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R Cammack

Publications and source records attributed to R Cammack.

At least 37 records · Page 2Linked to original sources
PubMed1997

Electron-nuclear double resonance spectroscopy investigation of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum: comparison with other flavin radical enzymes.

A FAD and [4Fe-4S]cluster-containing enzyme from Clostridium aminobutyricum catalyses the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA which involves the cleavage of an unactivated C-H bond at the beta-carbon. Transient oxidation of the substrate to an enoxy radical by FAD might facilitate the removal of this beta-proton, whereas no function could be attributed to the [4Fe-4S]cluster. In this paper the organic radical, which is formed by partial reduction of the enzyme with dithionite, was characterised as the neutral flavin semiquinone by EPR spectroscopy in H2O and D2O. The rapid electron-spin relaxation of the flavin semiquinone suggested a magnetic interaction with the [4Fe-4S]cluster. In order to obtain highly resolved information about nuclear spins in the vicinity of this paramagnetic centre, ENDOR spectroscopy was applied. The spectra were compared with those of the neutral semiquinone radicals of ferredoxin-NADP reductase and flavodoxin as well as with that of the anionic semiquinone radical of cholesterol oxidase. All ENDOR spectra showed strong couplings to the 8-methyl protons and to H-6 of the flavin. On addition of the substrates to the corresponding enzymes, the electron density changed significantly only at the 8-position. It decreased in the case of cholesterol oxidase and ferredoxin-NADP reductase, whereas an increase was observed with 4-hydroxybutyryl-CoA dehydratase. The results indicate an interaction of 4-hydroxybutyryl-CoA with the flavin as required by the proposed mechanism. Furthermore, the shift of electron density towards the benzoid ring of FAD in the dehydratase might be due to the location of the [4Fe-4S]cluster next to the 8-position as known from structurally characterised iron-sulfur flavoproteins.

Clostridium↗
PubMed1996

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.

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.

Binding Sites↗
PubMed1996

Detection of nitrosyl complexes in human substantia nigra, in relation to Parkinson's disease.

Idiopathic Parkinson's disease (PD) involves a documentable decline in the activity of mitochondrial complex I in substantia nigra (1-3). We have EPR spectroscopy to investigate complex I in human substantia nigra and globus pallidus. EPR signals characteristic of the iron-sulfur centers of complexes I and II were observed with globus pallidus, with no significant difference between control and PD. These complex 1 signals could not be clearly observed in substantia nigra. Instead, nitric oxide (NO.) radicals in PD nigra were detected at g approximately 2.08, 1.98 due to [haem-NO] formation. Although an EPR signal indicative of haem-NO was observed with control nigra, it lacked the distinctive g approximately 1.98 trough observed with PD nigra. As PD is associated with a reactive gliosis, the difference in the haem-NO EPR signal, between control and PD nigra, may result from cytotoxic NO. generated by microglia in PD substantia nigra.

Age of Onset↗
PubMed1996

Coordination of the [2Fe-2S] cluster in wild type and molecular variants of Clostridium pasteurianum ferredoxin, investigated by ESEEM spectroscopy.

