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1.
Desulfovibrio vulgaris Hildenborough cytochrome c3 contains four hemes in a low-spin state with bis-histidinyl coordination. High-spin forms of cytochrome c3 can be generated by protonation of the axial ligands in order to probe spin equilibrium (low-spin/high-spin). The spin alterations occurring at acid pH, the associated changes in redox potentials, as well as the reactivity towards external ligands were followed by the conjunction of square wave voltammetry and UV–visible, CD, NMR and EPR spectroscopies. These processes may be used for modelling the action of enzymes that use spin equilibrium to promote enzyme activity and reactivity towards small molecules. 相似文献
3.
Cytochrome c1 from a photosynthetic bacterium Rhodobacter sphaeroides R-26 has been purified to homogeneity. The purified protein contains 30 nmol heme per mg protein, has an isoelectric point of 5.7, and is soluble in aqueous solution in the absence of detergents. The apparent molecular weight of this protein is about 150 000, determined by Bio Gel A-0.5 m column chromatography; a minimum molecular weight of 30 000 is obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The absorption spectrum of this cytochrome is similar to that of mammalian cytochrome c1, but the amino acid composition and circular dichroism spectral characteristics are different. The heme moiety of cytochrome c1 is more exposed than is that of mammalian cytochrome c1, but less exposed than that of cytochrome c2. Ferricytochrome c1 undergoes photoreduction upon illumination with light under anaerobic conditions. Such photoreduction is completely abolished when p-chloromercuriphenylsulfonate is added to ferricytochrome c1, suggesting that the sulfhydryl groups of cytochrome c1 are the electron donors for photoreduction. Purified cytochrome c1 contains 3 ± 0.1 mol of the p-chloromercuriphenylsulfonate titratable sulfhydryl groups per mol of protein. In contrast to mammalian cytochrome c1, the bacterial protein does not form a stable complex with cytochrome c2 or with mammalian cytochrome c at low ionic strength. Electron transfer between bacterial ferrocytochrome c1 and bacterial ferricytochrome c2, and between bacterial ferrocytochrome c1 and mammalian ferricytochrome c proceeds rapidly with equilibrium constants of 49 and 3.5, respectively. The midpoint potential of purified cytochrome c1 is calculated to be 228 mV, which is identical to that of mammalian cytochrome c1. 相似文献
5.
- 1.
- 1. The spectral properties of ‘oxygenated’ cytochrome c oxidase, prepared by passing air through the dithionite-reduced enzyme solution, were compared with those of the ferric enzyme. 相似文献
6.
The low-spin ferric cyanide complex of beef heart cytochrome aa3 can be partially reduced by stoichiometric additions of ferrous cytochrome c or by similar additions of N, N, N′, N′-tetramethyl- p-phenylene diamine. In both cases the initial ratio of cytochrome c oxidized: cytochrome a reduced or Wurster's Blue: cytochrome a reduced approximates the value 2. It is concluded that the binding of a single HCN prevents the reduction of both cytochrome a3 and its associated EPR-invisible Cu atom. 相似文献
7.
Soluble cytochrome b5 of human erythrocytes was purified very effectively by hydrophobic chromatography using a butyl-Toyopearl 650 column. Cytochrome b5 was adsorbed tightly on the column in the presence of 60% saturated ammonium sulfate, and was eluted at 40% saturation of ammonium sulfate in the elution buffer. The chromatography gave a good yield of cytochrome b5 of the highest purity so far reported as estimated from the 414 nm to 280 nm absorbance ratio of the oxidized form of the cytochrome b5. The value obtained wit the cytochrome b5 purified in this study was 6.57, and is higher than the previously reported highest value of 6.4 (Hultquist, D.E., Dean, R.T. and Douglas, R.H. (1974) Biochem. Biophys. Res. Commun. 60, 28–34). Spectral properties including molecular absorption coefficients were determined using the cytochrome b5 purified by this method. 相似文献
9.
