Ferrochelatase of spinach chloroplasts

Spinach chloroplasts catalyse the incorporation of Fe(2+) 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 [(59)Fe]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 [(59)Fe]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 Fe(2+) incorporation into protoporphyrin was 8mum and that for Fe(2+) incorporation into mesoporphyrin was 36mum. At non-saturating Fe(2+) concentrations the K(m) for protoporphyrin was 0.2mum and that for mesoporphyrin was 0.4mum. 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 Fe(2+) to buffer solutions caused a rapid uptake of dissolved oxygen, believed to be due to the oxidation of Fe(2+) to Fe(3+); Fe(3+) is not a substrate for ferrochelatase.     

Iron targeting to mitochondria in erythroid cells

Immature erythroid cells have an exceptionally high capacity to synthesize haem that is, at least in part, the result of the unique control of iron metabolism in these cells. In erythroid cells the vast majority of Fe released from endosomes must cross both the outer and the inner mitochondrial membranes to reach ferrochelatase, which inserts Fe into protoporphyrin IX. Based on the fact that Fe is specifically targeted into erythroid mitochondria, we have proposed that a transient mitochondria-endosome interaction is involved in Fe transfer to ferrochelatase [Ponka (1997) Blood 89, 1-25]. In this study, we examined whether the inhibition of endosome mobility within erythroid cells would decrease the rate of (59)Fe incorporation into haem. We found that, in reticulocytes, the myosin light-chain kinase inhibitor, wortmannin, and the calmodulin antagonist, W-7, caused significant inhibition of (59)Fe incorporation from (59)Fe-transferrin-labelled endosomes into haem. These results, together with confocal microscopy studies using transferrin and mitochondria labelled by distinct fluorescent markers, suggest that, in erythroid cells, endosome mobility, and perhaps their contact with mitochondria, plays an important role in a highly efficient utilization of iron for haem synthesis.     

Mechanism of iron transport to the site of heme synthesis inside yeast mitochondria.

The import of metals, iron in particular, into mitochondria is poorly understood. Iron in mitochondria is required for the biosynthesis of heme and various iron-sulfur proteins. We have developed an in vitro assay to follow the uptake of iron into isolated yeast mitochondria. By measuring the incorporation of iron into porphyrin by ferrochelatase in the matrix, we were able to define the mechanism of iron import. Iron uptake is driven energetically by a membrane potential across the inner membrane but does not require ATP. Only reduced iron is functional in generating heme. Iron cannot be preloaded in the mitochondrial matrix but rather has to be transported across the inner membrane simultaneously with the synthesis of heme, suggesting that ferrochelatase receives iron directly from the inner membrane. Transport of iron is inhibited by manganese but not by zinc, nickel, and copper ions, explaining why in vivo these ions are not incorporated into porphyrin. The inner membrane proteins Mmt1p and Mmt2p proposed to be involved in mitochondrial iron movement are not required for the supply of ferrochelatase with iron. Iron transport can be reconstituted efficiently in a membrane potential-dependent fashion in proteoliposomes that were formed from a detergent extract of mitochondria. Our biochemical analysis of iron import into yeast mitochondria provides the basis for the identification of components involved in transport.     

The utilization of iron and its complexes by mammalian mitochondria

Sonicated mitochondria catalyse the reduction of ferric salts, and the subsequent incorporation of Fe(2+) into haem, when provided with a reducing substrate such as succinate or NADH. The rate of haem synthesis was low under aerobic conditions and, after a short lag period, accelerated once anaerobic conditions were achieved; it was insensitive to antimycin A. The lag period was decreased by preincubating the mitochondria with NADH and Fe(3+). Newly formed Fe(2+) was autoxidized rapidly and the consequent O(2) uptake was measured with an oxygen electrode to determine the rate of enzymic formation of Fe(2+) from FeCl(3); this reaction was rapid in sonicated mitochondria provided with NADH or succinate and was insensitive to antimycin A. The reaction was very slow in intact mitochondria, suggesting a permeability barrier to Fe(3+) ions. This system was used to test the permeability of the mitochondrial membrane to various iron complexes of biological importance. Of the compounds tested only ferrioxamine G appeared to penetrate readily and the iron of this complex was reduced when intact mitochondria were supplied with succinate or NADH-linked substrates. The reduction was insensitive to rotenone or antimycin A. Both ferrioxamine G and ferrioxamine B were, however, reduced by particles. The membrane fraction of sonicated mitochondria was necessary for the reduction. The rate of ferrioxamine B reduction by sonicated mitochondria was measured by a dual-wavelength spectrophotometric assay and was found to be stimulated in conditions where the Fe(2+) produced was utilized for haem synthesis. The addition of FeCl(3) to anaerobic particles caused an oxidation of cytochrome b when this region of the respiratory chain was isolated by treatment with rotenone and antimycin A. These results suggest that the reduction of ferric iron and its complexes occurs inside the inner mitochondrial membrane in proximity to ferrochelatase. Possible sites for this reduction are the flavoproteins, succinate and NADH dehydrogenase.     

