Richard Cammack - Academia.edu (original) (raw)
Papers by Richard Cammack
Febs Letters, Aug 14, 1989
Manganese was shown to be an essential trace metal for growth and thiosulphate oxidation by Thiob... more Manganese was shown to be an essential trace metal for growth and thiosulphate oxidation by Thiobacillus versutus in chemostat culture. In the thiosulphate-oxidizing enzyme system of T. versutus, protein B was the only component found to contain manganese in significant amounts. When it was examined by electron spin resonance (ESR) spectroscopy, protein B gave a broad, complex spectrum, indicative of the presence of a dimeric manganese cluster, with manganese in the Mn(II) oxidation state.
![Research paper thumbnail of Spin−Spin Interactions between the Ni Site and the [4Fe-4S] Centers as a Probe of Light-Induced Structural Changes in Active Desulfovibrio gigas Hydrogenase †](https://attachments.academia-assets.com/42830474/thumbnails/1.jpg)
](Biochemistry Usa, 1996
In typical NiFe hydrogenases like that from DesulfoVibrio gigas, the active state of the enzyme w... more In typical NiFe hydrogenases like that from DesulfoVibrio gigas, the active state of the enzyme which is obtained by incubation under hydrogen gas gives a characteristic Ni-C electron paramagnetic resonance (EPR) signal at g ) 2.19, 2.14, and 2.01. The Ni-C species is light-sensitive, being converted upon illumination at temperatures below 100 K in a mixture of different Ni-L species, the most important giving an EPR signal at g ) 2.30, 2.12, and 2.05. This photoprocess is considered to correspond to the dissociation of a hydrogen species initially coordinated to the Ni ion in the Ni-C state. When the [4Fe-4S] centers of the enzyme are reduced, the proximal [4Fe-4S] 1+ cluster interacts magnetically with the Ni center, which leads to complex split Ni-C or split Ni-L EPR spectra only detectable below 10 K. In order to probe the structural changes induced in the Ni center environment by the photoprocess, these spin-spin interactions were analyzed in D. gigas hydrogenase by simulating the split Ni-L spectra recorded at different microwave frequencies. We shown that, upon illumination, the relative arrangement of the Ni and [4Fe-4S] centers is not modified but that the exchange interaction between them is completely canceled. Moreover, the rotations undergone by the Ni center magnetic axes in the photoconversion were determined. Taken together, our results support a Ni-C structure in which the hydrogen species is not in the first coordination sphere of the Ni ion but is more likely bound to a sulfur atom of a terminal cysteine ligand of the Ni center.
Eur J Biochem, 1989
Pulsed electron-spin-resonance techniques were applied to the hydrogenase of the purple photosynt... more Pulsed electron-spin-resonance techniques were applied to the hydrogenase of the purple photosynthetic bacterium Thiocupsa roseopersicina, an enzyme which contains nickel and iron-sulphur clusters but no flavin. The linear electric field effect profile of the spectrum in the region of g = 2.01 indicated that the strong ESR signal in the oxidized protein is due to a [3Fe-4S] cluster. The electron spin-echo envelope of this spectrum was modulated by hyperfine interactions with 'H and 14N nuclei, probably from the polypeptide chain. The ESR spectrum of this species shows a complex pattern arising from spin-spin interaction with another paramagnet. When the protein was partially reduced by ascorbate plus phenazine methosulphate, the complexity of the spectrum was abolished but the form of the electron spin-echo envelope modulation (ESEEM) pattern was unchanged. This indicates that the reversible disappearance of the spin-spin interaction pattern on partial reduction is not due to cluster interconversion to a [4Fe-4S] cluster. In the ESR spectrum of nickel(III), weak hyperfine interactions with 'H and I4N were also observed by ESEEM. The nature of the interacting nuclei is discussed.
J Am Chem Soc, 2005
A new strategy is described for comparing, quantitatively, the ability of hydrogenases to tolerat... more A new strategy is described for comparing, quantitatively, the ability of hydrogenases to tolerate exposure to O2 and anoxic oxidizing conditions. Using protein film voltammetry, the inherent sensitivities to these challenges (thermodynamic potentials and rates of reactions) have been measured for enzymes from a range of mesophilic microorganisms. In the absence of O 2, all the hydrogenases undergo reversible inactivation at various potentials above that of the H + /H2 redox couple, and H2 oxidation activities are thus limited to characteristic "potential windows". Reactions with O2 vary greatly; the [FeFe]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757, an anaerobe, is irreversibly damaged by O2, surviving only if exposed to O2 in the anaerobically oxidized state (which therefore affords protection). In contrast, the membrane-bound [NiFe]-hydrogenase from the aerobe, Ralstonia eutropha, reacts reversibly with O2 even during turnover and continues to catalyze H2 oxidation in the presence of O2.
