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1.
The structure of haem in pyridine/water mixtures and its implication in studies of haem catabolism.
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1. A study of haem spectra in pyridine/water mixtures at low pyridine concentrations revealed changes in haemochrome structure consistent with an aggregation process. No corresponding change in the structure of the haemichrome species was observed. 2. This aggregation has been correlated with a previously observed sharp decrease in the rate of coupled oxidation (degradation) of haem as pyridine concentration is decreased. The decrease appears to be due primarily to haem aggregation and not to changes in the hydrophobic nature of the solvent. The effect of ethanol and butanone addition was examined and supports this conclusion. 3. Evidence is presented that coupled oxidation occurs via the iron (II) species (haemochrome). 相似文献
2.
O. T. G. Jones 《The Biochemical journal》1968,107(1):113-119
Spinach chloroplasts catalyse the incorporation of Fe(2+) into protoporphyrin, mesoporphyrin and deuteroporphyrin to form the corresponding haems. This ferrochelatase activity was detected by pyridine haemochrome formation with acetone-dried powders of chloroplasts, or from the formation of [(59)Fe]haems by intact chloroplasts. Decreasing the mitochondrial contamination of the chloroplasts by density-gradient centrifugation did not cause any loss of activity: spinach ferrochelatase appears to be principally a chloroplast enzyme. The characteristics of the enzyme were examined by using [(59)Fe]haem assay. The activity was pH-dependent: for both mesohaem and protohaem formation there were two pH maxima, a major peak at about pH7.8 and a smaller peak at about pH9.2. Lineweaver-Burk plots showed that the K(m) for Fe(2+) incorporation into protoporphyrin was 8mum and that for Fe(2+) incorporation into mesoporphyrin was 36mum. At non-saturating Fe(2+) concentrations the K(m) for protoporphyrin was 0.2mum and that for mesoporphyrin was 0.4mum. Ferrochelatase was not solubilized by treatment of chloroplasts with ultrasound but was solubilized by stirring in 1% (w/v) Tween 20 at pH10.4. Unlike the rat liver mitochondrial enzyme, chloroplast ferrochelatase was not stimulated by treatment with selected organic solvents. The spinach enzyme was inactive in aerobic conditions and it was shown by using an oxygen electrode that under such conditions the addition of Fe(2+) to buffer solutions caused a rapid uptake of dissolved oxygen, believed to be due to the oxidation of Fe(2+) to Fe(3+); Fe(3+) is not a substrate for ferrochelatase. 相似文献
3.
Thermostable peroxidase from Bacillus stearothermophilus 总被引:8,自引:0,他引:8
A peroxidase from Bacillus stearothermophilus was purified to homogeneity. The enzyme (Mr 175,000) was composed of two subunits of equal size, and showed a Soret band at 406 nm. On reduction with sodium dithionite, absorption at 434 nm and 558 nm was observed. The spectrum of reduced pyridine haemochrome showed peaks at 418, 526 and 557 nm; the reduced minus oxidized spectrum of pyridine haemochrome showed peaks of 418, 524 and 556 nm with a trough at 452 nm. These results indicate that the enzyme contained protohaem IX as a prosthetic group. The optimum pH was about 6 and the apparent optimum temperature was 70 degrees C. The enzyme was relatively stable up to 70 degrees C; at 30 degrees C it was stable for a month. The enzyme had peroxidase activity toward a mixture of 2,4-dichlorophenol and 4-aminoantipyrine with a Km for H2O2 of 1.3 mM. It also acted as a catalase with a Km for H2O2 of 7.5 mM. 相似文献
4.
Non-enzymic nature of the pyridine haemochrome-cleaving activity of mammalian tissue extracts (`haem α-methyl oxygenase'')
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1. The pyridine haemochrome-cleaving activity of extracts from mammalian liver and other tissues is shown conclusively to be entirely non-enzymic in nature and attributable to coupled oxidation with ascorbate. 2. Reduced glutathione probably contributes to the activity indirectly by continuously regenerating the ascorbate to the reduced form. 3. The cleavage shows no specificity for the alpha-methine bridge of pyridine haemochrome. 4. Results are presented suggesting some probable reasons for the erroneous characterization of the activity as an alpha-methine-specific haem-cleaving enzyme (;haem alpha-methenyl oxygenase') by Nakajima and co-workers (e.g. Nakajima, Takemura, Nakajima & Yamaoka, 1963; Nakajima & Gray, 1967). 相似文献
5.
