Iron-sulphur proteins: crosslinked by a cluster.
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Journal Article↗
Biomedical subjects
A J Thomson
Publications and source records attributed to A J Thomson.
Idiopathic lumbosacral plexus neuropathy in two children.
Two children diagnosed as having idiopathic lumbosacral plexopathy are presented. Although an apparently rare condition, it is probably underdiagnosed since it is difficult to recognise. Before such a diagnosis, careful exclusion of compressive and infiltrative lesions of the spinal cord, cauda equina and lumbosacral plexus is of utmost importance.
Blood Chemical Analysis↗
Neurocysticercosis--experience at the teaching hospitals of the University of Cape Town.
In the 15 years 1975-1989, 239 patients attending the associated teaching hospitals of the University of Cape Town have been identified retrospectively as having neurocysticercosis. One hundred and twenty-three (51.46%) were children 12 years of age or younger, 14 (5.86%) were adolescents aged 13-19 years, and 102 (42.68%) were adults 20 years of age or older. Two hundred and twelve (88.7%) of these patients were black, almost exclusively Xhosa-speakers originating from the eastern Cape homeland regions of Transkei and Ciskei. Although the clinical features of neurocysticercosis are protean, these patients could be divided into three clinicoradiological groups--a group with seizures, a group with raised intracranial pressure, and an asymptomatic group. One hundred and ninety patients (79.5%) presented with seizures, either alone or in combination with other neurological deficits. Eighty-six patients (36%) presented with features of raised intracranial pressure, due to hydrocephalus in 32 cases, to focal space-demanding lesions in 4 and to multifocal cysticercal encephalitis in 50. Of interest is the significant difference in the abnormality causing the raised intracranial pressure in the children as opposed to the adults--44 children had multifocal cysticercal encephalitis while only 6 had hydrocephalus, and 26 adults had hydrocephalus while only 6 young adults (including 2 adolescents) had multifocal encephalitis. In the other 4 adults the raised intracranial pressure was caused by space-demanding mass lesions. Ten adult patients (4.1%) were 'asymptomatic' and a computed tomography scan for investigation of head injury revealed neurocysticercosis.
Adolescent↗
Domains of the catalytically self-sufficient cytochrome P-450 BM-3. Genetic construction, overexpression, purification and spectroscopic characterization.
1. The gene CYP102 encoding cytochrome P-450 BM-3 and subgenes encoding the cytochrome P-450 and cytochrome P-450 reductase domains have been cloned in Escherichia coli. 2. The protein products of these genes have been overexpressed and purified to homogeneity. 3. The cytochrome P-450 domain is purified in the ferric low-spin state, but is readily converted into the high-spin state by addition of the substrate palmitate (Ks = 1 microM). The cytochrome P-450 reductase domain readily reduces cytochrome c. Mixing the two domains reconstitutes only about one-thousandth of the fatty acid hydroxylase activity associated with the intact cytochrome P-450 BM-3. 4. The X-band e.p.r. spectra of both the cytochrome P-450 domain and intact cytochrome P-450 BM-3 give g-values indicating low-spin ferric haem. The spectra are virtually identical with those of the equivalent form of cytochrome P-450 cam indicating that the haem ligation in cytochrome P-450 BM-3 is identical with that of cytochrome P-450 cam. 5. Resonance Raman spectra of the substrate-free and substrate-bound forms of the cytochrome P-450 domain are given. Spectral differences in comparison with cytochrome P-450 cam may reflect subtle electronic differences between the respective haem environments.
Bacillus megaterium↗
Bacterial nitrite-reducing enzymes.
The enzymic reduction of nitrite takes place in a wide range of bacteria and is found to occur in denitrifying, assimilatory and dissimilatory pathways. In this review we describe the major molecular characteristics of the various enzymes employed in each of these processes.
Bacteria↗
Spectroscopic identification of the haem axial ligands of haemoferritin and location of possible haem-binding sites in ferritin by molecular modelling.