![Research paper thumbnail of Electron spin-echo envelope modulation studies of the [3Fe–4S] cluster of Escherichia coli fumarate reductase](https://attachments.academia-assets.com/42830475/thumbnails/1.jpg)
](Journal of the Chemical Society Faraday Transactions, 1993
The dimeric form of purified Escherichia coli fumarate reductase contains three different iron-su... more The dimeric form of purified Escherichia coli fumarate reductase contains three different iron-sulfur clusters, termed centres 1, 2 and 3 ([2Fe-2S], [4Fe4S] and [3Fe-4S] clusters, respectively). W e have performed electron spin-echo envelope modulation (ESEEM) spectroscopy in order to obtain information about the environment of the [3F&S] cluster. Modulations from 14N were detected in the three-pulse ESEEM spectra from the cluster in t h e oxidised state. The low-frequency lines observed in t h e three-pulse ESEEM pattern were similar to those observed for t h e [3Fe-4S] cluster of bovine heart succinate dehydrogenase . . Determination of the hyperfine and quadrupolar coupling (A w 0.7 MHz, e29Q x 3.4 MHz) gave values that are similar to those determined for the [2Fe-2S] cluster of centre 1 constants indicate that t h e ' ' N -n~~l e~~ giving rise to t h e modulation pattern in the ESEEM spectrum is weakly coupled to centre 3, rather than directly coordinated to the iron.
The flavins of ferredoxin-NADP' reductase (FNR) and flavodoxin from the cyanobacterium Anabaena P... more The flavins of ferredoxin-NADP' reductase (FNR) and flavodoxin from the cyanobacterium Anabaena PCC 7119 were obtained in their semiquinone states at pH 7 by photoreduction of the pure proteins in the presence of EDTA and 5-deazariboflavin. For FNR, the ESR signal of the FAD semiquinone was centred at g = 2.005 with linewidths 2.0 mT in H,O and 1.48 mT in D,O. These data are in agreement with those reported for other neutral flavin semiquinones. The linewidths were the same when measured either at X-band (9.35 GHz) or at S-band (4 GHz), indicating that line broadening is due to unresolved nuclear hyperfine couplings, caused in part by exchangeable protons. When the substrate, NADP', was added to the semiquinone form of the protein no changes in the linewidth or shape of the spectra were detected, but a decrease in the ESR signal due to the FNR semiquinone was observed, consistent with the reduction of NADP+ to NADPH by reduced FNR and, subsequent displacement of the equilibrium. No changes in the shape or linewidth of the FNR ESR signals were observed when photoreduction of FNR was performed in the presence of either flavodoxin or ferredoxin. Electron nuclear double resonance (ENDOR) spectroscopy of FNR semiquinone from Anabaena PCC 711 9 provided further information about the interactions of the flavin radical with protons. A group of signals, with couplings of 5-9.5 MHz, is attributed to protons on C6 and on 8-CH3 of the flavin ring. No change in these hyperfine couplings was detected when the protein was studied in DzO, but the coupling A,,, attributed to protons on 8-CH3 decreased from 8.12 MHz to 7.72 MHz in the presence of NADP'. The decrease in the electron spin density distribution on this part of the flavin ring system was attributed to binding of the substrate, polarising the electron density distribution of the flavin towards the pyrimidine ring. A second group of signals was observed, with hyperfine couplings less than 3 MHz, some of which disappeared when the protein was transferred into D,O. Effects of NADP' binding to the protein were also observed in these weak couplings. These signals are attributed to displaced water protons, or to exchangeable protons from amino acid residues on the protein near the flavin-binding site, involved in substrate stabilization.
Biochimica et Biophysica Acta (BBA) - Protein Structure, 1976
A two-iron-two-sulphur non-haem iron protein, the ferredoxin from Spirulina maxima, has been stud... more A two-iron-two-sulphur non-haem iron protein, the ferredoxin from Spirulina maxima, has been studied by means of electron paramagnetic resonance (EPR) in the range where the spectrum loses resolution with increasing temperature. The spinlattice relaxation times were deduced from linewidths measured by spectral simulation and their variation as a function of temperature is interpreted in terms of an Orbach mechanism. On this basis, the exchange integral between the two iron atoms, assuming an antiferromagnetic interaction between them, is estimated to be --83 cm -~.
Biochemistry, Jan 11, 1990
The electron transfer centers in dimethyl sulfoxide reductase were examined by EPR spectroscopy i... more The electron transfer centers in dimethyl sulfoxide reductase were examined by EPR spectroscopy in membranes of the overproducing Escherichia coli strain HB101/pDMS159, and in purified enzyme. Iron-sulfur clusters of the [4Fe-4S] type and a molybdenum center were detected in the protein, which comprises three different subunits: DmsA, -B, and -C. The intensity of the reduced iron-sulfur clusters corresponded to 3.82 +/- 0.5 spins per molecule. The dithionite-reduced clusters were reoxidized by DMSO or TMAO. The enzyme, as prepared, showed a spectrum of Mo(V), which resembles the high-pH form of E. coli nitrate reductase. The Mo(V) detected by EPR was absent from a mutant which does not assemble the molybdenum cofactor. In these cases, the levels of EPR-detectable iron-sulfur clusters in the cells were increased. Extracts from HB101/pDMS159 enriched in DmsA showed more Mo(V) signals and considerably less iron-sulfur. These results are in agreement with predictions from amino acid seq...