The [2Fe-2S] ferredoxin from Clostridium pasteurianum contains five cysteine residues in positions 11, 14, 24, 56, and 60. This pattern is unique, and a combination of site-directed mutagenesis and spectroscopy is therefore being implemented to identify the ligands of the [2Fe-2S] cluster. The possible involvement of ligands other than cysteine in some molecular variants of this ferredoxin has been considered, histidines being likely candidates. Therefore, the three histidine residues in positions 6, 7, and 90 of the amino acid sequence have been individually and collectively replaced by alanine or valine. The mutated ferredoxins have been purified and were all found to contain [2Fe-2S] clusters of which the UV-visible absorption spectra were identical to that of the wild-type protein. The H6A/H7A/ H90A triply mutated ferredoxin was further characterized by EPR and by ESEEM spectroscopy and was found to differ only marginally from the wild-type protein. The ESEEM spectra of wild-type ferredoxin displayed weak 14N hyperfine interactions at the three principal g-factors of the [2Fe-2S] center. The estimated 14N coupling constants (Aiso = 0.6 MHz; e2qQ approximately 3.3 MHz) indicate that the ESEEM effect is most likely due to 14N from the polypeptide backbone. 2H2O ESEEM spectra showed that the [2Fe-2S] cluster is accessible for exchange with solvent deuterons. ESEEM spectra of the previously characterized C24A and C14A/C24A variants have been recorded and were also found to be very similar to those of the wild-type protein. There was no evidence for coordination of the [2Fe-2S] cluster by [14N]histidine or other 14N nuclei, in either wild-type or mutant forms of the ferredoxin. By these criteria, the environment of the [2Fe-2S] center is not distinguishable from those in plant-type ferredoxins. Non-cysteinyl coordination most probably occurs only in the C14A/C24A variant, which contains no more than three cysteine residues. The data shown here indicate that the fourth ligand of the [2Fe-2S] cluster is neither a histidine residue nor another nitrogenous ligand. The possibility of oxygenic coordination for this molecular variant is discussed.

Clostridium↗
PubMed1996

The relationship of intracellular iron chelation to the inhibition and regeneration of human ribonucleotide reductase.

The depletion of cellular iron can lead to the inhibition of ribonucleotide reductase, preventing new DNA synthesis and hence inhibiting cell proliferation. Electron paramagnetic resonance (EPR) spectroscopy has been used to examine simultaneously for the first time the relationship between chelation of intracellular iron and the rate of removal and regeneration of the tyrosyl radical of ribonucleotide reductase within intact human leukemia K562 cells. The different physiochemical characteristics of relatively hydrophobic low molecular weight bidentate hydroxypyridinone chelators and the higher molecular weight hexadentate ferrioxamine have been exploited to elucidate these interactions further. The base-line concentration of EPR-detectable mononuclear nonheme iron complexes was 3.15 =/- 1.05 microM, rising on incubation with chelators more rapidly with hydroxypyridinones than with desferrioxamine. Hydroxypyridinones also removed the tyrosyl radical more rapidly, apparently as a consequence of depletion of the intracellular iron pools necessary to regenerate the active enzyme and compatible with their reportedly greater cell toxicity. The radical decay rate is consistent with previous models, suggesting that iron is spontaneously removed from mammalian ribonucleotide reductase. Upon removal of extracellular chelator the regeneration of the tyrosyl radical was significantly faster for hydroxypyridinones than for desferrioxamine, consistent with their differential effects on cell cycle synchronization.

Deferoxamine↗
PubMed1996

Comparison of the "Rieske" [2Fe-2S] center in the bc1 complex and in bacterial dioxygenases by circular dichroism spectroscopy and cyclic voltammetry.

Two different types of "Rieske" [2Fe-2S] clusters have been observed in proteins, one in the bc complexes of the respiratory chain and the other in bacterial dioxygenases. We have compared the circular dichroic (CD) spectra and redox properties of the water soluble fragment of the Rieske center of the bovine heart mitochondrial bc1 complex (ISF) and of the ferredoxin from benzene dioxygenase in Pseudomonas putida ML2 (FDBED). Spinach ferredoxin was also measured for comparison. The redox potential of both proteins could be determined in solution by cyclic voltammetry (CV) and by CD-monitored spectroelectrochemistry using a specially constructed optically transparent thin layer (OTTLE) cell. Whereas the redox potential of the ISF (+312 +/- 5 mV at pH 7.0) depended both on the pH above pH 7 and on the ionic strength, the redox potential of the FDBED (-155 +/- 5 mV at pH 7.0) was observed to be independent of pH and ionic strength. The ISF showed a marked dependence of its redox potential on temperature, while the FDBED showed no temperature dependence. The entropy of the redox reaction delta S degrees rc was calculated as -88 +/- 11 J K-1 mol-1 for the bc1 Rieske center and approximately 0 J K-1 mol-1 for the FdBED. The CD spectra of Rieske type clusters are significantly different from those of plant type [2Fe-2S] ferredoxins. A strong negative CD band is present at 20 000 cm-1 (500 nm) in all reduced Rieske clusters. The possible assignment of this band is discussed as arising from the highest energy magnetically allowed d --> d transition (dz2 --> dxz) of the FeII site. If so, this band is highly indicative of the distortion of the ligand field of the FeII site.