Redox behaviour of the haem domain of flavocytochrome c3 from Shewanella frigidimarina probed by NMR
Pessanha M Rothery EL Louro RO Turner DL Miles CS Reid GA Chapman SK Xavier AV Salgueiro CA 《FEBS letters》2004,578(1-2):185-190
Flavocytochrome c3 from Shewanella frigidimarina (fcc3) is a tetrahaem periplasmic protein of 64 kDa with fumarate reductase activity. This work reports the first example of NMR techniques applied to the assignment of the thermodynamic order of oxidation of the four individual haems for such large protein, expanding its applicability to a wide range of proteins. NMR data from partially and fully oxidised samples of fcc3 and a mutated protein with an axial ligand of haem IV replaced by alanine were compared with calculated chemical shifts, allowing the structural assignment of the signals and the unequivocal determination of the order of oxidation of the haems. As oxidation progresses the fcc3 haem domain is polarised, with haems I and II much more oxidised than haems III and IV, haem IV being the most reduced. Thus, during catalysis as an electron is taken by the flavin adenosine dinucleotide from haem IV, haem III is eager to re-reduce haem IV, allowing the transfer of two electrons to the active site. 相似文献
6.
Electron conduction between b cytochromes of the mitochondrial respiratory chain in the presence of antimycin plus myxothiazol 总被引:1,自引:0,他引:1
The b haems of the bc1 complex of bovine heart mitochondria were poised with succinate and fumarate so that only the high-potential haem (b-562) was reduced, and then isolated from further redox exchange with the ubiquinone pool by adding antimycin and myxothiazol. A transmembrane electric potential difference was then developed, either by electron flow from [Ru(NH3)6]Cl2 to oxygen or by ATP hydrolysis. The small difference spectrum, caused by the electric field, indicated 32-55% oxidation of b-562 with concomitant reduction of b-566. No lag greater than 0.1 s was detectable between the initiation of respiration and the development of the difference spectrum, thus providing a direct demonstration of (fairly) rapid electron transfer between the b haems. 相似文献
7.
The unambiguous assignment of the nuclear magnetic resonance (NMR) signals of the alpha-substituents of the haems in the tetrahaem cytochrome isolated from Shewanella frigidimarina NCIMB400, was made using a combination of homonuclear and heteronuclear experiments. The paramagnetic (13)C shifts of the nuclei directly bound to the porphyrin of each haem group were analysed in the framework of a model for the haem electronic structure. The analysis yields g-tensors for each haem, which allowed the assignment of some electron paramagnetic resonance (EPR) signals to specific haems, and the orientation of the magnetic axes relative to each haem to be established. The orientation of the axial ligands of the haems was determined semi-empirically from the NMR data, and the structural results were compared with those of the homologous tetrahaem cytochrome from Shewanella oneidensis MR-1 showing significant similarities between the two proteins. 相似文献
8.
Two-dimensional NMR has been used to make specific assignments for the four haems in Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c3 and to determine their haem core architecture. The NMR signals from the haem protons were assigned according to type using two-dimensional NMR experiments which led to four sets of signals, one for each of the haems. Specific assignments were obtained by calculating the ring current shifts which arise from other haems and aromatic residues. Observation of interhaem NOEs confirmed the assignments and established that the relative orientation of the haems is identical to that found in the crystal structure of D. vulgaris (Miyazaki F.) ferricytochrome c3. Assignments were also made for all the aromatic residues except for the haem ligands and F20, which is shifted under the main envelope of signals. The NOEs observed between these aromatic protons and haem protons confirm the similarity between the structures in solution and in the crystal. The assignments reported here are the basis for the cross-assignments of the four microscopic haem redox potentials to specific haems in the protein structure [Salgueiro, C. A., Turner, D. L., Santos, H., LeGall, J. and Xavier, A. V. (1992) FEBS Lett., in the press] 相似文献
9.
The orientation of the two haems of the Escherichia coli ubiquinol oxidase:O2 reductase, cytochrome bo, has been determined by electron paramagnetic resonance studies on oriented multilayer preparations of cytoplasmic membrane fragments. The enzyme contains a low-spin b-like haem and a high-spin b-like haem, designated cytochromes b and o respectively. Both haems are oriented with their planes perpendicular to the membrane plane, further extending the catalogue of structural and functional similarities between this enzyme and the mammalian cytochrome c oxidase, cytochrome aa3. 相似文献
10.