European journal of biochemistry / FEBS, Jan 2, 1984
By preparative polyacrylamide gel electrophoresis at pH 8.5, and in the absence of nickel ions, t... more By preparative polyacrylamide gel electrophoresis at pH 8.5, and in the absence of nickel ions, two types of subunit dimers of the NAD-linked hydrogenase from Nocardia opaca 1b were separated and isolated, and their properties were compared with each other as well as with the properties of the native enzyme. The intact hydrogenase contained 14.3 +/- 0.4 labile sulphur, 13.6 +/- 1.1 iron and 3.8 +/- 0.1 nickel atoms and approximately 1 FMN molecule per enzyme molecule. The oxidized hydrogenase showed an absorption spectrum with maxima (shoulders) at 380 nm and 420 nm and an electron spin resonance (ESR) spectrum with a signal at g = 2.01. The midpoint redox potential of the Fe-S cluster giving rise to this signal was +25 mV. In the reduced state, hydrogenase gave characteristic low-temperature (10-20 K) and high-temperature (greater than 40 K) ESR spectra which were interpreted as due to [4Fe-4S] and [2Fe-2S] clusters, respectively. The midpoint redox potentials of these clusters wer...
Biochimica et biophysica acta, Jan 22, 1977
Electron paramagnetic resonance spectra were recorded of three forms of Desulphovibrio gigas ferr... more Electron paramagnetic resonance spectra were recorded of three forms of Desulphovibrio gigas ferredoxin, FdI, FdI' and FdII. The g = 1.94 signal seen in dithionite-reduced samples is strong in FdI, weaker in FdI' and very small in FdII. The g = 2.02 signal in the oxidized proteins is weak in FdI and strongest in FdII. It is concluded that most of the 4Fe-4S centres in FdI change between states C- and C2-; FdI' contain both types of centre. There is no evidence that any particular centre can change reversibly between all three oxidation states. Circular dichroism spectra show differences between FdI and FdII even in the diamagnetic C2- state. The redox potentials of the iron-sulphur centres of the three oligomers (forms) are different. After formation of the apo-protein of FdII and reconstitution with iron and sulphide, the protein behaves more like FdI, showing a strong g = 1.94 signal in the reduced states.
Journal of the Chemical Society, Faraday Transactions, 1991
Xanthine oxidase from milk contains two different [2Fe-2S] clusters, Fe-S(I) and Fe-S(II). The en... more Xanthine oxidase from milk contains two different [2Fe-2S] clusters, Fe-S(I) and Fe-S(II). The environment of each type of cluster has been examined by electron spin-echo envelope modulation (ESEEM) spectroscopy, where modulations were observed for the iron-sulphur clusters in the reduced state. The spectral contributions of the two clusters were distinguished (a) by poising the samples at redox potentials such that either Fe-S(1I) was predominantly reduced, or both iron-sulphur clusters were fully reduced and (b) by measurements at their characteristic g factors. Spectra of both [2Fe-2S] clusters showed weak 14N hyperfine interactions, similar to those seen in other [2Fe-2S] proteins, which were attributed to nitrogens of the polypeptide chain; there was no evidence for histidine imidazole coordination. Spectra of samples exchanged into 2H,0 gave evidence for exchangeable protons in close proximity to Fe-S clusters. By these criteria the environment of the [2Fe-2S] clusters in this complex protein is similar to those in the plant-type ferredoxins. In addition, ESEEM spectra of molybdenum (v) in the desulpho-inhibited form of the enzyme did not show modulations due to '*N.
Biochimica et Biophysica Acta (BBA) - Protein Structure, 1979
Desulphoviridin in the oxidized state showed EPR signals around g = 6, consistent with the siroha... more Desulphoviridin in the oxidized state showed EPR signals around g = 6, consistent with the sirohaem being in the high-spin ferric state. This was unreactive with sulphite, sulphide or cyanide; but readily reduced by methyl viologen. When the enzyme was treated with Na:S204 the sirohaem was slowly reduced and a spectrum of a reduced iron-sulphur cluster at g = 2.07, 1.93, 1.91 appeared over the course of an hour. An intermediate in this reaction was indicated by a free radical signal which appeared within seconds and then gradually disappeared. On treatment with nitrite and reduced methyl viologen, the enzyme gave a spectrum of a nitroxide derivative similar to that seen with plant nitrite reductase. The midpoint reduction potential of the haem was estimated to be --310 mV or less. The iron-sulphur cluster has a very low potential, being only reduced in the presence of free Na2SzO4 around--560 mV. Desulphoviridin can be classed with sirohaem-containing iron-sulphur proteins.
Biochimica et Biophysica Acta (BBA) - Protein Structure, 1977
1. Antibodies were produced in rabbits to the 4Fe-4S ferrodoxins from Bacillus stearothermophilus... more 1. Antibodies were produced in rabbits to the 4Fe-4S ferrodoxins from Bacillus stearothermophilus, the 2 [4Fe-4S] ferredoxin from Clostridium pasteurianum, and the 2Fe-2S ferredoxins from the blue-green alga Spirulina maxima, the green alga Scenedesmus obliquus, and the higher plant Beta vulgaris. The antibodies were tested for immunoprecipitation activity with seven bacterial, twelve blue-green algal, six eukaryotic algal and six higher plant ferredoxins.