1. Circular dichroism spectra of the cytochromes in membrane fragments derived from sonicated beef heart mitochondria have been obtained in the wavelength region 400–480 nm in which the major absorbance maxima of the heme prosthetic groups are found. 2. 2. Cytochrome oxidase in the mitochondrial membrane fragments has a band of positive ellipticity at 426 nm in the oxidized form and a pronounced band of positive ellipticity at 445 nm in the reduced form. The reduced-minus-oxidized difference molar ellipticity at 445 nm, Δ[θ]445 is 3.0·105 degree·cm−2·dmole−1 heme a for membrane-bound oxidase compared to 1.6·105 degree·cm−2·dmole−1 heme a for the purified oxidase. The membrane-bound oxidase in the reduced form also appears to have a band of negative ellipticity at 426 nm not found in the purified oxidase. 3. 3. When reduced with succinate in the presence of cyanide and oxygen, cytochrome oxidase in the membrane fragments has a positive band at 442 nm very similar to that observed with the purified oxidase. 4. 4. Cytochrome c, which has a positive band at 426 nm in the purified form when reduced, appears to have a negative band at this wavelength in the mito-chondrial membrane fragments which contributes to the pronounced negative band at 426 nm observed in the membrane fragments reduced with succinate in anaerobiosis. There is no evidence for a contribution to the CD spectra of the membrane fragments from cytochrome c1 or from cytochrome b561 in either the oxidized or the reduced form. 5. 5. Cytochrome b566 in the mitochondrial membrane fragments has no detectable CD spectrum in the oxidized form, but has a small positive band at 427 nm and a small negative band at 436 nm in the reduced form. The same CD spectrum is observed with cytochrome b566 reduced with succinate in the presence of antimycin A or 2-heptyl-4-hydroxyquinoline-N-oxide. The same increase in positive ellipticity is observed at 427 nm in the mitochondrial membrane fragments, treated with oligomycin to restore energy coupling, when cytochrome b566 is reduced with succinate in the energized membrane, as is observed in the inhibitor-treated membrane fragments. The absence of a pronounced conformational change in cytochrome b566 on energization, as revealed by its CD spectrum, favors the concept that its reduction by succinate in the energized state is due to reversed electron transport rather than an intrinsic shift in the cytochrome's midpoint redox potential.
Abbreviations: HOQNO, 2-heptyl-4-hydroxy quinoline-N-oxide; PMS, phenazine methosulfate 相似文献
11.
1. 1. Cycles of oxidation followed by reduction at pH 7.2 have been induced in uncoupled anaerobic mung bean mitochondria treated with succinate and malonate by addition of oxygen-saturated medium. Under the conditions used, cytochromes b557, b553, c549 (corresponding to c1 in mammalian mitochondria) and ubiquinone are completely oxidized in the aerobic state, but become completely reduced in anaerobiosis. 2. 2. The time course of the transition from fully oxidized to fully reduced in anaerobiosis was measured for cytochromes c549, b557, and b553. The intramitochondrial redox potential (IMPh) was calculated as a function of time for each of the three cytochromes from the time course of the oxidized-to-reduced transition and the known midpoint potentials of the cytochromes at pH 7.2. The three curves so obtained are superimposable, showing that the three cytochromes are in redox equilibrium under these conditions during the oxidized-to-reduced transition. 3. 3. This result shows that the slow reduction of cytochrome b557 under these conditions, heretofore considered anomalous, is merely a consequence of its more negative midpoint potential of +42 mV at pH 7.2, compared to +75 mV for cytochrome b553 and +235 mV for cytochrome c549. Cytochrome b557 is placed on the low potential side of coupling site II and transfers electrons to cytochrome c549 via the coupling site. 4. 4. The time course of the transition from fully oxidized to fully reduced was also measured for ubiquinone. Using the change in intramitochondrial potential IMPh with time obtained from the three cytochromes, the change in redox state of ubiquinone with IMPh was calculated. When replotted as IMPh versus the logarithm of the ratio (fraction oxidized)/(fraction reduced), two redox components with n = 2 were found. The major component is ubiquinone with a midpoint potential Em7.2 = + 70 mV. The minor component has a midpoint potential Em7.2 = − 12 mV; its nature is unknown.
Abbreviations: IMPh, intramitochondrial potential, referred to the normal hydrogen electrode; Em7.2, midpoint potential at pH 7.2 相似文献
12.
We have examined the thermodynamic properties of the physiological electron donor to ferricytochrome c2 in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides. This donor (Z), which is capable of reducing the ferri-cytochrome with a halftime of 1–2 ms under optimal conditions, has an oxidation-reduction midpoint potential of close to 150 mV at pH 7.0, and apparently requires two electrons and two protons for its equilibrium reduction. The state of reduction of Z, which may be a quinone · protein complex near the inner (cytochrome c2) side of the membrane, appears to govern the rate at which the cyclic photosynthetic electron transport system can operate. If Z is oxidized prior to the flash-oxidation of cytochrome c2, the re-reduction of the cytochrome takes hundreds of milliseconds and no third phase of the carotenoid bandshift occurs. In contrast if Z is reduced before flash activation, the cytochrome is rereduced within milliseconds and the third phase of the carotenoid bandshift occurs. The prior reduction of Z also has a dramatic effect on the uncoupler sensitivity of the rate of electron flow; if it is oxidized prior to activation, uncoupler can stimulate the cytochrome re-reduction after several turnovers by less than tenfold, but if it is reduced prior to activation, the stimulation after several turnovers can be as dramatic as a thousandfold. The results suggest that Z plays a central role in controlling electron and proton movements in the ubiquinone cytochrome b-c2 oxido-reductase. 相似文献
14.