Calorimetry↗
PubMed1996

Reversible hydrogenase of Anabaena variabilis ATCC 29413: catalytic properties and characterization of redox centres.

The catalytic and spectroscopic properties of the reversible hydrogenase from the cyanobacterium Anabaena variabilis have been examined. The hydrogenase required reductive activation in order to elicit hydrogen-oxidation activity. Carbon monoxide was a weak (Ki=35 microM), reversible and competitive inhibitor. A flavin with the chromatographic properties of FMN, and nickel were detected in the purified enzyme. A. variabilis hydrogenase exhibited electron paramagnetic resonance (EPR) spectra in its hydrogen-reduced state, indicative of [2Fe-2S] and [4Fe-4S] clusters. Although no EPR signals due to nickel were detected, the results are consistent with the enzyme being a flavin-containing hydrogenase of the nickel-iron type.

Anabaena↗
PubMed1996

Inhibition of tetrahydrobiopterin synthesis reduces in vivo nitric oxide production in experimental endotoxic shock.

Nitric oxide synthesis requires the cofactor tetrahydrobiopterin. We have examined the effect on nitric oxide synthesis in experimental endotoxic shock of 2,4- diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I, the first and rate limiting enzyme for tetrahydrobiopterin synthesis. Rats given lipopolysaccharide (LPS, 10 mg/kg) showed a large rise in plasma nitrate at 4 and 8 hours which was significantly reduced by DAHP (1 g/kg) given at the same time as LPS. There was a 40-50% reduction in the haem-NO signal detected in kidney by electron paramagnetic resonance spectroscopy. LPS produced hypotension at 3 hours and 6 hours and this was ameliorated at 6 hours in rats given DAHP. DAHP abolished the rise in kidney tetrahydrobiopterin levels seen 4 hours after LPS but no effect was seen on induction of inducible nitric oxide synthase (iNOS) as assessed by immunohistochemistry and reverse transcriptase PCR, consistent with the effect of DAPH being by reduction of tetrahydrobiopterin levels. The results show that inhibition of tetrahydrobiopterin synthesis is an effective strategy to reduce nitric oxide synthesis by iNOS in vivo.

Animals↗
PubMed1995

Coordination of the Rieske-type [2Fe-2S] cluster of the terminal iron-sulfur protein of Pseudomonas putida benzene 1,2-dioxygenase, studied by one- and two-dimensional electron spin-echo envelope modulation spectroscopy.