Journal of the Chemical Society, Faraday Transactions, 1994
Illumination of the Ni-C state of D. gigas hydrogenase at temperatures below 60 K caused the disa... more Illumination of the Ni-C state of D. gigas hydrogenase at temperatures below 60 K caused the disappearance of the single Ni-C signal (g = 2.19, 2.14, 2.01) and the simultaneous appearance of two overlapping signals with different g, and g, values. The overlapping spectra can be deconvoluted into two independent spectra with g = 2.26, 2.11, 2.044 for the signal here referred to as Ni-Ct and g = 2.29, 2.12, 2.044 for the signal referred to as Ni-C;. The rate of disappearance of the Ni-C signal upon illumination of D. gigas hydrogenase at 5 K was the same as at 30 K, but a complicated new spectrum appeared. This is interpreted as being due to the superposition of the spectra of the reduced [4Fe-4S] clusters and of the two overlapping photoilluminated Ni-C signals which, at this temperature, show splittings due to the interaction with a nearby paramagnet. The splittings are anisotropic. Upon light irradiation of D . gigas hydrogenase at temperatures above 60 K, only signal Ni-Cz appeared. This different behaviour of the two overlapping light-induced signals suggests that after illumination of the Ni-C species, two different forms of the Ni site are formed. The differences in the g values indicate slightly different coordination environments. The effects of illumination and temperature suggest that one of them is an intermediate in the formation of the other one, at least at low temperatures. Although irreversible below 100 K, the photolytic process was completely reversible at temperatures above 120 K.
Journal of the Chemical Society, Perkin Transactions 2, 1992
Solutions of reduced [2Fe-2S] ferredoxin from the cyanobacterium Spirulina platensis, are found t... more Solutions of reduced [2Fe-2S] ferredoxin from the cyanobacterium Spirulina platensis, are found t o be sensitive to visible light in the frozen state at 77 K, if trichloroacetate is also present. A n EPRdetectable radical is generated, which is stable for several hours at 77 K. The process is dependent on the presence of the protein and a reductant (dithionite). The shape of the radical spectrum is the same when the solvent is D,O. It has been identified as the dichloroacetate radical anion, 'CCI,CO,-. The ferredoxin becomes partially oxidized in the photochemical process, in parallel with the appearance of the radical. O n thawing the samples, the EPR spectrum of the reduced ferredoxin increases as the protein is partially re-reduced b y dithionite. This is an unusual situation in which photochemicallyinduced electron transfer can be observed from a metal site within a protein.
European Journal of Biochemistry, 1984
The non-ionic detergent lauryl dimethylamine N-oxide (LDAO) has been used to extract the NADH deh... more The non-ionic detergent lauryl dimethylamine N-oxide (LDAO) has been used to extract the NADH dehydrogenases of Arum /naculatun? mitochondria. Affinity chromatography on S'ADP-Sepharose 4B was used to separate the rotenone-sensitive (complex I) NADH dehydrogenase from thc rotcnone-insensitive NADH dchydrogenase. An 1 8-fold purification of the rolenonc-insensitive NADH dehydrogenase was achicvcd. The enzyme is specific for NADH with optimal activity around pH7.2. The apparent K, Tor NADH is 28pM, with dichloroindophcnol as acceptor at pH 7.2. The rotenone-insensitive NADH dehydrogenase appears to be a flavoprotein and no iron-sulphur centres were detected by electron spin resonance spectroscopy.
European Journal of Biochemistry, 1997
Nocurdiu corultina B-276 possesses a constitutive multi-component alkene monooxygenase which cata... more Nocurdiu corultina B-276 possesses a constitutive multi-component alkene monooxygenase which catalyses the epoxidation of terminal and sub-terminal alkenes. The epoxygenase component of this system has been purified with an overall yield of 35%. The electron paramagnetic resonance spectrum of the oxidised protein has a weak signal at g = 4.3, which we ascribe to rhombic iron and a free radical signal at g,,, = 2.01. Upon partial reduction with dithionite using methyl viologen as a mediator, a signal at g,,, = 1.9 appeared. Upon further reduction with excess dithionite a signal at g = 15 appeared with the concomitant disappearance of the g,,, = 1.9 signal. These results indicate that the epoxygenase contains a bridged dinuclear iron centre similar to that found in a variety of proteins involved in oxygen transport and activation as well as desaturation of fatty acids. Analysis of the products of the reaction indicates that A M 0 is capable of stereospecific epoxidation of alkenes producing the R-enantiomer in high yield, a reaction catalysed by very few oxygenase enzymes. Whole cells gave lower enantiomeric excess values for the epoxide and a stereospecific epoxidase enzyme has been proposed to account for this difference. Although alkene monooxygenase was not inhibited by ethyne, a potent inhibitor of soluble methane monooxygenase with which alkene monooxygenase shares many common features, it was weakly inhibited by propyne with an apparent K, value of 340 pM. The mechanistic implications of these physicochemical features of the enzyme are discussed.