Orientation of ferrochelatase in bovine liver mitochondria

The orientation of ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, was examined in bovine liver mitochondria. The ability of a membrane-impermeable sulfhydryl reagent, 4,4'-dimaleimidylstilbene-2,2'-disulfonic acid, to inactivate ferrochelatase in intact or disrupted mitochondria and mitoplasts was examined. Using succinate dehydrogenase as an internal marker, it was found that ferrochelatase was inactivated only in disrupted mitochondria and mitoplasts, suggesting an internal location for the active site of the enzyme. In addition, antibodies raised against purified ferrochelatase were found to inhibit activity only in disrupted but not in intact mitoplasts. These data demonstrate that in bovine liver mitochondria ferrochelatase is located on the matrix side of the inner mitochondrial membrane. Data obtained with the membrane-impermeable amino reagent isethionyl acetimidate indicate that ferrochelatase physically spans the inner mitochondrial membrane with portions of the protein exposed on both sides of the membrane.     

Mechanism and localization of cardiolipin biosynthesis revisited: evidence for the identical mechanism and different localization in mitochondrial and submitochondrial membranes isolated from guinea pig and rat liver

The mechanism of cardiolipin (diphosphatidylglycerol) biosynthesis was examined in mitochondria and outer and inner mitochondrial membranes prepared from guinea pig and rat livers to determine whether this formation from phosphatidylglycerol was absolutely dependent on cytidinediphosphodiglyceride, as previously reported for intact mitochondria. Experimental results confirmed that the biosynthesis of cardiolipin, from the membrane-bound radioactive phosphatidylglycerol in intact mitochondria isolated from guinea pig and rat liver, was absolutely dependent on CDP-diglycerides and required the addition of divalent cations. Furthermore, the same mechanism for the biosynthesis of cardiolipin was operational in the outer and inner mitochondrial membranes. This biosynthesis was associated with both the outer and inner mitochondrial membranes prepared from guinea pig liver, but only with the inner mitochondrial membranes prepared from rat liver. The release of radioactive glycerol was also measured, but the amount obtained did not satisfy the stoichiometric requirement for CDP-diglyceride-independent biosynthesis of cardiolipin from 2 mol of phosphatidylglycerol with the liberation of 1 mol of glycerol. Therefore, it was concluded that this mechanism is not involved in the biosynthesis of cardiolipin in mitochondrial and submitochondrial membranes prepared from guinea pig and rat liver.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

Synthesis of proteins in heart mitochondria during postnatal development of the rat

Proteins of inner mitochondrial membranes of the albino rat myocardium during postnatal development of 1, 3 and 6 months old animals were electrophoretically separated in 10% polyacrylamide gel. The rate of 14C-amino acids incorporation into examined proteins was determined in vitro. Specific radioactivity of the total mitochondrial fraction decreased in the course of the postnatal development. That of outer membranes remained unchanged, though it sharply increased in inner membranes of mature animals as compared with animals aged one month. Levels of radioactive precursor incorporation in separate protein fractions of inner membranes of the myocardium mitochondria were estimated.     

Properties of membrane bound ferrochelatase purified from baboon liver mitochondria

1. Baboon ferrochelatase was purified to apparent homogeneity. 2. The pH optimum was 7.85 and the pI 5.3. 3. The estimated molecular weight was 205 K made up by two 50 + 60 K heterodimers. 4. The Km values for proto- and mesoporphyrin were 18.5 and 10.8 microM with iron as co-substrate. With cobalt as co-substrate the Km values were 34.5 and 10.4 microM, respectively. The mean Km value for iron was 2.2 microM while cobalt acted as a complete inhibitor. 5. Lead played a dual role that of both pseudo substrate and inhibitor. As shown by inhibitor kinetics, Pb acted as a two-step two-site parabolic competitive inhibitor. The mean Ki value at low Pb levels was 0.65 mM and at high levels 0.17 mM. 6. Substrate inhibition occurred above 36 microM for proto- and 44 microM for mesoporphyrin with iron as co-substrate. For iron, with mesoporphyrin as co-substrate it occurred above 29 microM.     