One- and two-dimensional (1D and 2D) electron spin-echo envelope modulation (ESEEM) spectroscopy has been used to investigate the ligand environment of the [2Fe-2S] cluster from the terminal dioxygenase (ISPBED) of the Pseudomonas putida benzene dioxygenase complex. The modulation frequencies observed in the 0.5-8.5 MHz region of the Fourier transforms of 1D and 2D ESEEM spectra measured across the electron paramagnetic resonance (EPR) absorbance envelope (from gz through to gx) are consistent with their assignment to two 14N nuclei. Using hyperfine sublevel correlation spectroscopy (HYSCORE), two sets of correlated double quantum transitions sharing the same hyperfine coupling were observed and were identified as being due to the same two 14N nuclei. On the basis of the isotropic hyperfine and quadrupolar couplings estimated for these 14N nuclei [N(1), Aiso = 3.6 MHz and e2qQ = 2.2-2.8 MHz; N(2), Aiso = 4.8 MHz and e2qQ = 2.2-2.4 MHz], the ESEEM pattern of ISPBED is assigned to two histidine nitrogens which are directly coordinated to the reduced iron-sulfur cluster. Bonding parameters of the two [14N]histidine ligands were calculated from these hyperfine couplings. The primary covalent contributions to the hyperfine interaction arise from 14N-to-Fe2+ sigma bonds. For N(1), our analysis of the percentage of unpaired 2s and 2p electrons gave f2s approximately 1.3% and f2p approximately 0.2%, while values of f2s approximately 1.7% and f2p approximately 1.4% were estimated for N(2). Comparison of these values with those determined from electron nuclear double resonance (ENDOR) data of the Rieske-type [2Fe-2S] center of Pseudomonas cepacia phthalate dioxygenase [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871] indicates an apparent reduction in unpaired electron spin density residing on the two 14N ligands of ISPBED. Analysis of slices of the HYSCORE spectrum has provided evidence for another 14N nucleus (A approximately 1.1 MHz, e2qQ = 3.3 MHz), which we have attributed to a weakly coupled peptide nitrogen, similar to those observed for ferredoxin-type [2Fe-2S] clusters. This type of weak interaction has not been previously described by the detailed ENDOR and ESEEM studies of Rieske-type centers. The resolution of the spectra demonstrates the effectiveness of 2D ESEEM for the disentanglement of multiple hyperfine interactions to metalloprotein centers.

Binding Sites↗
PubMed1995

Cooperative manganese (II) activation of 3-phosphoglycerate mutase of Bacillus megaterium: a biological pH-sensing mechanism in bacterial spore formation and germination.

The conversion of 3-P-glycerate mutase of Bacillus megaterium from a catalytically inactive to an active form was markedly more effective with buffered Mn2+ than with just added Mn2+. The previously reported stimulation by threonine disappeared when buffered Mn2+ was used. Activation of mutase showed a sigmoid dependence on Mn2+ concentration when buffered with tetramethylenediamine tetraacetate. The curve obeyed Hill kinetics with a coefficient of 2.1 +/- 0.1. At 0.5 microM free Mn2+, buffered with trimethylenediamine tetraacetate, activation of mutase increased about 73-fold over the pH range 6.6 to 7.4. Plotted against [OH-], the activation showed a strongly sigmoid response with Hill coefficient of 3.5 +/- 0.1. When mutase activated at pH 6.4 and 0.5 microM free Mn2+ in the presence of substrate was transferred to a similar medium at pH 7.4, the rate of product accumulation increased 360-fold within a few minutes. The pH sensitivity conferred upon mutase by low [Mn2+] may account for its large activity decrease during sporulation, and later increase during spore germination, when spore pH, respectively, declines and rises by about 1 unit. These changes result in the accumulation, and later reutilization, of 3-P-glycerate reserves in the spore. Such a pH-sensing function of Mn2+ may have wider biological uses.

Bacillus megaterium↗
PubMed1995

L-arginine depletion by arginase reduces nitric oxide production in endotoxic shock: an electron paramagnetic resonance study.

Nitric oxide (NO) synthesis was measured in the liver, lung, spleen and kidney of lipopolysaccharide-treated male rats using the nitric oxide spin trap, iron (II)-diethyldithiocarbamate (FeDETC2). Nitric oxide formation in vivo was determined by the increase in intensity of the characteristic triplet hyperfine EPR spectrum of [NO-FeDETC2]. Intravenous bovine liver arginase, at a dose which completely depleted circulating arginine, significantly reduced the formation of nitric oxide in these tissues. The general decrease in NO levels was confirmed by the decrease in plasma nitrite levels. These results directly demonstrate that NO formation in endotoxic shock depends on extracellular arginine; depletion of plasma arginine may be a useful therapeutic strategy.

Animals↗
PubMed1995

EPR spectroscopic characterization of the iron-sulphur proteins and cytochrome P-450 in mitochondria from the insect Spodoptera littoralis (cotton leafworm).