European Journal of Biochemistry, 1975
The technique of electron paramagnetic resonance spectrometry has been applied to the study of pl... more The technique of electron paramagnetic resonance spectrometry has been applied to the study of plant microsomal electron-transport components. Only tulip-bulb microsomes were found to give strong enough signals to allow detailed study.
Febs Letters, Aug 14, 1989
Manganese was shown to be an essential trace metal for growth and thiosulphate oxidation by Thiob... more Manganese was shown to be an essential trace metal for growth and thiosulphate oxidation by Thiobacillus versutus in chemostat culture. In the thiosulphate-oxidizing enzyme system of T. versutus, protein B was the only component found to contain manganese in significant amounts. When it was examined by electron spin resonance (ESR) spectroscopy, protein B gave a broad, complex spectrum, indicative of the presence of a dimeric manganese cluster, with manganese in the Mn(II) oxidation state.
Biochemistry Usa, 1996
In typical NiFe hydrogenases like that from DesulfoVibrio gigas, the active state of the enzyme w... more In typical NiFe hydrogenases like that from DesulfoVibrio gigas, the active state of the enzyme which is obtained by incubation under hydrogen gas gives a characteristic Ni-C electron paramagnetic resonance (EPR) signal at g ) 2.19, 2.14, and 2.01. The Ni-C species is light-sensitive, being converted upon illumination at temperatures below 100 K in a mixture of different Ni-L species, the most important giving an EPR signal at g ) 2.30, 2.12, and 2.05. This photoprocess is considered to correspond to the dissociation of a hydrogen species initially coordinated to the Ni ion in the Ni-C state. When the [4Fe-4S] centers of the enzyme are reduced, the proximal [4Fe-4S] 1+ cluster interacts magnetically with the Ni center, which leads to complex split Ni-C or split Ni-L EPR spectra only detectable below 10 K. In order to probe the structural changes induced in the Ni center environment by the photoprocess, these spin-spin interactions were analyzed in D. gigas hydrogenase by simulating the split Ni-L spectra recorded at different microwave frequencies. We shown that, upon illumination, the relative arrangement of the Ni and [4Fe-4S] centers is not modified but that the exchange interaction between them is completely canceled. Moreover, the rotations undergone by the Ni center magnetic axes in the photoconversion were determined. Taken together, our results support a Ni-C structure in which the hydrogen species is not in the first coordination sphere of the Ni ion but is more likely bound to a sulfur atom of a terminal cysteine ligand of the Ni center.
Eur J Biochem, 1989
Pulsed electron-spin-resonance techniques were applied to the hydrogenase of the purple photosynt... more Pulsed electron-spin-resonance techniques were applied to the hydrogenase of the purple photosynthetic bacterium Thiocupsa roseopersicina, an enzyme which contains nickel and iron-sulphur clusters but no flavin. The linear electric field effect profile of the spectrum in the region of g = 2.01 indicated that the strong ESR signal in the oxidized protein is due to a [3Fe-4S] cluster. The electron spin-echo envelope of this spectrum was modulated by hyperfine interactions with 'H and 14N nuclei, probably from the polypeptide chain. The ESR spectrum of this species shows a complex pattern arising from spin-spin interaction with another paramagnet. When the protein was partially reduced by ascorbate plus phenazine methosulphate, the complexity of the spectrum was abolished but the form of the electron spin-echo envelope modulation (ESEEM) pattern was unchanged. This indicates that the reversible disappearance of the spin-spin interaction pattern on partial reduction is not due to cluster interconversion to a [4Fe-4S] cluster. In the ESR spectrum of nickel(III), weak hyperfine interactions with 'H and I4N were also observed by ESEEM. The nature of the interacting nuclei is discussed.
J Am Chem Soc, 2005
A new strategy is described for comparing, quantitatively, the ability of hydrogenases to tolerat... more A new strategy is described for comparing, quantitatively, the ability of hydrogenases to tolerate exposure to O2 and anoxic oxidizing conditions. Using protein film voltammetry, the inherent sensitivities to these challenges (thermodynamic potentials and rates of reactions) have been measured for enzymes from a range of mesophilic microorganisms. In the absence of O 2, all the hydrogenases undergo reversible inactivation at various potentials above that of the H + /H2 redox couple, and H2 oxidation activities are thus limited to characteristic "potential windows". Reactions with O2 vary greatly; the [FeFe]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757, an anaerobe, is irreversibly damaged by O2, surviving only if exposed to O2 in the anaerobically oxidized state (which therefore affords protection). In contrast, the membrane-bound [NiFe]-hydrogenase from the aerobe, Ralstonia eutropha, reacts reversibly with O2 even during turnover and continues to catalyze H2 oxidation in the presence of O2.
