Studies on ferrochelatase: The effects of thiols and other factors on the determination of activity

1. Haems are unstable under aerobic conditions in the presence of thiols, which are used to activate the ferrochelatase enzyme; catalase inhibits this degradation of haem. In addition, thiols interfere with the determination of protohaem as its pyridine haemochromogen derivative. 2. Three ferrochelatase assays are described that minimize interference by these two reactions. Two of these assays involve measurement of porphyrin utilization, one spectrophotometrically and the second spectrofluorimetrically. The third assay measures haem formation by a pyridine haemochromogen technique. Results obtained with these three methods were in close agreement at a GSH concentration of 4mm. 3. The stimulatory effect of GSH on ferrochelatase has been confirmed. The spectrum of the haem formed is dependent on GSH concentration; at high GSH concentrations (20mm) the haem is in the reduced state, but at low concentration (4mm) the spectrum of the product resembles that of an oxidized haemoprotein such as ferrihaemoglobin. 4. The inhibitory effect of oxygen on ferrochelatase activity has been confirmed by spectrophotometric assay of porphyrin disappearance.     

The effect of exogenous δ-aminolaevulinate on rat liver haem and cytochromes

The activity of delta-aminolaevulinate synthetase is generally regarded as rate-limiting for hepatic haem biosynthesis. It has been suggested that cytochrome synthesis may also be regulated by changes in delta-aminolaevulinate synthetase activity. This hypothesis was studied by injecting product, delta-aminolaevulinate, into adult rats over a 4-240h period. The concentrations of hepatic mitochondrial cytochromes a, b, c and c(1) were unchanged by treatment with delta-aminolaevulinate, allylisopropylacetamide or phenobarbital. In control animals, total microsomal haem content equalled the sum of cytochromes b(5) plus P-450. After delta-aminolaevulinate administration the total amount of microsomal haem, measured as the pyridine haemochromogen, exceeded these components, indicating the formation of a ;free' haem pool. Haem synthesis does not appear rate-limiting for hepatic cytochrome synthesis in the adult rat.     

The structure of haem in pyridine/water mixtures and its implication in studies of haem catabolism.

1. A study of haem spectra in pyridine/water mixtures at low pyridine concentrations revealed changes in haemochrome structure consistent with an aggregation process. No corresponding change in the structure of the haemichrome species was observed. 2. This aggregation has been correlated with a previously observed sharp decrease in the rate of coupled oxidation (degradation) of haem as pyridine concentration is decreased. The decrease appears to be due primarily to haem aggregation and not to changes in the hydrophobic nature of the solvent. The effect of ethanol and butanone addition was examined and supports this conclusion. 3. Evidence is presented that coupled oxidation occurs via the iron (II) species (haemochrome).     

Electron exchange in pyridine solutions of iron(III) protoporphyrin IX chloride

Proton magnetic resonance and absorption spectroscopy have been used to examine solutions of mixtures of reduced and oxidised iron protoporphyrin IX chloride in deuterated pyridine. The Fe(II) species are low spin but the Fe(III) complex is an equilibrium mixture of high and low spin forms. The movement to high field of the ring protons of the low-spin Fe(III) signals alone increases regularly with the amount of diamagnetic Fe(II) relative to the paramagnetic Fe(III) haem. The low spin Fe(III) must be in rapid exchange with the low-spin Fe(II) complex but not with the high-spin form. The addition of carbon monoxide to the Fe(II)/Fe(III) mixture effectively blocks electron exchange between the complexes as shown by a return of the proton resonances of the Fe(III) complex to positions seen in the absence of any Fe(II).     

A comparison by magnetic circular dichroism of compound X and compound II of horseradish peroxidase

The chlorite product of horseradish peroxidase, compound X, is shown by magnetic circular dichroism (MCD) spectroscopy in the temperature range 1.6-50 K to have a very similar haem structure to compound II under the same conditions (pH 10.7). Both are concluded to contain the Fe(IV) = 0 group. The MCD spectrum also detects an unusual species, absorbing at wavelengths between 600 and 750 nm, that has magnetic properties different from those of the ferryl haem group. It is suggested that this is a species at the same oxidation level as ferryl haem but with the porphyrin ring having suffered a one-electron oxidation, i.e. [Fe(III) P.+].     