EPR spectroscopy was used to investigate the cytochrome P-450-dependent steroid hydroxylase ecdysone 20-mono-oxygenase of the cotton leafworm (Spodoptera littoralis) and the redox centres associated with membranes from the fat-body mitochondrial fraction. Intense features at g = 2.42, 2.25 and 1.92 from oxidized mitochondrial membranes have been assigned to the low-spin haem form of ferricytochrome P-450, probably of ecdysone 20-mono-oxygenase. High-spin cytochrome P-450 (substrate-bound) was tentatively assigned to a signal at g = 8.0, which was detectable from membranes as prepared. An EPR signal characteristic of a [2Fe-2S] cluster detected from the soluble mitochondrial matrix fraction has been shown to be distinct from the signals associated with mitochondrial NADH dehydrogenase and succinate dehydrogenase, and has therefore been attributed to a ferredoxin. We conclude that the S. littoralis fat-body mitochondrial electron-transport system involved in steroid 20-hydroxylation comprises both ferredoxin and cytochrome P-450 components, and thus resembles the enzyme systems of adrenocortical mitochondria. EPR signals characteristic of the respiratory chain were also observed from fat-body mitochondria and assigned to the iron-sulphur clusters associated with Complex I (Centres N1, N2), Complex II (Centres S1, S3), Complex III (the Rieske centre), and the copper centre of Complex IV, demonstrating similarities to mammalian mitochondria. The reduced membrane fraction also yielded a major resonance at g = 2.09 and 1.88 characteristic of the [4Fe-4S] cluster of electron-transferring flavoprotein: ubiquinone oxidoreductase. As the fat-body is the major metabolic organ of insects, this protein is presumably required for the beta-oxidation of fatty acids in mitochondria. High-spin haem signals in the low-field region of spectra also demonstrated that the mitochondrial fraction contains relatively high concentrations of catalase.

Animals↗
PubMed1995

Structural organization of the Ni and (4Fe-4S) centers in the active form of Desulfovibrio gigas hydrogenase. Analysis of the magnetic interactions by electron paramagnetic resonance spectroscopy.

The Desulfovibrio gigas hydrogenase is a typical (NiFe) hydrogenase containing a Ni center and three FeS centers, one [3Fe-4S] and two [4Fe-4S] clusters. When the enzyme is activated under hydrogen gas, the Ni center becomes paramagnetic, giving a characteristic electron paramagnetic resonance (EPR) signal with g values at 2.19, 2.14 and 2.01, the Ni-C signal. Two redox states of the enzyme can be prepared, in which the [4Fe-4S] clusters are either diamagnetic or paramagnetic. In this latter state, the magnetic coupling between metal centers induces both the appearance at low temperature of a complex EPR spectrum, the split Ni-C signal, and a significant enhancement of the relaxation rates of the Ni center. Good simulations of the split Ni-C signal recorded at three different microwave frequencies (X-band, Q-band, and S-band) are obtained by using a model based on a point dipole approximation of the dipolar and exchange interactions between paramagnets. The spectral analysis demonstrates that only one [4Fe-4S]1+ cluster is significantly coupled to the Ni site and provides a detailed description of the relative arrangement of the two centers. In addition, the magnetic characteristics of this [4Fe-4S]1+ cluster can be deduced from the simulations. Moreover, the spin-spin and spin-lattice relaxation times of the interacting centers were measured in the two redox states of the enzyme, either by power saturation and pulsed EPR experiments at low temperature or from the broadening of the EPR lines at higher temperature. The relaxation behavior of the Ni center is well explained by using in the theoretical analysis, the set of structural and magnetic parameters deduced from the spectral simulations. Our structural conclusions on the active D. gigas hydrogenase are compared to the preliminary data of a low-resolution crystal structure of the oxidized enzyme [Volbeda, A., Piras, C., Charon, M. H., Hatchikian, E. C., Frey, M., & Fontecilla-Camps, J. C. (1993) News Lett. Protein Crystallogr. 28, 30-33].