Journal of the Chemical Society Faraday Transactions, 1993
The dimeric form of purified Escherichia coli fumarate reductase contains three different iron-su... more The dimeric form of purified Escherichia coli fumarate reductase contains three different iron-sulfur clusters, termed centres 1, 2 and 3 ([2Fe-2S], [4Fe4S] and [3Fe-4S] clusters, respectively). W e have performed electron spin-echo envelope modulation (ESEEM) spectroscopy in order to obtain information about the environment of the [3F&S] cluster. Modulations from 14N were detected in the three-pulse ESEEM spectra from the cluster in t h e oxidised state. The low-frequency lines observed in t h e three-pulse ESEEM pattern were similar to those observed for t h e [3Fe-4S] cluster of bovine heart succinate dehydrogenase . . Determination of the hyperfine and quadrupolar coupling (A w 0.7 MHz, e29Q x 3.4 MHz) gave values that are similar to those determined for the [2Fe-2S] cluster of centre 1 constants indicate that t h e ' ' N -n~~l e~~ giving rise to t h e modulation pattern in the ESEEM spectrum is weakly coupled to centre 3, rather than directly coordinated to the iron.
The flavins of ferredoxin-NADP' reductase (FNR) and flavodoxin from the cyanobacterium Anabaena P... more The flavins of ferredoxin-NADP' reductase (FNR) and flavodoxin from the cyanobacterium Anabaena PCC 7119 were obtained in their semiquinone states at pH 7 by photoreduction of the pure proteins in the presence of EDTA and 5-deazariboflavin. For FNR, the ESR signal of the FAD semiquinone was centred at g = 2.005 with linewidths 2.0 mT in H,O and 1.48 mT in D,O. These data are in agreement with those reported for other neutral flavin semiquinones. The linewidths were the same when measured either at X-band (9.35 GHz) or at S-band (4 GHz), indicating that line broadening is due to unresolved nuclear hyperfine couplings, caused in part by exchangeable protons. When the substrate, NADP', was added to the semiquinone form of the protein no changes in the linewidth or shape of the spectra were detected, but a decrease in the ESR signal due to the FNR semiquinone was observed, consistent with the reduction of NADP+ to NADPH by reduced FNR and, subsequent displacement of the equilibrium. No changes in the shape or linewidth of the FNR ESR signals were observed when photoreduction of FNR was performed in the presence of either flavodoxin or ferredoxin. Electron nuclear double resonance (ENDOR) spectroscopy of FNR semiquinone from Anabaena PCC 711 9 provided further information about the interactions of the flavin radical with protons. A group of signals, with couplings of 5-9.5 MHz, is attributed to protons on C6 and on 8-CH3 of the flavin ring. No change in these hyperfine couplings was detected when the protein was studied in DzO, but the coupling A,,, attributed to protons on 8-CH3 decreased from 8.12 MHz to 7.72 MHz in the presence of NADP'. The decrease in the electron spin density distribution on this part of the flavin ring system was attributed to binding of the substrate, polarising the electron density distribution of the flavin towards the pyrimidine ring. A second group of signals was observed, with hyperfine couplings less than 3 MHz, some of which disappeared when the protein was transferred into D,O. Effects of NADP' binding to the protein were also observed in these weak couplings. These signals are attributed to displaced water protons, or to exchangeable protons from amino acid residues on the protein near the flavin-binding site, involved in substrate stabilization.
Biochimica et Biophysica Acta (BBA) - Protein Structure, 1976
A two-iron-two-sulphur non-haem iron protein, the ferredoxin from Spirulina maxima, has been stud... more A two-iron-two-sulphur non-haem iron protein, the ferredoxin from Spirulina maxima, has been studied by means of electron paramagnetic resonance (EPR) in the range where the spectrum loses resolution with increasing temperature. The spinlattice relaxation times were deduced from linewidths measured by spectral simulation and their variation as a function of temperature is interpreted in terms of an Orbach mechanism. On this basis, the exchange integral between the two iron atoms, assuming an antiferromagnetic interaction between them, is estimated to be --83 cm -~.
Biochemistry, Jan 11, 1990
The electron transfer centers in dimethyl sulfoxide reductase were examined by EPR spectroscopy i... more The electron transfer centers in dimethyl sulfoxide reductase were examined by EPR spectroscopy in membranes of the overproducing Escherichia coli strain HB101/pDMS159, and in purified enzyme. Iron-sulfur clusters of the [4Fe-4S] type and a molybdenum center were detected in the protein, which comprises three different subunits: DmsA, -B, and -C. The intensity of the reduced iron-sulfur clusters corresponded to 3.82 +/- 0.5 spins per molecule. The dithionite-reduced clusters were reoxidized by DMSO or TMAO. The enzyme, as prepared, showed a spectrum of Mo(V), which resembles the high-pH form of E. coli nitrate reductase. The Mo(V) detected by EPR was absent from a mutant which does not assemble the molybdenum cofactor. In these cases, the levels of EPR-detectable iron-sulfur clusters in the cells were increased. Extracts from HB101/pDMS159 enriched in DmsA showed more Mo(V) signals and considerably less iron-sulfur. These results are in agreement with predictions from amino acid seq...