NMR and electron-paramagnetic-resonance studies of a dihaem cytochrome from Pseudomonas stutzeri (ATCC 11607) (cytochrome c peroxidase)

A dihaem cytochrome (Mr 37 400) with cytochrome c peroxidase activity was purified from Pseudomonas stutzeri (ATCC 11 607). The haem redox potentials are far apart: one of the haems is completely ascorbate-reducible and the other is only reduced by dithionite. The coordination, spin states and redox properties of the covalently bound haems were probed by visible, NMR and electron paramagnetic resonance (EPR) spectroscopies in three oxidation states. In the oxidized state, the low-temperature EPR spectrum of the native enzyme is a complex superimposition of three components: (I) a low-spin haem indicating a histidinyl-methionyl coordination; (II) a low-spin haem indicating a histidinyl-histidinyl coordination; and (III) a minor high-spin haem component. At room temperature, NMR and optical studies indicate the presence of high-spin and low-spin haems, suggesting that for one of the haems a high-spin to low-spin transition is observed when temperature is decreased. In the half-reduced state, the component I (high redox potential) of the EPR spectrum disappears and induces a change in the g-values and linewidth of component II; the high-spin component II is no longer detected at low temperature. Visible and NMR studies reveal the presence of a high-spin ferric and a low-spin (methionyl-coordinated) ferrous state. The NMR data fully support the haem-haem interaction probed by EPR. In the reduced state, the NMR spectrum indicates that the low-potential haem is high-spin ferrous.     

The formation of ferric haem during low-temperature photolysis of horseradish peroxidase Compound I.

Illumination at low temperature of the peroxide compound of horseradish peroxidase (HRP-I) causes partial conversion of the haem electronic structure from a ferryl-porphyrin radical species into a low-spin ferric state. Magnetic-c.d. (m.c.d.) and e.p.r. spectral features of the photolysis product are almost identical with those of the alkaline form of ferric HRP, proposed on the basis of its near-i.r. m.c.d. spectrum to be a Fe(III)-OH species. The ferric product of HRP-I photolysis also contains free-radical e.p.r. signals. Conversion of HRP-I into the Fe(III)-OH species, which requires transfer of a proton and two electrons from the protein, is shown to be a two-step process.     

Formation of bile pigments by coupled oxidation of cobalt-substituted haemoglobin and myoglobin.

Treatment of cobalt-substituted haemoglobin and myoglobin with ascorbate and molecular O2 (coupled oxidation) resulted in biliverdin formation from the cobalt(II) derivatives but not from the cobalt(III) derivatives. This was apparently due to the inability of ascorbate to reduce cobalt(III) haemoproteins. Isomer analysis of the biliverdins produced from coupled oxidation of cobalt(II) oxyhaemoglobin suggested that the orientation of the cobalt protoporphyrin IX in the haem pocket differed slightly from that of the haem in native haemoglobin.     

Redox potentials of the pyridine-haem c system

1. Haem c was synthesized and purified. It was shown unequivocally that the method gives a product with the cysteine residues on the -carbon atoms at the 2 and 4 positions of the haem.

2. Redox potentials of haem c in the presence of 2.5 M pyridine were determined in the pH range 1.5–13; it was found necessary to add cetyl trimethyl ammonium bromide (CTAB) to prevent precipitation in the acid range below about pH 4. The E_m vs pH curve shows three slopes (−dE/dpH) of value, 0.18, 0.01 and 0.06 with points of inflexion at pH 3.8 and 10.6. The potentials are intermediate between those of protohaem and mesohaem obtained under similar conditions.

3. With constant haem c concentration (a) 10⁻⁴ M and (b) 10⁻⁵ M and varying pyridine concentration (0.12–5 M) it was found at pH 9.0 that E_m values increased as the pyridine concentration was increased and there was a tendency to reach a plateau value. The explanation appears to be that pyridine binds more firmly to ferroporphyrin c than to ferriporhyrin c.

4. When the pyridine concentration was kept constant (2.5 M) and the haem c concentration was varied in the range 7 · 10⁻⁴–7 · 10⁻⁶ M, it was found that a decrease in haem c concentration brought about an increase in redox potential. The results are explained as being due to dimerization of the oxidized form.