Isabel B. Coutinho David L. Turner Ming Y. Liu Jean LeGall António V. Xavier 《Journal of biological inorganic chemistry》1996,1(4):305-311
The trihaem cytochrome c
551.5, formerly known as cytochrome c
7, from the organism Desulfuromonas
acetoxidans, has been studied in the reduced state by 2D proton NMR. The haem proton resonances were assigned, and several nuclear Overhauser
enhancements (NOEs) between resonances arising from different haems were detected and assigned. The relative orientations
of the three haems were calculated by fitting both the intensities of the interhaem NOEs and the magnitudes of the ring current
shifts of the haem resonances, following the strategy previously used by the authors to reassess the X-ray structure of the
haem core in tetrahaem cytochrome c
3 from Desulfumicrobium baculatum.
It is concluded that, although the comparison of the protein sequence with those of the tetrahaem cytochromes c
3 shows that in cytochrome c
551.5 about 40% of the sequence is deleted, including the region involved in the attachment of the second of the four haems, this
does not induce any significant rearrangement of the remaining three haems other than a slight decrease in the iron-iron distance
between two of the haems, namely those corresponding to haems I and IV of cytochrome c
3.
Received: 2 February 1996 / Accepted: 26 March 1996 相似文献
11.
Addition of NaHSO3 or HCN to the formyl group of heme alpha was greatly accelerated by freezing reaction mixtures prepared in aq. Na2CO3, and freezing resulted in characteristic color and spectral changes of the solutions. Similar changes were observed on decreasing the pH of alkaline reaction mixtures with HCl at room temperature, indicating that the effective pH of certain salt solutions is greatly lowered by freezing. The reactivity of the formyl group changed depending on the redox state of the heme iron and the species of ligand. 相似文献
12.
The effects of pH and inhibitors on the spectra and redox properties of the haems b of the bc1 complex of beef heart submitochondrial particles were investigated. The major findings were: (1) both haems have a weakly redox-linked protonatable group with pKox and pKred of around 6 and 8; (2) at pH values above 7, haem bH becomes heterogeneous in its redox behaviour. This heterogeneity is removed by the Qi site inhibitors antimycin A, funiculosin and HQNO, but not by the Qo site inhibitors myxothiazol or stigmatellin; (3) of all inhibitors tested only funiculosin had a large effect on the Em/pH profile of either haem b. In all cases where definite effects were found, the haem most affected was that thought to be closest to the site of inhibitor binding; (4) spectral shifts of haem groups caused by inhibitor binding were usually, but not always, of the haem group closest to the binding site; (5) titrations with succinate/fumarate were in reasonable agreement with redox-mediated data provided that strict anaerobiosis was maintained. Apparent large shifts of haem midpoint potentials with antimycin A and myxothiazol could be produced in aerobic succinate/fumarate titrations in the presence of cyanide, as already reported in the literature, but these were artefactual; (6) the heterogeneous haem bH titration behaviour can be simulated with a model similar to that proposed by Salerno et al. (J. Biol. Chem. (1989) 264, 15398-15403) in which there is redox interaction between haem bH and ubiquinone species bound at the Qi site. Simulations closely fit both the haem bH data and known semiquinone data only if it is assumed that semiquinone bound to oxidised haem bH is EPR-silent. 相似文献
13.
Ingeborg M. Kooter Nicole Moguilevsky Alex Bollen Nanna M. Sijtsema Cees Otto R. Wever 《Journal of biological inorganic chemistry》1997,2(2):191-197
The optical absorbance spectrum of reduced myeloperoxidase is red-shifted with respect to that of other haemoproteins, and
has the Soret band at 472 nm and the α band at 636 nm. The origin of the red shift is poorly understood, but the interaction
of the protein matrix with the chromophore is thought to play an important role. Met243 is one of the three residues in close
proximity to the prosthetic group of the enzyme, and we have examined the effect of a Met243Gln mutation on the spectroscopic
properties and catalytic activity of the enzyme. The mutation has a large effect on the position of the Soret band in the
optical absorbance spectrum of the reduced mutated enzyme, which shifts from 472 nm to 445 nm. The alkaline pyridine haemochrome
spectrum is greatly affected and similar to that of protohaem. The mutation also drastically affects the resonance Raman (RR)
spectrum, which is indicative of an iron porphyrin-like chromophore. The mutant enzyme is unable to peroxidise chloride to
hypochlorous acid. We conclude that there are two factors involved which account for the red-shifted Soret band. One of them
is the interaction of Met243 with the prosthetic group via a special sulfonium linkage. The other factor which contributes
is the presence of ester linkages between hydroxylated methyl groups on the haem and glutamate and aspartate residues, respectively.
The results, combined with those of previous studies, now give us a comprehensive picture of the various factors which contribute
to the unusual optical properties of myeloperoxidase.
Received: 17 July 1996 / Accepted: 28 November 1996 相似文献
14.
