Biomedical subjects
Publications and source records attributed to A J Thomson.
Peritonitis is a rare extra-articular manifestation of juvenile chronic arthritis (JCA) which, although mentioned in passing, seems not to have been documented previously. Two patients with systemic-onset JCA, 1 of whom had peritonitis in the early weeks of onset while the second developed peritonitis 10 years after onset, are described.
Magnetic-c.d., e.p.r. and optical-absorption spectra are reported for the half-reduced form of Pseudomonas aeruginosa cytochrome c-551 peroxidase, a di-haem protein, and its fluoride derivative. Comparison of this enzyme species with oxidized peroxidase shows the occurrence of spin-state changes at both haem sites. The high-potential haem changes its state from partially high-spin to low-spin upon reduction. This is linked to a structural alteration at the ferric low-potential haem group, causing it to change from low-spin to high-spin. Low-temperature spectra demonstrate photolysis of an endogenous ligand of the high-potential haem. In addition, an inactive form of enzyme is examined in which the structural change at the ferric low-potential haem does not occur on reduction of the high-potential haem.
The six-coordinate monohaem ferricytochrome b-562 from Escherichia coli exhibits two haem-linked pH-dependent transitions detected by NMR and optical spectroscopy. Only one of these transitions, that of the Fe(III)-coordinated His-102, is detected by EPR and MCD; the ionisation of a haem propionate is not. Both ionisations are redox-state-dependent and the midpoint redox potential of the protein is markedly pH-dependent. Over the pH range 5.0 to 8.5 the potential drops from 260 mV to 110 mV and at least five single proton ionisations are responsible for this. In addition to the two spectroscopically identified ferricytochrome ionisations, there are at least three unidentified ionisations, two of which occur in the ferrous protein. From a consideration of the X-ray structure, together with NMR data, it seems probable that at least one of these ionisations involves an amino acid carboxylate. The X-ray structure also suggests that the relatively low pKa of His-102 is a result of its proximity to Arg-98. However, an appreciable interaction between these groups requires that the solution conformation differs slightly from the X-ray structure. The fast rate of electron self-exchange, over 4 X 10(6) M-1 X s-1 at 315 K and pH* 7, may be a reflection of the fact that, as shown by the X-ray structure, a large amount of the haem and axial histidine ligand are exposed at the molecular surface with an asymmetric distribution of charged groups surrounding them.
The m.c.d. spectrum of the oxidized state of hydrogenase from Megasphaera elsdenii has been measured at liquid-helium temperatures. This oxidation state of the enzyme displays a characteristic rhombic e.p.r. signal with g-values of 2.101, 2.052 and 2.005 assigned previously to a [4Fe-4S]3+ cluster as in oxidized HiPIP (high-potential iron-sulphur protein) [Van Dijk, Grande, Mayhew & Veeger (1980) Eur. J. Biochem. 107, 251-261]. The low-temperature m.c.d. spectrum shows no features attributable to an oxidized four-iron cluster of the HiPIP type, but does reveal broad, positive peaks at 460 and 730 nm, which magnetize in a manner untypical of a spin S = 1/2 cluster with g-values close to 2. The m.c.d. spectrum is most closely similar to that of dye-oxidized P-clusters known in the enzyme nitrogenase. It is therefore proposed that the rhombic e.p.r. spectrum at a g-value close to 2 arises from an m.c.d.-silent radical species that may be related chemically to the cysteine persulphide species, RS-S., recently found in the hexacyanoferrate-oxidized seven-iron ferredoxin of Azotobacter vinelandii [Morgan, Stephens, Devlin, Stout, Melis & Burgess (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1931-1935].
Cerebrospinal fluid (CSF) from 221 patients with multiple sclerosis (MS) and 85 patients with other neurological disorders (OND) was examined using a competitive radioimmunoassay for myelin basic protein (MBP) immunoreactivity. MBP was found in 46 of 55 MS patients (84%) examined within six weeks of relapse but in only 11 of 85 patients (13%) with OND. There was a significant correlation between the concentration of MBP in the CSF and relapse severity in patients seen within four weeks of the onset of symptoms (p less than 0.01). Of 44 patients in remission, MBP was detected in 12, and these patients had a significantly higher tendency to subsequent relapse (p less than 0.05). In 72 patients with progressive disease the presence of MBP in the CSF reflected the confidence of clinical diagnosis. The results of this study suggest that measurement of MBP in the CSF gives an objective method of monitoring disease activity in patient with MS.