Phospholipid metabolism in plant mitochondria: submitochondrial sites of synthesis

Intact mitochondria from the endosperm of castor bean were isolated on linear sucrose gradients. These mitochondria were ruptured and the membranes separated on discontinuous sucrose gradients into outer membrane, intact inner membrane, and ruptured inner membrane fractions. Each membrane fraction was examined for its capacity to synthesize phosphatidylglycerol, CDP-diglyceride, phosphatidylcholine via methylation, and phosphatidic acid. The syntheses of phosphatidylglycerol, CDP-diglyceride, and phosphatidylcholine were localized exclusively in the inner mitochondrial membrane fractions while phosphatidic acid synthesis occurred in both the inner and outer mitochondrial membranes.     

The functional size of ferrochelatase determined in situ by radiation inactivation.

Ferrochelatase (EC 4.99.1.1) catalyzes the final step of heme biosynthesis, the insertion of iron(II) into protoporphyrin. It is an integral protein of the inner mitochondrial membrane. The functional size of bovine hepatic ferrochelatase has been studied in situ using radiation inactivation analysis. The functional unit required for enzymic activity in intact mitochondria was found to have a mass of 82 +/- 13 kDa. In contrast, the structural unit (evaluated in immunoblots following sodium dodecyl sulfate-polyacrylamide gel electrophoresis) has a mass of 40 +/- 10 kDa. Similar results were obtained when irradiation was performed on sodium cholate-solubilized mitochondria. The presence or absence of dithiothreitol during irradiation had no effect on target sizes obtained from either intact or solubilized mitochondria. Pairwise comparison of the functional and structural target sizes from each set of irradiated samples yielded a ratio of 2.0 +/- 0.4. Previous studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography have shown that a Mr 40,000 peptide is associated with ferrochelatase activity. This study shows that the functional size of bovine ferrochelatase is approximately 80 kDa; the data are most consistent with a model for active ferrochelatase composed of two structural subunits of about 40 kDa each.     

Gazing at translocation in the mitochondrion

Most mitochondrial proteins are synthesized in the cytosol and imported into the mitochondrion via molecular machines called translocons on the outer and inner mitochondrial membranes. Alder et al. (2008b) examine protein translocation into intact mitochondria by adapting fluorescent techniques first used to study translocation in the endoplasmic reticulum.     

Purification and properties of ferrochelatase from the yeast Saccharomyces cerevisiae. Evidence for a precursor form of the protein

Ferrochelatase was purified to homogeneity from yeast mitochondrial membranes and found to be a 40-kDa polypeptide with a pI at 6.3. Fatty acids were absolutely necessary to measure the activity in vitro. The Michaelis constants for protoporphyrin IX (9 x 10(-8) M), ferrous iron (1.6 x 10(-7) M), and zinc (9 x 10(-6) M) were determined on purified enzyme preparations in the presence of dithiothreitol. However, the Km for zinc was lower when measured in the absence of dithiothreitol (K-m(Zn2+) = 2.5 x 10(-7) M, Km(protoporphyrin) unchanged). The maximum velocities of the enzyme were 35,000 nmol of heme/h/mg of protein and 27,000 nmol of zinc-protoporphyrin/h/mg of protein. Antibodies against yeast ferrochelatase were raised in rabbits and used in studies on the biogenesis of the enzyme. Ferrochelatase is synthesized as a higher molecular weight precursor (Mr = 44,000) that is very rapidly matured in vivo to the Mr = 40,000 membrane-bound form. This precursor form of ferrochelatase was immunoprecipitated from in vitro translation (in a rabbit reticulocyte lysate system) of total yeast RNAs. The antibodies were used to characterize two yeast mutant strains deficient in ferrochelatase activity as being devoid of immunodetectable protein in vivo and ferrochelatase mRNA in vitro translation product. The N-terminal amino acid sequence of the purified protein has been established and was found to be frayed.     

Mitochondrial iron not bound in heme and iron-sulfur centers and its availability for heme synthesis in vitro