The photoinduced linear dichroism of absorption changes resulting from photolysis of the complex between heme a3 of the cytochrome oxidase and CO is studied. The experiments started from isotropic solutions or suspensions of the enzyme both in its isolated form and in mitochondria. The anisotropy responsible for the linear dichroism was induced by excitation with a flash of linearly polarized light. The dichroic ratios observed with various systems; polymerized enzyme in solution, enzyme in mitochondria and in submitochondrial particles (at 20 °C as well as at liquid N 2-temperature) all approached a value of 4/3 which characterizes a chromophore which is circularly degenerate. Therefrom we conclude that the interaction of heme a3 with its microenvironment within the protein does not break its four-fold symmetry. The experiments with mitochondria and submitochondrial particles suspended in aqueous buffer revealed similarly high dichroic ratios without any dichroic relaxation other than a rather slow one which could be attributed to the rotation of the whole organelle in the suspending medium. Therefrom we conclude that the cytochrome oxidase either is totally immobilized in the membrane, or that it carries out only limited rotational diffusion around a single axis coinciding with the symmetry axis of heme a3. In the light of independent evidence for a transmembrane arrangement of the oxidase and for the general fluidity of the inner mitochondrial membrane we consider anisotropic mobility of the cytochrome oxidase around an axis normal to the plane of the membrane as the most likely interpretation. Then our experimental results imply that the plane of heme a3 is coplanar to the membrane. 相似文献
15.
The interaction of a series of mixed ligand complexes of the type [Ru(NH 3) 4(diimine)]Cl 2, where diimine=2,2 ′-bipyridine (bipy), 1,10-phenanthroline (phen), 5,6-dimethyl-1,10-phenanthroline (5,6-dmp), 4,7-dimethyl-1,10-phenanthroline (4,7-dmp), 2,9-dimethyl-1,10-phenanthroline (2,9-dmp), 3,4,7,8-tetra-methyl-1,10-phenanthroline (Me 4phen), with calf thymus DNA has been studied using absorption, emission and circular dichroic spectral measurements and viscometry and electrochemical techniques. On interaction with DNA the complexes show hypochromism and red-shift in their MLCT band suggesting that the complexes bind to DNA. The magnitude of the binding constant ( Kb) obtained from absorption spectral titration varies depending upon the nature of the diimine ligand: Me 4phen > 5,6-dmp > 4,7-dmp > phen suggesting the use of diimine ‘face’ of the octahedral complexes in binding to DNA. The interaction of phen complex possibly involves phen ring partially inserted into the DNA base pairs. In contrast, the methyl-substituted phen complexes would involve hydrophobic interaction of the phen ring in the grooves of DNA, which is supported by hydrogen bonding interactions of the ammonia ligands with the intrastrand nucleobases. Also the shape and size of the phen ligand as modified by the methyl substituents determine the DNA binding site sizes (0.12-0.45 base pairs). The relative emission intensities ( I/ I0) of the DNA-bound complexes parallel the variation in Kb values. Almost all the metal complexes exhibit induced CD bands on binding to B DNA, with the 4,7-dmp and Me 4phen complexes inducing certain structural modifications on the biopolymer. DNA melting curves obtained in the presence of metal complexes reveal a monophasic melting of the DNA strands, the Me 4phen complex exhibiting a slightly enhanced tendency to stabilize the double-stranded DNA. There were slight to appreciable changes in the relative viscosities of DNA, which are consistent with enhanced hydrophobic interaction of the methyl-substituted phen rings. Upon interaction with CT DNA, the Me 4phen, 4,7-dmp and 5,6-dmp complexes, in contrast to bipy, phen and 2,9-dmp complexes, show a decrease in anodic peak current in their cyclic voltammograms suggesting that they exhibit enhanced DNA binding. DNA cleavage experiments show that all the complexes induce cleavage of pBR322 plasmid DNA, the Me 4phen and 5,6-dmp complexes being remarkably more efficient than other complexes. 相似文献
16.