The effect of sulphide on resting oxidized cytochrome c oxidase was studied by both e.p.r. and optical-absorption spectroscopy. Excess sulphide causes some reduction of cytochrome a, CuA and CuB, and the formation of the cytochrome a3-SH complex after about 1 min. After several hours in the presence of excess sulphide only the e.p.r. signals due to low-spin ferricytochrome a3-SH persist, giving a partially reduced species. Re-oxidation of this partially reduced sulphide-bound enzyme by ferricyanide makes all of the metal centres except CuB detectable by e.p.r. We conclude that sulphide has reduced and binds to CuB as well as to ferricytochrome a3. Sulphide binding to cuprous CuB may raise its mid-point potential and make re-oxidation difficult. Addition of reductant (ascorbate + NNN'N'-tetramethyl-p-phenylenediamine) and sulphide together to the oxidized resting enzyme produces a species in which cytochrome a and CuA are nearly completely reduced and cytochrome a3 is e.p.r.-detectable as approx. 80% of one haem in the low-spin sulphide-bound complex. The g = 12 signal of this partially reduced derivative is almost unchanged in magnitude relative to that of the resting enzyme; this suggests that the g = 12 signal may arise from less than 20% of the enzyme and that it may be relatively unreactive to both ligation and reduction. Such a reactivity pattern of the g = 12 form of the oxidase is also demonstrated with the ligands F- and NO, which are thought to bind to cytochrome a3 and CuB respectively.
Preparations of nitrate reductase in the resting state from Pseudomonas aeruginosa exhibit an Mo(V) e.p.r. signal. Progressive reduction of the enzyme results at first in the intensification and then in the disappearance of the signal. Three different species of Mo(V) were detected by e.p.r. These are the high-pH species (g1 = 1.9871; g2 = 1.9795; g3 = 1.9632) and nitrate and nitrite complexes of a low-pH species (respectively g1 = 2.0004; g2 = 1.9858; g3 = 1.9670; and g1 = 1.9975; g2 = 1.9848; g3 = 1.9652). These signals are closely analogous to those for the enzyme from Escherichia coli described by Vincent & Bray [(1978) Biochem. J. 171, 639-647]. Signals typical of iron-sulphur clusters were also detected. In the oxidized enzyme these are believed to arise from a [3Fe-4S] cluster (g = 2.01) and in the reduced enzyme from an unusual low-potential [4Fe-4S]+ cluster (g1 = 2.054; g2 = 1.952; g3 = 1.878). The iron-sulphur centres were also studied in a 'high-catalytic-activity' form of the enzyme. Reduction with Na2S2O4 resulted in the formation of a complex signal with g values at 2.054, 1.952, 1.928, 1.903 and 1.878. The signal could be deconvoluted by reductive titration of the enzyme into two species (g1 = 2.054; g2 = 1.952; g3 = 1.878; and g1 = 2.036; g2 = 1.928; g3 = 1.903). The degradation of a [4Fe-4S] into a [3Fe-4S] cluster in the enzyme is suggested by these studies, the process being dependent on the method used to purify the enzyme. The addition of nitrate to the reduced enzyme results in the oxidation of Mo(IV) to Mo(V) and of all the iron-sulphur centres.
Ferredoxin from Azotobacter chroococcum has been studied by low-temperature magnetic-circular-dichroism and electron-paramagnetic-resonance spectroscopy. When aerobically isolated ferredoxin contains a [3Fe-4S] and [4Fe-4S] cluster. Anaerobic treatment with dithionite in the presence of ethanediol reduces the [3Fe-4S] cluster to give two spectroscopically distinct forms RI and RII which are reversibly interconvertible with a pKa approximately 7.5. The higher-pH form, RII, has a high affinity for ferrous ion and converts readily to a [4Fe-4S]1+ cluster, scavenging iron from the medium. The presence of the iron chelator EDTA inhibits this conversion.