Rat liver mitochondrial fractions have previously been shown to contain a pool of iron which was bound neither in cytochromes nor in iron-sulfur centers (Tangerås, A., Flatmark, T., Bäckström, D. and Ehrenberg, A. (1980) Biochim. Biophys. Acta 589, 162–175), and in the present study the availability of this iron pool for heme synthesis has been studied in isolated mitochondria. A minor fraction of this iron is here shown to originate from iron-rich lysosomes present as a contaminant in mitochondrial fractions isolated by differential centrifugation, and a method for the selective quantitation of this iron pool was developed. The availability of the mitochondrial iron pool for heme synthesis by mitochondria in vitro was studied using a recently developed HPLC method for the assay of ferrochelatase activity. When deuteroporphyrin was used as the substrate, 1.04±0.13 nmol/mg protein of deuteroheme was formed after 6 h incubation at 37°C when a plateau was approached, and the initial rate of heme synthesis was 0.3 nmol/h per mg protein. Heme formation from the physiological substrate protoporphyrin was also seen. The heme synthesis increased with the amount of mitochondria used and was blocked by both Fe(II) and Fe(III) chelators. The heme synthesis was independent of mitochondrial oxidizable substrates and no difference was observed between pH 7.4 and 6.5. FMN slightly stimulated the formation of heme from endogenous iron, probably by mobilization of a small amount of contaminating lysosomal iron present in the preparations. The possibility that the mitochondrial iron pool functions as the proximate iron donor for heme synthesis by ferrochelatase in vivo is discussed.     

Cytochrome c sorption-desorption effects on the external NADH oxidation by mitochondria: experimental and computational study

The rupture of the outer mitochondrial membrane is known to be critical for cell death, but the mechanism, specifically its redox-signaling aspects, still needs to be studied in more detail. In this work, the external NADH oxidation by rat liver mitochondria was studied under the outer membrane rupture induced by the mitochondria hypotonic treatment or the inner membrane permeability transition. The saturation of the oxidation rate was observed as a function of mitochondrial protein concentration. This effect was shown to result from cytochrome c binding to the mitochondrial membranes. At a relatively high concentration of mitochondria, the oxidation rate was strongly activated by 4 mm Mg(2+) due to cytochrome c desorption from the membranes. A minimal kinetic model was developed to explain the main phenomena of the external NADH oxidation modulated by cytochrome c and Mg(2+) in mitochondria with the ruptured outer membrane. The computational behavior of the model closely agreed with the experimental data. We suggest that the redox state of the released cytochrome c, considered by other authors to be important for apoptosis, may strongly depend on its oxidation by the fraction of mitochondria with the ruptured outer membrane and on the cytoplasmic cytochrome c reductase activity.     

Translocation and insertion of precursor proteins into isolated outer membranes of mitochondria

Nuclear-encoded proteins destined for mitochondria must cross the outer or both outer and inner membranes to reach their final sub- mitochondrial locations. While the inner membrane can translocate preproteins by itself, it is not known whether the outer membrane also contains an endogenous protein translocation activity which can function independently of the inner membrane. To selectively study the protein transport into and across the outer membrane of Neurospora crassa mitochondria, outer membrane vesicles were isolated which were sealed, in a right-side-out orientation, and virtually free of inner membranes. The vesicles were functional in the insertion and assembly of various outer membrane proteins such as porin, MOM19, and MOM22. Like with intact mitochondria, import into isolated outer membranes was dependent on protease-sensitive surface receptors and led to correct folding and membrane integration. The vesicles were also capable of importing a peripheral component of the inner membrane, cytochrome c heme lyase (CCHL), in a receptor-dependent fashion. Thus, the protein translocation machinery of the outer mitochondrial membrane can function as an independent entity which recognizes, inserts, and translocates mitochondrial preproteins of the outer membrane and the intermembrane space. In contrast, proteins which have to be translocated into or across the inner membrane were only specifically bound to the vesicles, but not imported. This suggests that transport of such proteins involves the participation of components of the intermembrane space and/or the inner membrane, and that in these cases the outer membrane translocation machinery has to act in concert with that of the inner membrane.     

Mitochondrial iron not bound in heme and iron-sulfur centers and its availability for heme synthesis in vitro

Rat liver mitochondrial fractions have previously been shown to contain a pool of iron which was bound neither in cytochromes nor in iron-sulfur centers (Tanger?s, A., Flatmark, T., B?ckstr?m, D. and Ehrenberg, A. (1980) Biochim. Biophys. Acta 589, 162-175), and in the present study the availability of this iron pool for heme synthesis has been studied in isolated mitochondria. A minor fraction of this iron is here shown to originate from iron-rich lysosomes present as a contaminant in mitochondrial fractions isolated by differential centrifugation, and a method for the selective quantitation of this iron pool was developed. The availability of the mitochondrial iron pool for heme synthesis by mitochondria in vitro was studied using a recently developed HPLC method for the assay of ferrochelatase activity. When deuteroporphyrin was used as the substrate, 1.04 +/- 0.13 nmol/mg protein of deuteroheme was formed after 6 h incubation at 37 degrees C when a plateau was approached, and the initial rate of heme synthesis was 0.3 nmol/h per mg protein. Heme formation from the physiological substrate protoporphyrin was also seen. The heme synthesis increased with the amount of mitochondria used and was blocked by both Fe(II) and Fe(III) chelators. The heme synthesis was independent of mitochondrial oxidizable substrates and no difference was observed between pH 7.4 and 6.5. FMN slightly stimulated the formation of heme from endogenous iron, probably by mobilization of a small amount of contaminating lysosomal iron present in the preparations. The possibility that the mitochondrial iron pool functions as the proximate iron donor for heme synthesis by ferrochelatase in vivo is discussed.     