The photosynthetic membranes from Rhodopseudomonas palustris contained one species of membrane-bound c-type cytochrome, presumably cytochrome c1, and a b-type cytochrome with two heme centers. The molecular weight and midpoint potential of cytochrome c1 were 30000 and 275 mV, respectively. The peak of the reduced-minus-oxidized difference spectrum of cytochrome c1 was at 552 nm. Molecular weight of the b-type cytochrome was 32000 and the cytochrome had two midpoint potentials of 60 mV and −55 mV. The peaks of the reduced-minus-oxidized difference spectra of the high and low midpoint potential heme centers were at 560 and 562 nm, respectively. These results suggested that there was a cytochrome b- c1 complex in Rps. palustris. 相似文献
17.
The prosthetic group of low-spin haem proteins is an iron porphyrin with two axial ligands, typically histidine, methionine or lysine. Determining the geometry of the axial ligands is an important step in structural characterisation, particularly in the paramagnetic oxidised forms. This work extends earlier studies of the hyperfine nuclear magnetic resonance (NMR) shifts of haem substituents in bis-His and His–Met cytochromes to His–Lys co-ordination in the M100K mutant of Paracoccus versutus cytochrome c550. The electronic structure of the His–Lys haem is shown to be similar to that produced by His–cyanide co-ordination, such that NMR can be used to determine the geometry of the His ligand. 相似文献
18.
Spectroscopic studies on trans-[Ru(NH 3) 4(SO 4)L] + where L=imidazole, histidine, pyridine and substituted pyridines were undertaken to understand the effect of various ligands on the Ru-N bonding in these complexes. The sulfate complexes show two major bands in the 250-270 and 310-350 nm region of the UV-Vis spectrum. Based on quantum chemical calculations the lowest energy band has been assigned to a LMCT (SO 4 2− → Ru III) transition. The energy of the LMCT transition decreases as the order of the axial ligand L basicity: Him > L-hist > 4-NH2-py > 4-Cl-py > 4-pic > py > nia > 4-Cn-py > isn > pz. EPR spectra give only two g values showing that the two LUMO containing the metal d π orbitals are degenerate and the energy separation between the LUMO and HOMO, calculated from the g values correlates linearly with the charge transfer energy and electrochemical properties. These correlations suggest extensive π donation from L to the Ru(III) d orbitals. An X-ray study of the 4-pic complex shows a bent S-O-Ru bond of 127.5° and MO calculations for three other complexes predict similar angles due to extensive σ and π bonding interaction between the sulfate oxygen and the Ru(III) ion. Surprisingly, the MO calculations do not predict the observed degeneracy in the LUMO orbital found by EPR studies. We shall argue that these discrepancies can be reconciled by insisting that the orientation of the L ring be coplanar with the S-O-Ru plane as is the case in the one X-ray study. 相似文献
20.
Summary The mitochondria of the cyt-2-1, cya-3-16, cya-4-23 and 299-1 nuclear mutants and the [ mi-3] and [ exn-5] cytoplasmic mutants of Neurospora crassa are deficient in cytochrome aa
3, while the cyb-1-1 and cyb-2-1 mutants have mitochondrial b-cytochrome dificiencies. However, the mitochondria from cyb-1-1 cyt-2-1, cyb-1-1 [mi-3] and cyb-2-1 [mi-3] double mutants contain 30% to 50% of the amount of cytochrome aa
3 that is present in mitochondria from wild-type; i.e. cyb-1-1 and cyb-2-2 act as suppressors of the cytochrome aa
3 deficiency phenotypes that are associated with the cyt-2-1 and [ mi-3] mutations.The production of cytochrome aa
3 can be induced in cyt-2-1 and [ mi-3] by growing cells in medium containing antimycin A, an inhibitor of electron transport in the cytochrome bc
1 segment of the mitochondrial electrontransport chain. Moreover, the growth of the [ mi-3] mutant is strongly stimulated by low concentrations of antimycin A. The induction of cytochrome aa
3 by antimycin treatments does not occur in [ exn-5], cya-4-23 and 299-1 cells, but does take place in cya-3-16 cells.Although some of the seven constituent polypeptides of cytochrome aa
3 are present the mitochondria of [ mi-3], the holoenzyme complex is not formed in the mutant. In contrast, the mitochondria of cyb-1-1 [ mi-3] and cyb-2-2 [ mi-3] double mutants contain a fully assembled cytochrome oxidase complex as well as some unassembled subunit polypeptides.The observations are indicative of the existence of at least two regulatory systems controlling the production of cytochrome aa
3. One of the circuits appears to control the basal or constitutive production of cytochrome oxidase, the other seems to coordinate the level of cytochrome aa
3 with some function of the mitochondrial cytochrome bc
1 complex, possibly electron transport. 相似文献
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