Effects of lipids on the activity of ferrochelatase.

Removal of lipids from submitochondrial particles or detergent-solubilized mitochondrial preparations of rat liver resulted in a 90% loss of ferrochelatase (protochemeferro-lyase, EC 4.99.1.1) activity. The addition of either a fatty acid or phospholipid restored enzyme activity; the extent of reactivation being correlated with the degree of unsaturation of the fatty acid or acyl chain and independent of the polar head group of the phospholipid, Arrhenius plots of the ferrochelatase activities of submitochondrial particles and detergent-solubilized mitochondrial preparations showed transition temperatures of 37 and 28.5 degrees C, respectively. Ferrochelatase of submitochondrial particles or detergent-solubilized preparations had an absolute requirement for Ca2+. The ferrous salt of oxalic acid, a Ca2+ chelator, was a very poor substrate for these preparations. In contrast, ferrochelatase activities of fatty acid- or lipid-supplemented acetone extracts of these preparations were not dependent on the presence of Ca2+ and ferrous oxalate served as substrate for these extracts.20     

Biosynthesis of porphyrins in Rhodopseudomonas palustris--VI. The effect of metals, thiols and other reagents on the activity of uroporphyrinogen decarboxylase

The effect of several metals and reagents on the decarboxylation rate of uroporphyrinogen I by using a 16-fold purified preparation of Uroporphyrinogen Decarboxylase from Rhodopseudomonas palustris, was studied. 1 mM Hg2+ and Cu2+ were strong inhibitors, 1 mM Zn2+ and Fe2+ under certain conditions and 1 mM Fe3+ and Cr3+ also inactivated the enzyme, but Pb2+, Cd2+ and Al3+ did not. Metals inhibition was reversed by 1 mM GSH or CySH. 0.1 mM DTNB and PCMB, 1 mM pyridoxal phosphate and 100 mM chloral hydrate, as well as 1 mM 2-methoxy-5-nitrotropone and 0.2 mM diethylpyrocarbonate inhibited Uroporphyrinogen Decarboxylase; while GSH, CySH, N-ethylmaleimide, sodium thioglycolate, 1,4-dithioerythritol, EDTA and O-phenantroline did not modify activity. Data obtained would indicate that one cysteine, one or two histidine residues and probably a lysine group are required for enzyme activity.     

The effects of ionizing radiation on DNA: the role of thiols as radioprotectors

Electron loss from N-(2-mercaptopropionyl) glycine (PSH) gave an EPR detectable radical anion, PS-.SP(-). When the PSH derivative was frozen in aqueous DNA solutions to 77 K and exposed to ionizing radiation, normal damage to the DNA was detected by EPR spectroscopy. However, on annealing above 77 K, central EPR features for the DNA base radical cations and anions gave central features assigned to PS-.SP(-) sigma*-radical anions, together with outer features for 5-6-dihydro-5-thymyl radicals, TH.. It is proposed that on freezing, the PSH molecules are constrained into a glassy region around the DNA, and that, on annealing, electron donation gives PS. radicals, with loss of quanine radical-cations, G(.+). The PS. radicals were not detectable, but on reaction with another PSH molecule, gave good EPR spectra for PS-.SP(-) radical-anions. These results indicate that PSH had little effect on the yield of the other base radicals C(.-)/T(.-). Also, growth of TH. radicals, formed from protonated thymine radical-anions, T(.-), were detected. We conclude that the primary effect of PSH is to capture the G(.+) centers, and thus could either prevent or repair radiation damage to DNA.     

Effect of sulfhydryl group modification on the activity of bovine ferrochelatase

The role of sulfhydryl groups in the activity of the terminal enzyme of the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), has been examined by using a variety of sulfhydryl group-specific reagents. The enzyme is rapidly inactivated in a pseudo-first order reaction by N-ethylmaleimide and monobromobimane and more slowly by iodoacetamide and bromotrimethylammoniobimane. Reaction with [3H]N-ethylmaleimide indicates that modification of a single sulfhydryl group is sufficient to inactivate bovine ferrochelatase. The enzyme is protected from inactivation by one substrate, ferrous iron, but not by the porphyrin substrate. Mercury and arsenite are reversible inhibitors. The fluorescence of the bound bimane is blue shifted 8 nm from that obtained in aqueous solutions and is sensitive to quenching by iodide.     

Cloning and characterization of chironomidae ferrochelatase: Copper activation of the purified ferrochelatase

All organisms utilize ferrochelatase (EC 4.99.1.1) to catalyze the insertion of ferrous iron into protoposphyrin IX in the terminal step of the heme biosynthetic pathway. Different metal-binding affinity for the enzyme leads to changes in enzyme activity. In this work, we have cloned and over-expressed the enzyme from chironomidae in E. coli. The enzyme was purified and characterized. The recombinant enzyme showed higher enzymatic activity (four-fold increase) in the presence of copper ions and unaffected by calcium ions. Other divalent metal ions including magnesium, manganese, lead, reduced the enzyme activity by >60%. Over 90% of the enzyme activity was inhibited by Zn2+. The sequence alignment of amino acid residues reveals 83% homology with other ferrochelatases. The results of electron proton resonance (EPR) analysis showed that Fe2+ ion was present in the cluster of the recombinant enzyme complex. The recombinant enzyme also contained the [2Fe-2S] center with two-fold higher enzymatic activity than human ferrochelatase.     

Studies on the ferrochelatase activity of mitochondria and submitochondrial particles with special reference to the regulatory function of the mitochondrial inner membrane

The question addressed in the title was examined by measuring fluorescence emission spectra and light-induced fluorescence-yield changes of chloroplasts which had been frozen to ?196 °C rapidly, as very thin samples adsorbed into substrates which were plunged directly into liquid nitrogen, or slowly by the cooling action of liquid nitrogen through the wall of the cuvette. Contrary to previous reports, we found that the rate of cooling had no influence on the shape of the emission spectrum, the extent of the variable fluorescence or the fraction of the absorbed quanta which are delivered initially to Photosystem I.     

Redox control of hydrogenase activity in the green alga Scenedesmus obliquus by thioredoxin and other thiols.

The activity of the NiFe-hydrogenase from the green alga Scenedesmus obliquus is inhibited by both algal thioredoxins f and I+II, and by Escherichia coli thioredoxin. The strongest inhibition was observed with homologous chloroplastic thioredoxin f (I50 = 21 nM) and E. coli thioredoxin (I50 = 83 nM). For the homologous cytoplasmic thioredoxins I+II an I50 of 667 nM was determined. Glutathione shows a similar but much less pronounced inhibitory effect whereas dithiothreitol had no effect. In addition to glucose-6-phosphate dehydrogenase, NiFe-hydrogenase is only the second enzyme known to be inhibited by reduced thioredoxin.     

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.