The magnetic properties at different temperatures of oxidized Pseudomonas aeruginosa cytochrome c-551 peroxidase were studied, with the use of the technique of magnetic-circular-dichroism spectroscopy. At 4.2K, both constituent haems were found to be low-spin, and the axial ligand pairs were identified as histidine-histidine and histidine-methionine. At room temperature high-spin signals were observed, amounting to less than 25% of the total haem present. These signals are concluded to arise mainly from a temperature-dependent spin-state equilibrium in the methionine-ligated haem.
The half-reduced, fully reduced and fully reduced CO-bound forms of the enzyme cytochrome c-551 peroxidase isolated from Pseudomonas aeruginosa were examined by a combination of low-temperature absorption and magnetic-circular-dichroism spectroscopy. Deliberate low-temperature (4.2K) photolysis of these forms of the enzyme, in all of which the high-potential haem is in the ferrous state, revealed that this haem group, assigned to have a histidine-methionine ligand set, is photosensitive. The photolabile ligand is most likely to be the methionine residue, and the product of photolysis, namely the high-spin (S = 2) ferrous form, is stable at low temperature (4.2K). Warming to approx. 20K allows thermal recombination to occur, restoring the low-spin (S = 0) state. The low-potential haem (bis-histidine ligation) is photoinert in both ferric and ferrous states; however, the photosensitive CO adduct of this centre cannot be maintained as the photolysed (S = 2) product at 4.2K. This surprising observation may be due to quantum-mechanical tunnelling of the CO through the activation barrier even at 4.2K, implying that the activation barrier to thermal recombination is both narrow and low. Low-temperature absorption spectroscopy reveals that the high-potential haem has a very characteristic low-spin ferrous spectrum with intense highly structured beta- and split alpha-bands, whereas the spectrum of the low-potential ferrous haem contains alpha- and beta-bands devoid of fine structure.
The major metal clusters of the MoFe protein, Kpl , of Klebsiella pneumoniae nitrogenase were characterized separately by low-temperature magnetic-circular-dichroism spectroscopy. The spectra and magnetization curves of the extracted iron-molybdenum cofactor, FeMoco , and of 'P' clusters in NifB - Kpl , the inactive, FeMoco -less, MoFo protein from an nifB mutant, were measured and compared with those of the holoprotein. (When FeMoco and NifB - Kpl are combined, active Kpl is formed.) Reduced NifB - Kpl had a spectrum with a weak, paramagnetic, component superimposed on a diamagnetic background. The paramagnetic component was assigned to a contaminating, e.p.r.-active, species. Thionine-oxidized NifB - Kpl had a spectrum and magnetization properties very similar to those of thionine-oxidized Kpl , demonstrating that the 'P' clusters are not significantly affected by the absence of the FeMoco clusters. The spectra of reduced isolated FeMoco had similar magnetization curves but sharper features and higher intensities than those of this centre in dithionite-reduced Kpl . Furthermore, a shoulder near 580 nm in the Kpl spectrum was absent from that of FeMoco . This may be due to the loss of a ligand or to a change in symmetry of the FeMoco cluster on extraction.
Beef heart aconitase has been studied by low temperature magnetic circular dichroism (MCD) spectroscopy in the wavelength region 300 to 1900 nm. Together with parallel electron paramagnetic resonance and activity measurements, these data enable correlations between Fe-S cluster-type and enzymic activity in aconitase. In samples not exposed to extraneous Fe, the Fe-S cluster in aconitase exhibits the characteristic properties of a 3Fe center in both the as isolated and dithionite-reduced states. On the basis of the detailed form of the low temperature MCD spectra, three types of 3Fe center can be distinguished in biological samples. These are typified by the 3Fe centers in aconitase, Desulfovibrio gigas FdII, and Azotobacter crooccocum Fd. In aconitase, maximal enzymic activity is found to be associated with the build-up of [4Fe-4S]2+ clusters in good agreement with the Mössbauer studies of Kent et al. (Kent, T. A., Dreyer, J. L., Kennedy, M. C., Huynh, B. H., Emptage, M. H., Beinert, H., and Münck, E. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1096-1100). However, significant catalytic activity (approximately 60%) was obtained by reduction of the 3Fe center with dithionite in the absence of added Fe. The form and intensity of the resultant MCD spectrum are consistent with the majority of the Fe being in the form of reduced 3Fe clusters. The possibility that a reduced 3Fe cluster is capable of promoting partial catalytic activity in aconitase is discussed in light of these results.