5. The results are discussed in comparison with a number of related haem systems.     

Redox behaviour of the haem domain of flavocytochrome c3 from Shewanella frigidimarina probed by NMR

Flavocytochrome c3 from Shewanella frigidimarina (fcc3) is a tetrahaem periplasmic protein of 64 kDa with fumarate reductase activity. This work reports the first example of NMR techniques applied to the assignment of the thermodynamic order of oxidation of the four individual haems for such large protein, expanding its applicability to a wide range of proteins. NMR data from partially and fully oxidised samples of fcc3 and a mutated protein with an axial ligand of haem IV replaced by alanine were compared with calculated chemical shifts, allowing the structural assignment of the signals and the unequivocal determination of the order of oxidation of the haems. As oxidation progresses the fcc3 haem domain is polarised, with haems I and II much more oxidised than haems III and IV, haem IV being the most reduced. Thus, during catalysis as an electron is taken by the flavin adenosine dinucleotide from haem IV, haem III is eager to re-reduce haem IV, allowing the transfer of two electrons to the active site.     

Structure of soybean seed coat peroxidase: a plant peroxidase with unusual stability and haem-apoprotein interactions

Soybean seed coat peroxidase (SBP) is a peroxidase with extraordinary stability and catalytic properties. It belongs to the family of class III plant peroxidases that can oxidize a wide variety of organic and inorganic substrates using hydrogen peroxide. Because the plant enzyme is a heterogeneous glycoprotein, SBP was produced recombinant in Escherichia coli for the present crystallographic study. The three-dimensional structure of SBP shows a bound tris(hydroxymethyl)aminomethane molecule (TRIS). This TRIS molecule has hydrogen bonds to active site residues corresponding to the residues that interact with the small phenolic substrate ferulic acid in the horseradish peroxidase C (HRPC):ferulic acid complex. TRIS is positioned in what has been described as a secondary substrate-binding site in HRPC, and the structure of the SBP:TRIS complex indicates that this secondary substrate-binding site could be of functional importance. SBP has one of the most solvent accessible delta-meso haem edge (the site of electron transfer from reducing substrates to the enzymatic intermediates compound I and II) so far described for a plant peroxidase and structural alignment suggests that the volume of Ile74 is a factor that influences the solvent accessibility of this important site. A contact between haem C8 vinyl and the sulphur atom of Met37 is observed in the SBP structure. This interaction might affect the stability of the haem group by stabilisation/delocalisation of the porphyrin pi-cation of compound I.     

Effect of inorganic salts on hemochromogen aggregation

The absorbance and difference absorbance spectra of pyridine-hemochromogen at different concentrations showed the presence of two different types of pyridine-hemochromogens, one unstable form (compound III) at low concentration and a more stable form (compound II) at higher concentrations, suggesting that there is an interaction between hemochromogen molecules at high concentration. In the presence of inorganic salts, the unstable compound III is converted to stable compound II. The effect of various inorganic ions on the formation of compound II has been studied. The order of increasing effectiveness of the anions on compound II formation was HPO₄²⁻SO₄²⁻F⁻Cl⁻Br⁻NO₃⁻SCN⁻ and that of cation was Li⁺Na⁺K⁺. The results are discussed on the basis of the effect of these ions on the structure of water. It appears compound II is an aggregate of compound III and the aggregation is due to hydrophobic interaction.     

Characterization of the extracellular haemoglobins of Artemia salina.

The following factors were measured for extracellular haemoglobins of Artemia salina: a minimal molecular weight of globin chain per haem group (based on the iron and haem contents), the absorption coefficients, the absorption spectra of various derivatives and the amino acid compositions. These were compared with those of the haemoglobins of other invertebrates. Three Artemia haemoglobins (I, II and III) had similar molecular structures, constructed from two-globin subunits of 122000-130000mol.wt. Since the minimal mol.wt. was determined to be 18000, this suggests that one globin subunit was bound by seven haem groups, and hence one haemoglobin molecule (240000-260000mol.wt.) should contain 14 haem groups. A successful identification of this high-molecular-weight subunit required first the denaturation of haemoglobin in 1% sodium dodecyl sulphate before sodium dodecyl sulphate gel electrophoresis. Denaturation by prolonged incubation (12-36 h) at room temperature in the presence of 0.1% sodium dodecyl sulphate [Bowen, Moise, Waring & Poon (1976) Comp. Biochem. Physiol. B55, 99-103] was accompanied by extensive proteolysis, resulting in low recovery of the stainable protein and heterogeneous gel patterns. Regardless of which electrophoretic system was used, the high-molecular-weight subunit was always present provided that 1% sodium dodecyl sulphate was present during denaturation. These results contrast with those obtained by Bowen et al. (1976). However, preferential cleavage of the globin subunit (alpha) seemed to occur in vitro when standard conditions were used, producing two specific fragments having mol.wts. of 80000 (beta) and 50000 (gamma).     