Purification of a hexokinase-binding protein from the outer mitochondrial membrane.

Brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) binds selectively to the outer membrane of rat liver mitochondria but not to inner mitochondrial or microsomal membranes nor to the plasma membrane of human erythrocytes. A protein having subunit molecular weight of 31,000, determined by sodium dodecyl sulfate-gel electrophoresis, has been highly purified from the outer mitochondrial membrane by repetitive solubilization with octyl-beta-D-glucopyranoside followed by reconstitution into membranous vesicles when the detergent is removed by dialysis. When incorporated into lipid vesicles, the protein confers the ability to bind brain hexokinase in a Glc-6-P-sensitive manner as is seen with the intact outer mitochondrial membrane. Hexokinase binding ability and the 31,000 subunit molecular weight protein co-sediment during sucrose density gradient centrifugation. Both hexokinase binding ability and the 31,000 subunit molecular weight protein are resistant to protease treatment of the intact outer mitochondrial membrane while other membrane proteins are extensively degraded. It is concluded that this protein, designated the hexokinase-binding protein (HBP), is an integral membrane protein responsible for the selective binding of hexokinase by the outer mitochondrial membrane.     

Hepatic mitochondrial membrane lipid environment and protein nutrition

Effect of feeding rice diet with and without lysine and threonine supplementation on hepatic mitochondria and its inner and outer membrane proteins, enzymes and phospholipids has been studied. The exchange of phosphatidylcholine and phosphatidylethanolamine between microsomes and mitochondria has also been studied under these conditions. Deficient diet lead to significant decrease in proteins as well as activities of monoamine oxidase, succinate dehydrogenase, cytochrome a + a3 and cytochrome c in mitochondria and its inner and outer membranes. Feeding of the deficient diet also significantly reduced total phospholipids and PC in mitochondria and its outer mitochondrial membrane. In the inner mitochondrial membrane, only PE and cardiolipin were reduced. The incorporation (DPM/microgram PLP) of [methyl-3H]choline and [methyl-14C]methionine into PC of mitochondria and its outer membrane and that of 32Pi into PC and PE of outer mitochondrial membrane but only into PC of inner mitochondrial membrane were significantly reduced in the deficient group. The exchange rates of PC and PE between microsomes and mitochondria were reduced in the deficient group. Supplementation of the deficient diet with lysine and threonine profoundly improved the above biochemical lesions as compared to casein fed rats.     

Mitochondrial iron not bound in heme and iron-sulfur centers. Estimation, compartmentation and redox state

A method is described for the assay of total mitochondrial non-heme iron and a fraction which does not belong to the iron-sulfur proteins (FeS centers) of the outer and inner membrane. The assay of the latter fraction, which is termed 'non-heme non-FeS iron', is based on the formation of a chelate of Fe(II) with bathophenanthroline sulfonate in osmotically swollen mitochondria under conditions where the FeS centers are quite stable as determined by EPR spectroscopy at 20.4 K, 93 K and 123 K. The 'non-heme non-FeS iron', which in normal rat liver mitochondria amounts to approx. one third of the total mitochondrial iron (i.e. 1.7 +/- 0.3 nmol . mg-1 protein), does not represent a homogeneous pool of iron. Based on studies of its reaction with bathophenanthroline sulfonate and the dependency of this reaction on reducing agents in mitochondria and mitoplasts, evidence is presented that this non-heme iron is present in two major pools in which the inner membrane constitutes the barrier. A minor fraction (i.e. 0.4 +/- 0.2 nmol . mg-1 protein) is localized to the 'outer' compartment and a major fraction (i.e. 1.1 +/- 0.1 nmol . mg-1 protein) is localized to the 'inner' compartment and is equally distributed between the inner membrane and the matrix. The experiments described in this study also indicate that approximately half of the 'non-heme non-FeS iron' of the 'inner' pool is in the ferrous form in mitochondria as isolated, and this was not increased when oxidizable substrates were added to the mitochondria. Although the biological significance of this iron pool is not yet clear, it is likely that it represents a transit iron pool being the proximate iron donor for heme synthesis catalyzed by the enzyme ferrochelatase.