Desulfovibrio↗
PubMed1995

Electron spin resonance and electron nuclear double resonance studies of flavoproteins involved in the photosynthetic electron transport in the cyanobacterium Anabaena sp. PCC 7119.

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 H2O and 1.48 mT in D2O. 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 7119 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 D2O, but the coupling Aiso 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 D2O. 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Anabaena↗
PubMed1994

ESR and electron nuclear double resonance characterization of the cholesterol oxidase from Brevibacterium sterolicum in its semiquinone state.

Reduction of the flavin of cholesterol oxidase from Brevibacterium sterolicum, at pH values above 7, by sodium dithionite or light irradiation in the presence of EDTA (either in the presence or absence of deazariboflavin) was found to occur through a stable intermediate state. This intermediate had an optical spectrum characteristic of a flavin anionic semiquinone. The rate and extent of reduction were pH-dependent. No semiquinone intermediate was detected during reduction by these agents at pH values of 6.5 or below or at any pH when dehydroisoandrosterone, a protein substrate analogue, was used as reductant. No intermediate radical was detected during the reoxidation process. Treatment of cholesterol oxidase semiquinone with dehydroisoandrosterone did not convert the semiquinone intermediate to the fully reduced state. The absorption coefficient of oxidised cholesterol oxidase at 470 nm is 10.3 M-1 cm-1. The ESR signal of Brevibacterium sterolicum cholesterol oxidase semiquinone is centred at g = 2.004. The linewidth of the signal was 1.48 mT when the protein was studied in H2O or D2O. These data are in agreement with those reported for anionic semiquinones. The linewidths were the same when measured either at X-band or at S-band frequencies, indicating that line broadening is due to hyperfine interactions. The linewidth decreased to 1.43 mT when the substrate, dehydroisoandrosterone, was added. Electron nuclear double resonance (ENDOR) spectroscopy of cholesterol oxidase semiquinone provided further information about the interactions of the flavin radical with protons. A group of signals, with couplings of 9-12 MHz, is attributed to protons on 8-CH3 (Aiso = 10.9 MHz) and on C6 (Aiso = 9 MHz) of the flavin ring. No change in these hyperfine coupling constants was detected when the protein was studied in D2O. However, the hyperfine coupling constant attributed to protons on 8-CH3 decreased by 0.98 MHz when the ENDOR spectrum of the cholesterol oxidase semiquinone was studied in the presence of dehydroisoandrosterone (Aiso = 9.92 MHz). A second group of signals was observed with hyperfine couplings less than 2.5 MHz. Some of these weak couplings disappeared when the protein was transferred to D2O, or when the substrate, dehydroisoandrosterone, was present. 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 stabilisation.

Binding Sites↗
PubMed1994

ESEEM and ENDOR studies of the Rieske iron-sulphur protein in bovine heart mitochondrial membranes.

Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) were applied to the respiratory-chain iron-sulphur clusters in natural bovine heart mitochondrial membranes. By using specific reduction, signals were observed from the Complex III Rieske [2Fe-2S] cluster. In ENDOR, 1H hyperfine couplings in the range 0.5-7 MHz were observed. In ESEEM, modulations were obtained which were assigned to two 14N nuclei of directly-coupled imidazole ligands. The ESEEM spectra are similar to previous observations on purified iron-sulphur proteins of this type, in which the iron-sulphur cluster is coordinated by two cysteine and two histidine ligands. They confirm that the coordination of the cluster in the purified proteins, with two cysteinyl sulphur and two histidine nitrogens, is unchanged from its natural mitochondrial membrane environment. In order to investigate the possible interaction of the membrane-bound Rieske protein with quinones, measurements were conducted on membranes preincubated with 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), and in the pH range 6-7.5. No significant changes were detected, either in the proton hyperfine couplings as detected by ENDOR, or in the nitrogen couplings to the histidines as detected by ESEEM.

Animals↗