M Degli Esposti M Crimi C K?rtner A Kr?ger T Link 《Biochimica et biophysica acta》1991,1056(3):243-249
The fumarate reductase from Wolinella succinogenes contains two haem groups with markedly different midpoint potentials (-20 mV and -200 mV). The enzyme is made up of three subunits, the lipophilic one of which (cytochrome b) ligates the haems. Circular dichroism (CD) spectroscopy has been applied to the reductase in order to obtain information on the structure of the haems and of their environment. This approach is integrated with amino acid sequence comparison of the cytochrome b with other quinone-reacting membrane haemoproteins for predicting the axial ligands of the haems as well as their location relative to the membrane. The following results have been obtained: (1) the CD spectra in the Soret region show exciton coupling indicating haem-haem interaction, which is particularly evident in the reduced state and disappears upon denaturation of the enzyme; (2) The apoprotein of cytochrome b is predicted to consist of five hydrophobic helices (helices A-D and cd), four of which should span the membrane. Helices A, B, C and cd contain a histidine residue each which possibly forms one of the ligands of the haems. It is proposed that haem b (-20 mV) is ligated by H44 and H93, and haem b (-200 mV) by H143 and H182. 相似文献
15.
J Villalaín I Moura M C Liu W J Payne J LeGall A V Xavier J J Moura 《European journal of biochemistry》1984,141(2):305-312
A dihaem cytochrome (Mr 37 400) with cytochrome c peroxidase activity was purified from Pseudomonas stutzeri (ATCC 11 607). The haem redox potentials are far apart: one of the haems is completely ascorbate-reducible and the other is only reduced by dithionite. The coordination, spin states and redox properties of the covalently bound haems were probed by visible, NMR and electron paramagnetic resonance (EPR) spectroscopies in three oxidation states. In the oxidized state, the low-temperature EPR spectrum of the native enzyme is a complex superimposition of three components: (I) a low-spin haem indicating a histidinyl-methionyl coordination; (II) a low-spin haem indicating a histidinyl-histidinyl coordination; and (III) a minor high-spin haem component. At room temperature, NMR and optical studies indicate the presence of high-spin and low-spin haems, suggesting that for one of the haems a high-spin to low-spin transition is observed when temperature is decreased. In the half-reduced state, the component I (high redox potential) of the EPR spectrum disappears and induces a change in the g-values and linewidth of component II; the high-spin component II is no longer detected at low temperature. Visible and NMR studies reveal the presence of a high-spin ferric and a low-spin (methionyl-coordinated) ferrous state. The NMR data fully support the haem-haem interaction probed by EPR. In the reduced state, the NMR spectrum indicates that the low-potential haem is high-spin ferrous. 相似文献
16.
NMR studies of electron transfer mechanisms in a protein with interacting redox centres: Desulfovibrio gigas cytochrome c3 总被引:1,自引:0,他引:1
H Santos J J Moura I Moura J LeGall A V Xavier 《European journal of biochemistry》1984,141(2):283-296
The proton NMR spectra of the tetrahaem cytochrome c3 from Desulfovibrio gigas were examined while varying the pH and the redox potential. The analysis of the NMR reoxidation pattern was based on a model for the electron distribution between the four haems that takes into account haem-haem redox interactions. The intramolecular electron exchange is fast on the NMR time scale (larger than 10(5) s-1). The NMR data concerning the pH dependence of the chemical shift of haem methyl resonances in different oxidation steps and resonance intensities are not compatible with a non-interacting model and can be explained assuming a redox interaction between the haems. A complete analysis at pH* = 7.2 and 9.6, shows that the haem-haem interacting potentials cover a range from -50 mV to +60 mV. The midpoint redox potentials of some of the haems, as well as some of their interacting potentials, are pH-dependent. The physiological relevance of the modulation of the haem midpoint redox potentials by both the pH and the redox potential of the solution is discussed. 相似文献
17.
1. Titration of haem with pyridine in alkaline media of low ionic strength yields a true pyridine haemochromogen, compound III, at very low concentrations of pyridine. 2. Graphical analysis of this titration gives the first spectrophotometric evidence for a dimeric haem. 3. Compound III is unstable and tends to aggregate to a second compound, compound II, whose formation is enhanced under those conditions favourable to hydrophobic bonding. 4. At higher concentrations of pyridine, compound II is dispersed to yield the classical pyridine haemochromogen, compound I, whose spectral properties are essentially those of pyridine haemochromogen in a non-aqueous medium. 相似文献
18.