Heme a3+ isolated from bovine cardiac muscle cytochrome oxidase has been converted to the bis-imidazole species and studied by magnetic circular dichroism (MCD) spectroscopy. Spectra have been recorded down to 1.5 degrees K, enabling the MCD magnetization curves to be measured at a number of wavelengths in the visible and near infrared regions. The experimentally determined curves show excellent correlation to a curve using the g-values determined by electron paramagnetic resonance spectroscopy to be gz = 2.96, gy = 2.29, and gx = 1.73. The data show that the bis-imidazole derivative of extracted heme a3+ is an excellent model of cytochrome a in the enzyme, confirming the presence of two histidine residues in the protein as the fifth and sixth ligands. The spectral features of heme a3+ bis-imidazole in the near infrared region are consistent with transitions of the porphyrin ( a1u , a2u ) to ferric (eg) charge transfer type.
Sixty-one children with cerebral cysticercosis are presented. The commonest clinical manifestation was epilepsy with or without focal signs in 43% of children, followed by the syndrome of raised intracranial pressure in 34% and meningoencephalitis in 13%. The CT scan was positive in 93% of cases and the HAI test in 86% of children so examined. Both were positive in 80%. Skull X-ray revealed evidence of calcification and/or raised intracranial pressure in 60%. Two children died, one each with subacute sclerosing panencephalitis and tuberculous meningitis. Of those who improved on discharge, 43% had epilepsy and 32% neurological deficit of an obvious nature. The parasitology, pathogenesis, pathology and diagnosis of cerebral cysticercosis are briefly reviewed. Present therapy and the suggested use of a specific anti-cysticeral drug are discussed. Case histories are presented to draw attention to the variability of the clinical picture and the particular importance of recognising the acute parenchymatous form of the disease in children which may present with critically elevated intracranial pressure.
The visible-near-i.r.-region m.c.d. (magnetic-circular-dichroism) spectrum recorded at low temperature in the range 450-900 nm is reported for oxidized resting mammalian cytochrome c oxidase. M.c.d. magnetization curves determined at different wavelengths reveal the presence of two paramagnetic species. Curves at 576, 613 and 640 nm fit well to those expected for an x,y-polarized haem transition with g values of 3.03, 2.21 and 1.45, i.e. cytochrome a3+. The m.c.d. features at 515, 785 and 817 nm magnetize as a S = 1/2 paramagnet with average g values close to 2, and simulated m.c.d. magnetization curves obtained by using the observed g values of CuA2+, i.e. 2.18, 2.03 and 1.99, fit well to the experimental observations. The form of the m.c.d. magnetization curve at 466 nm is curious, but it can be explained if CuA2+ and cytochrome a3+ contribute with oppositely signed bands at this wavelength. By comparing the m.c.d. spectrum of the enzyme with that of extracted haem a-bisimidazole complex it has been possible to deconvolute the m.c.d. spectrum of CuA2+, which shows transitions throughout the spectral region from 450 to 950 nm. The m.c.d.-spectral properties of CuA2+ were compared with those of a well-defined type I blue copper centre in azurin isolated from Pseudomonas aeruginosa. The absolute intensities of the m.c.d. signals at equal fields and temperatures for CuA2+ are 10-20-fold greater than those for azurin. The optical spectrum of CuA2+ strongly suggests an assignment as a d9 ion rather than Cu(I) bound to a thiyl radical.