Horse spleen ferritin will bind up to 16 protoporphyrin IX haem groups per 24 subunits in vitro [Kadir & Moore (1990) FEBS Lett. 276, 81-84] at a site that causes the haem to be low spin for both ferric and ferrous states. E.p.r. spectra at 10 K of the oxidized form of the resulting haemoferritin gives g values of 2.93, 2.26 and 1.55, characteristic of low-spin haem. The near-i.r. magnetic circular dichroism spectrum shows a porphyrin-to-ferric charge-transfer band at 1590 nm. The spectroscopic parameters indicate that the haem group is probably bound by two histidine ligands. Molecular modelling studies reveal one type of potential haem-binding site in horse L-chain ferritin with bis-histidine co-ordination. This is an intersubunit site which lies in a pocket within the ferritin protein shell in the region of the 3-fold channel. The ligands are His-114 and His-124 in horse L-chain. A second possible set of sites in human H-chain ferritin involves His-60 residues in the pockets between pairs of subunits. These are considered less likely sites of haem occupancy. There are three of the intersubunit sites in horse L-chain ferritin at each of the eight 3-fold channels. We propose that conformational crowding between haem-binding sites at a given channel prevents more than two haems per channel being bound.
Animals↗
E.p.r. and magnetic circular dichroism spectroscopic characterization of bacterioferritin from Pseudomonas aeruginosa and Azotobacter vinelandii.
The e.p.r. and magnetic circular dichroism (m.c.d.) spectra of bacterioferritin (BFR) extracted from Pseudomonas aeruginosa and Azotobacter vinelandii have been studied over a wide temperature range down to liquid-helium temperature. The e.p.r. spectra show the presence of low-spin Fe3+ haem with g values of 2.86, 2.32, 1.48 (P. aeruginosa) and 2.88, 2.31, 1.46 (A. vinelandii), in both the presence and absence of the BFR core. Together with evidence from the porphyrin-to-Fe3+ charge-transfer band at 2240 and 2270 nm the axial haem ligands are identified as two methionines. The low-temperature m.c.d. spectra in the region 300-1000 nm of P. aeruginosa and A. vinelandii BFR are identical with one another and unaffected by removal of the iron core. Hence it can be concluded that the presence of the iron core has no detectable effect on the electronic states and on the stereochemistry of the haem group. This was unexpected, in view of the observations by Watt, Frankel, Papaefthymiou, Spartalian & Stiefel [(1986) Biochemistry 25, 4330-4336] that the redox potential of the haem group in A. vinelandii BFR shifts from -475 mV to -225 mV on removal of the core. The e.p.r. spectra of holoBFR show a broad symmetrical derivative-shaped band centred at g = 2.0 which decreases in bandwidth as the temperature is raised. This signal is assigned to the uncompensated electron spins of the iron core.
Azotobacter vinelandii↗
Detection of the optical bands of molybdenum(V) in DMSO reductase (Rhodobacter capsulatus) by low-temperature MCD spectroscopy.
Dimethylsulphoxide (DMSO) reductase from R. capsulatus contains a molybdenum-pterin cofactor at its active site. As prepared the molybdenum is in the 6+ oxidation state, devoid of EPR signals. Stepwise reduction generates an EPR signal characteristic of Mo(V) having hyperfine coupling to a single proton and integrating to less than 25% of the total molybdenum. The low temperature MCD spectrum shows oppositely signed bands between approximately 550-700 nm. These bands are assigned as dithiolene-to-Mo(V) charge transitions. A simple theoretical model can satisfactorily account for the bands in the MCD spectrum. No evidence is found for cysteine coordination to Mo(V).
Circular Dichroism↗
Spectroscopic identification of the haem ligands of cellobiose oxidase.
A spectroscopic study of the flavocytochrome b enzyme, cellobiose oxidase, employing optical, NMR, EPR and near infra-red MCD techniques, has identified the axial ligands of the b-type haem. These are a histidine and a methionine, and this ligation set is discussed in relation to the functional role of the haem group.
Carbohydrate Dehydrogenases↗
Spectroscopic studies of Rhodobacter capsulatus cytochrome c' in the isolated state and in intact cells.