Lígia M. Saraiva Carlos A. Salgueiro Jean LeGall Walter M. A. M. van Dongen António V. Xavier 《Journal of biological inorganic chemistry》1996,1(6):542-550
Reduction of the haems in tetrahaem cytochromes c
3 is a cooperative process, i.e., reduction of each of the haems depends on the redox states of the other haems. Furthermore,
electron transfer is coupled to proton transfer (redox-Bohr effect). Two of its haems and a strictly conserved nearby phenylalanine
residue, F20, in Desulfovibrio vulgaris (Hildenborough) cytochrome c
3 form a structural motif that is present in all cytochromes c
3 and also in cytochrome c oxidase. A putative role for this phenylalanine residue in the cooperativity of haem reduction was investigated. Therefore,
this phenylalanine was replaced, with genetic techniques, by isoleucine and tyrosine in D. vulgaris (Hildenborough) cytochrome c
3. Cyclic voltammetry studies revealed a small increase (30 mV) in one of the macroscopic redox potentials in the mutated cytochromes.
EPR showed that the main alterations occurred in the vicinity of haem I, the haem closest to residue 20 and one of the haems
responsible for positive cooperativities in electron transfer of D. vulgaris cytochrome c
3. NMR studies of F20I cytochrome c
3 demonstrated that the haem core architecture is maintained and that the more affected haem proton groups are those near the
mutation site. NMR redox titrations of this mutated protein gave evidence for only small changes in the relative redox potentials
of the haems. However, electron/electron and proton/electron cooperativity are maintained, indicating that this aromatic residue
has no essential role in these processes. Furthermore, chemical modification of the N-terminal amino group of cytochrome c
3 backbone, which is also very close to haem I, had no effect on the network of cooperativities.
Received: 25 June 1996 / Accepted: 26 August 1996 相似文献
19.
The structure is based on a difference Fourier synthesis at 2.8 Å resolution, using observed structure amplitudes and calculated phases, derived from a refinement of horse methaemoglobin at 2.0 Å resolution. Carbonmonoxyhaemoglobin has the same quaternary structure as methaemoglobin, but differs from it by slight changes in tertiary structure in the immediate vicinity of the haems. On transition from met- to carbonmonoxyhaemoglobin the distal histidines move away from the haem ligands towards the molecular surface, and both the haems and F-helices rotate slightly and shift towards the distal side. In methaemoglobin the sulphydryl group of cysteine F9(93)β is in equilibrium between two alternative positions: one external and the other half-buried in the “tyrosine pocket” between helices F and H. In carbonmonoxyhaemoglobin all the electron density for the sulphydryl group is in the half-buried position, so that the side chain of tyrosine HC2(145)β is completely displaced from its pocket. The difference map shows that the CO oxygen lies off the haem axis in both subunits, but the carbon cannot be seen as it coincides with the water molecule in methaemoglobin. A preliminary refinement of carbonmonoxyhaemoglobin suggests that the carbon may be displaced from the haem axis in the same direction as the oxygen. The haem pocket is so constructed that it fits an oxygen molecule in the bent conformation, but not a CO molecule which has its axis normal to the haem plane, because of steric hindrance by N? of the distal histidine and by Cγ2 of the distal valine. These two side chains apparently push the CO oxygen off the haem axis. The difference map indicates that in methaemoglobin the α-haem is ruffled and that on transition from met- to carbonmonoxyhaemoglobin it becomes flattened; in the β-haem the iron appears to move towards the porphyrin plane. The resolution is not sufficient to determine the exact position of the iron atoms and the proximal histidines relative to the porphyrins. 相似文献
20.
Structure of haemoglobin in the deoxy quaternary state with ligand bound at the alpha haems 总被引:2,自引:0,他引:2
We report the X-ray crystal structure of two analogues of human haemoglobin in the deoxy quaternary (T) state with ligand bound exclusively at the alpha haems. These models were prepared from symmetric, mixed-metal hybrid haemoglobin molecules. The structures of alpha Fe(II) beta Co(II), its carbonmonoxy derivative alpha Fe(II)CO beta Co(II), and alpha Fe(II)O2 beta Ni(II) are compared with native deoxy haemoglobin by difference Fourier syntheses at 2.8, 2.9 and 3.5 A resolution, respectively, and the refined alpha Fe(II)CO beta Co(II) structure is analysed. In both the native deoxy and liganded T molecules, the mean plane of the alpha-subunit haem is parallel with the axis of the F helix, but this plane is tilted with respect to the helix axis in the oxy-quaternary R state. The side-chains of LeuFG3 and ValFG5 sterically restrict haem tilting in the T state. We propose that strain energy develops at the contact between the haem and these residues in the liganded T-state haemoglobin, and that the strain is, in part, responsible for the low affinity of the T-state alpha haem. 相似文献