Optical-absorption-, e.p.r.- and m.c.d. (magnetic-circular-dichroism)-spectroscopic measurements were made on liganded derivatives of oxidized and partially reduced cytochrome c oxidase. When NO was added to oxidized cyanide-bound cytochrome c oxidase, no changes occurred in the optical-absorption difference spectrum. In contrast, NO induced reduction of cytochrome a3 and formation of the nitrosylferrohaem species when the oxidized resting enzyme was the starting material. E.p.r. spectroscopy of the NO-treated oxidized cyanide-bound enzyme revealed the presence of a low-spin haem signal at g = 3.40, whereas the g = 3.02 and g = 2.0 signals of the oxidized enzyme remained unchanged. Both haem groups in this species are e.p.r.-detectable simultaneously. Examination of an identical sample by m.c.d. spectroscopy in the near-i.r. region identified two distinct low-spin species at 1565 and 1785 nm. Irradiation with white light of the NO-treated cyanide-bound sample at 10K resulted in the disappearance of the g = 3.40 e.p.r. signal and the m.c.d. signal at 1785 nm, whereas a band at 1950nm increased in intensity. When the photolysed sample was warmed to 50K and held in the dark for 15 min, the original spectrum returned. Magnetization studies of the 1785nm m.c.d. band support the assignment of this signal to the same metal centre that gives rise to the g = 3.40 e.p.r. signal. The effect of NO on the oxidized cyanide-bound enzyme was compared with that obtained when the oxidized cyanide-bound species was taken to the partially reduced state. Cytochrome a3 is e.p.r.-detectable with a g-value of 3.58 [Johnson, Eglinton, Gooding, Greenwood & Thomson (1981) Biochem. J. 193, 699-708]. Its near-i.r. m.c.d. spectrum shifts from 1950nm in the oxidized cyanide-bound enzyme to 1545nm on addition of reductant. A scheme is advanced for the structure of the cytochrome a3-CuB site that allows for cyanide binding to Fea3 and NO binding to CuB. Cyanide is the bridging ligand in the ferromagnetically coupled cytochrome a3-CuB pair of oxidized cyanide-bound cytochrome c oxidase. The bridged structure and the magnetic interaction are broken when the enzyme is partially reduced. However, when NO binds to CuB the cyanide bridge remains intact, but now the odd spins of NO and CuB are magnetically coupled.
Contents. 1. Introduction and history. 2. Characteristic spectroscopic features of 3Fe clusters. 1. General considerations. 2. Mössbauer spectroscopy. 3. Magnetic circular dichroism (MCD) spectroscopy. 4. Electron paramagnetic resonance (EPR) spectroscopy. 5. Resonance Raman (RR) spectroscopy. 6. Extended X-ray fine-structure (EXAFS) spectroscopy. 3. Results of X-Ray diffraction studies. 4. Proteins containing or showing features characteristic of 3Fe clusters 1. Overview. 2. Ferredoxin I of Azotobacter vinelandii. 3. Ferredoxin II of Desulfovibrio gigas. 4. Aconitase from beef heart. 5. Other observations and considerations relevant to 3Fe clusters or cluster interconversions 1. Oxidative degradation of [4Fe-4S] clusters to 3Fe clusters. 2. Extrusion studies on 3Fe clusters. 3. Reconstitution of 3Fe clusters. 4. Disposition of iron ligands in cluster interconversions. 6. Do all 3Fe clusters have the same structure? Evidence for [3Fe-4S] clusters. 7. Are 3Fe clusters artifacts or biologically significant structures?
The magnetic circular dichroism (MCD) spectra of three horse heart metmyoglobin compounds, the cyanide, azide and hydroxide forms, have been measured in the visible and near infrared spectral regions at temperatures down to 1.5 K. The three compounds are all virtually completely low-spin at low temperatures with ground g factors of decreasing rhombicity in the order CN- greater than N3- greater than OH-. The MCD magnetization curves have been constructed at selected wavelengths throughout the visible and near infrared regions. The curves are independent of wavelength, showing that all the bands studied are x,y polarized and can, moreover, be satisfactorily fitted to the g factors determined by EPR spectroscopy with theoretical expressions (Thomson, A.J. and Johnson, M.K. (1980) Biochem. J. 191, 411-420). This confirms the assignment and polarizations of the near infrared region low-spin ferric haem charge-transfer bands. The energies of these transitions are markedly dependent upon the added axial ligand, ranging from 1595 to 1295, and 1050 nm for the compounds CN-, N3- and OH-. The MCD spectra of bovine liver catalase and its cyanide adduct have been recorded in the Soret, visible and near infrared regions. Catalase is know to have phenolate anion as the proximal ligand of the haem group. The forms of the spectra make an interesting comparison with those of the analogous metmyoglobin derivatives, in which histidine is the proximal ligand. The MCD spectra of catalase at 4.2 K is an example of a fully high-spin haemoprotein. The cyanide compound is completely low-spin at 4.2 K. The near infrared charge-transfer band is at 1300 nm, showing the effect on the energy of this band of changing from imidazole to phenolate ion as the proximal ligand to haem.