Cobalt toxicity and iron metabolism in Neurospora crassa

1. Increasing concentrations of cobalt in the medium result in increased production of an iron-binding compound and a corresponding fall in catalase activity of Neurospora crassa. 2. Cobalt rapidly depletes the medium of iron by enhancing the rate of iron uptake by the mycelium. 3. With toxic amounts of cobalt there is a fall in bound (59)Fe and haem (59)Fe as well as a decreased incorporation of [2-(14)C]glycine into the mycelial haem fraction. The production of the iron-binding compound precedes the fall in the iron-dependent systems mentioned. 4. The (59)Fe bound to the iron-binding compound acts as a better iron source for haem synthesis in cell-free extracts as compared with (59)FeSO(4). 5. Cobalt inhibits iron incorporation into protoporphyrin in cell-free extracts but is not itself incorporated to an appreciable extent.     

A mechanism for NADPH inhibition of catalase compound II formation.

Catalase-bound NADPH both prevents and reverses the accumulation of inactive bovine liver catalase peroxide compound II generated by 'endogenous' donors under conditions of steady H2O2 formation without reacting rapidly with either compound I or compound II. It thus differs both from classical 2-electron donors of the ethanol type, and from 1-electron donors of the ferrocyanide/phenol type. NADPH also inhibits compound II formation induced by the exogenous one-electron donor ferrocyanide. A catalase reaction scheme is proposed in which the initial formation of compound II from compound I involves production of a neighbouring radical species. NADPH blocks the final formation of stable compound II by reacting as a 2-electron donor to compound II and to this free radical. The proposed behaviour resembles that of labile free radicals formed in cytochrome c peroxidase and myoglobin. Such radical migration patterns within haem enzymes are increasingly common motifs.     

The identification of cation-binding domains on the surface of microsomal cytochrome b5 using 1H-NMR paramagnetic difference spectroscopy.

One-dimensional and two-dimensional 1H-NMR methods and paramagnetic difference spectroscopy have defined cation binding domains on the surface of the tryptic fragment of microsomal cytochrome b5. The addition of tris(ethylenediamine) chromium(III) [Cr(en)3(3+)] to solutions of ferricytochrome b5 reveals at least three distinct sites on the surface of the protein to which highly charged cations may bind (20 mM phosphate pH 7.0, T = 300 K). Surprisingly only one of these sites is located close to the haem edge region of the protein, whilst the remaining two sites are more remote. Site I contains the exposed haem C13 propionate and a series of carboxylate residues that includes glutamates 37, 38, 43, 44, and 48. Sites II and III are located away from the haem edge region and are delineated by the broadening of aromatic resonances of histidines 26 and 80. Further investigation of the interaction between Cr(en)3(3+) and cytochrome b5 using two-dimensional double-quantum-filtered correlated spectroscopy shows that resonances assigned to Glu59, Asp60, Glu79, Asp82 and Asp83 are broadened with the distribution of these charged side chains correlating with the relaxation broadening observed from one-dimensional experiments. In a binary complex with ferricytochrome c, Cr(en3(3+) broadens many cytochrome b45 resonances including the haem propionates, His26, Ala54, Thr55 and His80. Although the pattern of line-broadening of resonances at sites II and III is unaltered by complex formation, cytochrome c shields residues at site I, the haem edge site. The results indicate that the interaction between cytochrome b5 and c in a binary complex involves multiple protein configurations.     

Inhibition of the bovine branched-chain 2-oxo acid dehydrogenase complex and its kinase by arylidenepyruvates

A novel class of inhibitors for the branched-chain 2-oxo acid dehydrogenase (BCOAD) complex has been synthesized and studied. The sodium salts of arylidenepyruvates: e.g., furfurylidenepyruvate (compound I), 4-(3-thienyl)-2-oxo-3-butenoate (compound II), cinnamalpyruvate (compound III) and 4-(2-thienyl)-2-oxo-3-butenoate (compound IV) inhibit the overall and kinase reactions of the BCOAD complex from bovine liver. Inhibitions of the overall reaction occur at the decarboxylase (E1) step as determined by a spectrophotometric assay with 2,6-dichlorophenolindophenol as an electron acceptor. Inhibition of the E1 reaction by compound I (Ki = 0.5 microM) is competitive, whereas inhibitions by compounds II (Ki = 150 microM) and III (Ki = 500 microM) are non-competitive with respect to the substrate 2-oxoisovalerate. The Km value for 2-oxoisovalerate is 6.7 microM as measured by the E1 assay. Inhibition of the E1 step by compounds I, II and III are reversible at low inhibitor concentrations based on the Michaelis-Menten kinetics observed. By comparison, compound I does not significantly inhibit pyruvate and 2-oxoglutarate dehydrogenase complexes. The arylidenepyruvates (compounds I, II and IV) inhibit the BCOAD kinase reaction in a manner similar to the substrate 2-oxo acids. The inhibition of the kinase reaction by compound I is non-competitive with respect to ATP, with an apparent Ki value of 4.5 mM. The results suggest that arylidenepyruvates may be useful probes for elucidating the reaction mechanisms of the BCOAD complex and its kinase.     