European journal of biochemistry / FEBS, Jan 2, 1984
By preparative polyacrylamide gel electrophoresis at pH 8.5, and in the absence of nickel ions, t... more By preparative polyacrylamide gel electrophoresis at pH 8.5, and in the absence of nickel ions, two types of subunit dimers of the NAD-linked hydrogenase from Nocardia opaca 1b were separated and isolated, and their properties were compared with each other as well as with the properties of the native enzyme. The intact hydrogenase contained 14.3 +/- 0.4 labile sulphur, 13.6 +/- 1.1 iron and 3.8 +/- 0.1 nickel atoms and approximately 1 FMN molecule per enzyme molecule. The oxidized hydrogenase showed an absorption spectrum with maxima (shoulders) at 380 nm and 420 nm and an electron spin resonance (ESR) spectrum with a signal at g = 2.01. The midpoint redox potential of the Fe-S cluster giving rise to this signal was +25 mV. In the reduced state, hydrogenase gave characteristic low-temperature (10-20 K) and high-temperature (greater than 40 K) ESR spectra which were interpreted as due to [4Fe-4S] and [2Fe-2S] clusters, respectively. The midpoint redox potentials of these clusters wer...
Biochimica et biophysica acta, Jan 22, 1977
Electron paramagnetic resonance spectra were recorded of three forms of Desulphovibrio gigas ferr... more Electron paramagnetic resonance spectra were recorded of three forms of Desulphovibrio gigas ferredoxin, FdI, FdI' and FdII. The g = 1.94 signal seen in dithionite-reduced samples is strong in FdI, weaker in FdI' and very small in FdII. The g = 2.02 signal in the oxidized proteins is weak in FdI and strongest in FdII. It is concluded that most of the 4Fe-4S centres in FdI change between states C- and C2-; FdI' contain both types of centre. There is no evidence that any particular centre can change reversibly between all three oxidation states. Circular dichroism spectra show differences between FdI and FdII even in the diamagnetic C2- state. The redox potentials of the iron-sulphur centres of the three oligomers (forms) are different. After formation of the apo-protein of FdII and reconstitution with iron and sulphide, the protein behaves more like FdI, showing a strong g = 1.94 signal in the reduced states.
Journal of the Chemical Society, Faraday Transactions, 1991
Xanthine oxidase from milk contains two different [2Fe-2S] clusters, Fe-S(I) and Fe-S(II). The en... more Xanthine oxidase from milk contains two different [2Fe-2S] clusters, Fe-S(I) and Fe-S(II). The environment of each type of cluster has been examined by electron spin-echo envelope modulation (ESEEM) spectroscopy, where modulations were observed for the iron-sulphur clusters in the reduced state. The spectral contributions of the two clusters were distinguished (a) by poising the samples at redox potentials such that either Fe-S(1I) was predominantly reduced, or both iron-sulphur clusters were fully reduced and (b) by measurements at their characteristic g factors. Spectra of both [2Fe-2S] clusters showed weak 14N hyperfine interactions, similar to those seen in other [2Fe-2S] proteins, which were attributed to nitrogens of the polypeptide chain; there was no evidence for histidine imidazole coordination. Spectra of samples exchanged into 2H,0 gave evidence for exchangeable protons in close proximity to Fe-S clusters. By these criteria the environment of the [2Fe-2S] clusters in this complex protein is similar to those in the plant-type ferredoxins. In addition, ESEEM spectra of molybdenum (v) in the desulpho-inhibited form of the enzyme did not show modulations due to '*N.
Biochimica et Biophysica Acta (BBA) - Protein Structure, 1979
Desulphoviridin in the oxidized state showed EPR signals around g = 6, consistent with the siroha... more Desulphoviridin in the oxidized state showed EPR signals around g = 6, consistent with the sirohaem being in the high-spin ferric state. This was unreactive with sulphite, sulphide or cyanide; but readily reduced by methyl viologen. When the enzyme was treated with Na:S204 the sirohaem was slowly reduced and a spectrum of a reduced iron-sulphur cluster at g = 2.07, 1.93, 1.91 appeared over the course of an hour. An intermediate in this reaction was indicated by a free radical signal which appeared within seconds and then gradually disappeared. On treatment with nitrite and reduced methyl viologen, the enzyme gave a spectrum of a nitroxide derivative similar to that seen with plant nitrite reductase. The midpoint reduction potential of the haem was estimated to be --310 mV or less. The iron-sulphur cluster has a very low potential, being only reduced in the presence of free Na2SzO4 around--560 mV. Desulphoviridin can be classed with sirohaem-containing iron-sulphur proteins.
Biochimica et Biophysica Acta (BBA) - Protein Structure, 1977
1. Antibodies were produced in rabbits to the 4Fe-4S ferrodoxins from Bacillus stearothermophilus... more 1. Antibodies were produced in rabbits to the 4Fe-4S ferrodoxins from Bacillus stearothermophilus, the 2 [4Fe-4S] ferredoxin from Clostridium pasteurianum, and the 2Fe-2S ferredoxins from the blue-green alga Spirulina maxima, the green alga Scenedesmus obliquus, and the higher plant Beta vulgaris. The antibodies were tested for immunoprecipitation activity with seven bacterial, twelve blue-green algal, six eukaryotic algal and six higher plant ferredoxins.