Ferricytochrome c' from Rhodobacter capsulatus was investigated by 1H-NMR, EPR and optical spectroscopies. A haem-linked ionisation, occurring with a pKa of 8.4 at 25 degrees C, was observed and assigned to the ionisation of the axial histidine ligand by comparison with data for related proteins. At pH values below this pKa the spin-state of the haem Fe3+ is shown to be a quantum mechanically admixed S = 3/2, 5/2 state. Above the pKa the Fe3+ is high-spin. EPR studies of intact cells grown photoheterotrophically reveal that in situ cytochrome c' exists largely in the ferrous state. Upon the addition of [Fe(CN)6]3- the protein becomes oxidised and EPR spectra reveal that the Fe3+ spin-state is a quantum mechanically admixed S = 3/2, 5/2 state. These data indicate that the unusual spin-state of ferricytochrome c' is not a consequence of changes to the protein on its isolation, as had been suggested previously. They also indicate that in situ cytochrome c' is located in an environment with a pH less than 7.
Cytochrome c Group↗
A model of the copper centres of nitrous oxide reductase (Pseudomonas stutzeri). Evidence from optical, EPR and MCD spectroscopy.
Nitrous oxide reductase (N2OR), Pseudomonas stutzeri, catalyses the 2 electron reduction of nitrous oxide to di-nitrogen. The enzyme has 2 identical subunits (Mr approximately 70,000) of known amino acid sequence and contains approximately 4 Cu ions per subunit. By measurement of the optical absorption, electron paramagnetic resonance (EPR) and low-temperature magnetic circular dichroism (MCD) spectra of the oxidised state, a semi-reduced form and the fully reduced state of the enzyme it is shown that the enzyme contains 2 distinct copper centres of which one is assigned to an electron-transfer function, centre A, and the other to a catalytic site, centre Z. The latter is a binuclear copper centre with at least 1 cysteine ligand and cycles between oxidation levels Cu(II)/Cu(II) and Cu(II)/Cu(I) in the absence of substrate or inhibitors. The state Cu(II)/Cu(I) is enzymatically inactive. The MCD spectra provide evidence for a second form of centre Z, which may be enzymatically active, in the oxidised state of the enzyme. Centre A is structurally similar to that of CuA in bovine and bacterial cytochrome c oxidase and also contains copper ligated by cysteine. This centre may also be a binuclear copper complex.
Circular Dichroism↗
Does ferredoxin I (Azotobacter) represent a novel class of DNA-binding proteins that regulate gene expression in response to cellular iron(II)?
Azotobacter vinelandii (Av) and chroococcum (Ac) ferredoxin I contain [3Fe-4S]1 + 0 and [4Fe-4S]2+1+ clusters, when isolated aerobically, which undergo one-electron redox cycles at potentials of -460 +/- 10 mV (vs SHE) at pH 8.3 and -645 +/- 10 mV, respectively. The X-ray structure of Fd I (Av) reveals that the N-terminal half of the polypeptide folds as a sandwich of beta-strands which enclose the iron-sulphur clusters. The C-terminal sequence contains an amphiphilic alpha-helix of four turns which lies on the surface of the beta-barrel. Fd I (Av) controls expression of an unknown protein of Mr approximately 18,000. Fd I (Ac) will complex iron(II) avidly above pH approximately 8.0 only when the [3Fe-4S] cluster is reduced and provided that cellular nucleic acid is bound. Fd I (Ac) rigorously purified from nucleic acid does not undergo iron(II) uptake. These facts, together with recent evidence that the interconversion process [3Fe-4S]0 + Fe2+----[4Fe-4S]2+ in the iron-responsive element binding protein (IRE-BP) of eukaryotic cells is controlling protein expression at the level of mRNA [1991, Cell 64, 4771; 1991, Nucleic Acid Res. 19, 1739] leads to the following hypothesis. Fd I is a DNA-binding protein which interacts by single alpha-helix binding in the wide groove of DNA. The binding is regulated by iron(II) levels in the cell. The 7Fe form binds to DNA and represses gene expression. Only the DNA-bound form of the 7Fe Fd I will take up iron(II), not the form free in solution. Iron(II) becomes bound when the [3Fe-4S] cluster is reduced. The 8Fe Fd I thus generated no longer binds DNA and the gene is de-repressed. Sequence comparisons and the crystal structure suggests that the two central turns of the alpha-helix are important elements of the DNA-recognition process and that residues Gln69 and Glu73, which lie on the outer surface of the helix, hydrogen-bond with specific base pairs.
