyrin, mesoporphyrin and deuteroporphyrin to form the corresponding haems. This ferrochelatase activity was detected by pyridine haemochrome formation with acetone-dried powders of chloroplasts, or from the formation of [⁵⁹Fe]haems by intact chloroplasts. Decreasing the mitochondrial contamination of the chloroplasts by density-gradient centrifugation did not cause any loss of activity: spinach ferrochelatase appears to be principally a chloroplast enzyme. The characteristics of the enzyme were examined by using [⁵⁹Fe]haem assay. The activity was pH-dependent: for both mesohaem and protohaem formation there were two pH maxima, a major peak at about pH7·8 and a smaller peak at about pH9·2. Lineweaver–Burk plots showed that the K_m for Fe²⁺ incorporation into protoporphyrin was 8μm and that for Fe²⁺ incorporation into mesoporphyrin was 36μm. At non-saturating Fe²⁺ concentrations the K_m for protoporphyrin was 0·2μm and that for mesoporphyrin was 0·4μm. Ferrochelatase was not solubilized by treatment of chloroplasts with ultrasound but was solubilized by stirring in 1% (w/v) Tween 20 at pH10·4. Unlike the rat liver mitochondrial enzyme, chloroplast ferrochelatase was not stimulated by treatment with selected organic solvents. The spinach enzyme was inactive in aerobic conditions and it was shown by using an oxygen electrode that under such conditions the addition of Fe²⁺ to buffer solutions caused a rapid uptake of dissolved oxygen, believed to be due to the oxidation of Fe²⁺ to Fe³⁺; Fe³⁺ is not a substrate for ferrochelatase.