Journal of the Chemical Society, Faraday Transactions, 1994
Illumination of the Ni-C state of D. gigas hydrogenase at temperatures below 60 K caused the disa... more Illumination of the Ni-C state of D. gigas hydrogenase at temperatures below 60 K caused the disappearance of the single Ni-C signal (g = 2.19, 2.14, 2.01) and the simultaneous appearance of two overlapping signals with different g, and g, values. The overlapping spectra can be deconvoluted into two independent spectra with g = 2.26, 2.11, 2.044 for the signal here referred to as Ni-Ct and g = 2.29, 2.12, 2.044 for the signal referred to as Ni-C;. The rate of disappearance of the Ni-C signal upon illumination of D. gigas hydrogenase at 5 K was the same as at 30 K, but a complicated new spectrum appeared. This is interpreted as being due to the superposition of the spectra of the reduced [4Fe-4S] clusters and of the two overlapping photoilluminated Ni-C signals which, at this temperature, show splittings due to the interaction with a nearby paramagnet. The splittings are anisotropic. Upon light irradiation of D . gigas hydrogenase at temperatures above 60 K, only signal Ni-Cz appeared. This different behaviour of the two overlapping light-induced signals suggests that after illumination of the Ni-C species, two different forms of the Ni site are formed. The differences in the g values indicate slightly different coordination environments. The effects of illumination and temperature suggest that one of them is an intermediate in the formation of the other one, at least at low temperatures. Although irreversible below 100 K, the photolytic process was completely reversible at temperatures above 120 K.
Journal of the Chemical Society, Perkin Transactions 2, 1992
Solutions of reduced [2Fe-2S] ferredoxin from the cyanobacterium Spirulina platensis, are found t... more Solutions of reduced [2Fe-2S] ferredoxin from the cyanobacterium Spirulina platensis, are found t o be sensitive to visible light in the frozen state at 77 K, if trichloroacetate is also present. A n EPRdetectable radical is generated, which is stable for several hours at 77 K. The process is dependent on the presence of the protein and a reductant (dithionite). The shape of the radical spectrum is the same when the solvent is D,O. It has been identified as the dichloroacetate radical anion, 'CCI,CO,-. The ferredoxin becomes partially oxidized in the photochemical process, in parallel with the appearance of the radical. O n thawing the samples, the EPR spectrum of the reduced ferredoxin increases as the protein is partially re-reduced b y dithionite. This is an unusual situation in which photochemicallyinduced electron transfer can be observed from a metal site within a protein.
European Journal of Biochemistry, 1984
The non-ionic detergent lauryl dimethylamine N-oxide (LDAO) has been used to extract the NADH deh... more The non-ionic detergent lauryl dimethylamine N-oxide (LDAO) has been used to extract the NADH dehydrogenases of Arum /naculatun? mitochondria. Affinity chromatography on S'ADP-Sepharose 4B was used to separate the rotenone-sensitive (complex I) NADH dehydrogenase from thc rotcnone-insensitive NADH dchydrogenase. An 1 8-fold purification of the rolenonc-insensitive NADH dehydrogenase was achicvcd. The enzyme is specific for NADH with optimal activity around pH7.2. The apparent K, Tor NADH is 28pM, with dichloroindophcnol as acceptor at pH 7.2. The rotenone-insensitive NADH dehydrogenase appears to be a flavoprotein and no iron-sulphur centres were detected by electron spin resonance spectroscopy.
European Journal of Biochemistry, 1997
Nocurdiu corultina B-276 possesses a constitutive multi-component alkene monooxygenase which cata... more Nocurdiu corultina B-276 possesses a constitutive multi-component alkene monooxygenase which catalyses the epoxidation of terminal and sub-terminal alkenes. The epoxygenase component of this system has been purified with an overall yield of 35%. The electron paramagnetic resonance spectrum of the oxidised protein has a weak signal at g = 4.3, which we ascribe to rhombic iron and a free radical signal at g,,, = 2.01. Upon partial reduction with dithionite using methyl viologen as a mediator, a signal at g,,, = 1.9 appeared. Upon further reduction with excess dithionite a signal at g = 15 appeared with the concomitant disappearance of the g,,, = 1.9 signal. These results indicate that the epoxygenase contains a bridged dinuclear iron centre similar to that found in a variety of proteins involved in oxygen transport and activation as well as desaturation of fatty acids. Analysis of the products of the reaction indicates that A M 0 is capable of stereospecific epoxidation of alkenes producing the R-enantiomer in high yield, a reaction catalysed by very few oxygenase enzymes. Whole cells gave lower enantiomeric excess values for the epoxide and a stereospecific epoxidase enzyme has been proposed to account for this difference. Although alkene monooxygenase was not inhibited by ethyne, a potent inhibitor of soluble methane monooxygenase with which alkene monooxygenase shares many common features, it was weakly inhibited by propyne with an apparent K, value of 340 pM. The mechanistic implications of these physicochemical features of the enzyme are discussed.
European Journal of Biochemistry, 1975
The technique of electron paramagnetic resonance spectrometry has been applied to the study of pl... more The technique of electron paramagnetic resonance spectrometry has been applied to the study of plant microsomal electron-transport components. Only tulip-bulb microsomes were found to give strong enough signals to allow detailed study.