Amino Acid Sequence↗
Misdiagnosis of intraspinal lesions in childhood.
Three children with intraspinal mass lesions in whom the diagnosis was initially missed are described. Their case histories highlight the specific clinical features of and diagnostic difficulties with the syndromes produced by lesions of the craniocervical junction, the mid-thoracic spinal cord and the cauda equina.
Cauda Equina↗
Iron-sulphur clusters in electron transfer, catalysis and control.
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Amino Acid Sequence↗
Low temperature EPR and MCD studies on cytochrome b-558 of the Bacillus subtilis succinate: quinone oxidoreductase indicate bis-histidine coordination of the heme iron.
Bacillus subtilis cytochrome b-558 was expressed in high amounts in Escherichia coli, solubilized from membranes with detergent and purified free from other hemoproteins. The cytochrome possibly contains two heme groups. To determine the axial ligands to the low-spin heme and the heme rhombicity, the cytochrome was analyzed using low-temperature electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopy. The combined results exclude bis-methionine, bis-lysine and histidine-methionine coordination. Bis-histidine coordination of the heme(s) with a near perpendicular orientation of the imidazole planes is strongly suggested by the highly axial low-spin EPR signals and the intense near infrared MCD spectrum (delta epsilon = 380 M-1.cm-1 at 4.2 K and 5 T) of the charge-transfer band at 1600 nm.
Bacillus subtilis↗
The effect of haem ligands on the redox states of the hexa-haem nitrite reductase from Wolinella succinogenes.
The nitrite reductase of Wolinella succinogenes containing six covalently bound haem groups has one haem group that will not reduce fully in the presence of excess Na2S2O4. The effect of the extrinsic ligands CO and cyanide on the redox state of this haem was studied by e.p.r. and magnetic c.d. spectroscopy. It was found that both ligands increased the extent of reduction of this haem group, and that in the case of CO binding the level of reduction was correlated with the extent of CO saturation of the enzyme. Stopped-flow studies of the effect of cyanide binding on the rate of dithionite reduction showed that the rate of reduction of the ligand-binding site was increased in the presence of cyanide. This suggests that reduction of the haem groups at the active site is thermodynamically unfavourable in the absence of an extrinsic ligand. The role of the 'non-reducing' haem group and the effect of ligands on this centre and on the rate of reduction are discussed in relation to the reduction of nitrite by this enzyme.
Bacteroidaceae↗
Two structurally and kinetically distinct forms of Wolinella succinogenes nitrite reductase.
It is shown that the oxidized form of the hexa-haem nitrite reductase of Wolinella succinogenes exists in two structurally and functionally distinct forms, termed 'resting' and 'redox-cycled'. The nitrite reductase as initially isolated, termed 'resting', has five low-spin ferrihaem groups and one high-spin ferrihaem group. The reduction of these haem groups by Na2S2O4 occurs in two kinetically and spectrally distinct phases. In the slower phase the haem groups are reduced by dithionite with a limiting rate of 4 s-1. If the enzyme is re-oxidized after reduction with dithionite or with methyl viologen, the resulting ferric form, termed 'redox-cycled', possesses only low-spin haem centres and a rate of reduction in the slower phase that is no longer limited. In the resting form of the enzyme the high-spin ferrihaem group is weakly exchange-coupled to a low-spin haem group. It is proposed that in the redox-cycled form the exchange coupling occurs between two low-spin ferric haem groups. This change in spin state allows a more rapid rate of electron transfer to the coupled pair.
Bacteroidaceae↗