Radical intermediates in the oxidation of octaethylheme to octaethylverdoheme

Iron(III) oxyoctaethylporphyrin was isolated and purified as a dimer. The addition of tosylmethyl isocyanide to a solution of the dimer produced a monomer species, which was isolated and identified as bis(tosylmethyl isocyanide)iron(II) 5-oxyoctaethylporphyrin pi-neutral radical. The product of dissociation of the dimer by imidazole was bis(imidazole)iron(III) 5-oxyoctaethylporphyrin. The spectral properties of the product of dissociation of the dimer by pyridine and published data on bis(pyridine)oxymesoheme and bis(pyridine)oxyprotoheme were consistent with its identification as bis(pyridine)iron(II) 5-oxyoctaethylporphyrin pi-neutral radical. When this product was exposed to oxygen, a weak radical signal appeared in its electron spin resonance spectrum, which was attributed to the displacement of one of its pyridine ligands by O2 to form (pyridine)(dioxygen)iron(II) 5-oxyoctaethylporphyrin pi-neutral radical. The pyridine oxygen radical converted spontaneously to octaethylverdohemochrome, which was purified and identified as bis-(tosylmethyl isocyanide)iron(II) octaethylverdohemochrome hydroxide. The yield of verdohemochrome from iron oxyporphyrin was increased by the addition of phenylhydrazine or ascorbate. A scheme for the oxidation of iron(III) oxyporphyrin to iron(II) verdoheme by O2 that proposes a mechanism for the expulsion of CO and the replacement of a methene bridge of the porphyrin ring by an oxa bridge is presented.     

Studies on cytochrome c oxidase, I. Purification and characterization of bovine myocardial enzyme and identification of peptide chains in the complex]

As part of the preliminary work for the structural elucidation of cytochrome c oxidase, the enzyme complex was isolated from bovine heart muscle and characterised chemically. The enzyme contains 10-11 nmol haem a, and 12-13 nmol copper per mg protein. The solubilised active enzyme also contains 5% phospholipid, comprising about 2 mol each of cardiolipin and phosphatidylethanolamine per mol haem a. In addition, the preparation contains a small number of detergent molecules (Tween-80). Eight polypeptide components were isolated by preparative dodecylsulphate gel electrophoresis, gel filtration on Biogel P-60, and counter current distribution. The apparent molecular weights of these components were I - 36 000, II - 28 000 (21 000), III - 19 000, IV - 14 000, V - 12 500, VI - 11 000, VII - 10 000 and VIII - 6000. At least seven intact polypeptide chains contribute to the structure of the enzyme complex of the terminal oxidase. On the basis of amino acid analysis and end group determination, they can be divided into two groups. The high molecular weight peptides I -III are hydrophobic and their amino acid compositions differ markedly from those of known enzyme proteins, especially with respect to their contents of leucine and methionine. Components I and II have formyl methionine at their N-termini. They are therefore possibly mitochondrial membrane components from complex 4 of the respiratory chain. Polypeptides IV - VII resemble functional enzyme subunits in their amino acid composition. Some of them possess free N-termini (alanine). The low molecular weight component VIII is heterogeneous and contains the N-terminal amino acids isoleucine, serine and phenylelanine in non-stoichiometric amounts. Analysis gives a minimal protein molecular weight of 130 000 (65 000 per haem a) for the two haem and two copper-containing "monomers". The molecular weight of the moiety preliminarily defined as enzymatic is about 48 000. The chemical characterisation provides data for the strategy of the subsequent sequence analysis of the polypeptides.     

The mechanism of haem degradation in vitro. Kinetic evidence for the formation of a haem-oxygen complex.

The rate of haem degradation in aqueous pyridine shows a saturation dependence on O2 concentration. CO competitively inhibits the reaction. This is evidence for formation of an iron-O2 complex and is consistent with an intramolecular pathway for haem degradation.