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1.
The light in the near-infrared region (700-900 nm) was illuminated on the rat head, and absorption spectra were measured with the transmitted light under various conditions. The absorbance changes less than 780 nm were attributable to hemoglobin in the brain tissue, whereas those greater than 780 nm were associated with both hemoglobin and cytochrome oxidase. The changes of oxy- and total (oxy- plus deoxy-) hemoglobin content in the rat head could be monitored quantitatively by expressions of delta A700--1.20 delta A730 and delta A700--1.52 delta A730, respectively. The oxyhemoglobin content in the tissue was decreased as the O2 tension in inspired gas was lowered. At 10% O2 approximately 50% of hemoglobin was deoxygenated. The total hemoglobin content was increased under anoxic conditions. Inhalation of 5% CO2 and intravenous injection of a Ca2+ blocker nicardipine increased the O2 saturation of hemoglobin in the brain. These conclusions were confirmed by measuring the difference absorption spectra in the near-infrared region.  相似文献   

2.
W J Ingledew  M Bacon  P R Rich 《FEBS letters》1992,305(3):167-170
The bacterial quinol oxidase, cytochrome o, is an enzyme which is highly analogous to the better known cytochrome c oxidase, cytochrome aa3, but with the important difference that it lacks the near infra-red absorbing pigment CuA. In this article we report an absorption band in the near IR spectrum of cytochrome o with a maximal absorption at 758 nm, and which is attributable to the ferrous high-spin haem. The 758 nm band has an extinction coefficient of 0.2-0.3 mM-1.cm-1 at 758-800 nm. This region in cytochrome aa3 is dominated by the CuA absorption. The 758 nm absorption is lost on addition of CO or cyanide to the reduced enzyme. The carbon monoxide compound of cytochrome o also has absorbance bands in the near infra-red, and these may be attributable to a low-spin ferrous haem compound.  相似文献   

3.
The reaction of cyanide with cytochrome aa3 in intact mitochondria is known to differ significantly from the reaction with the isolated enzyme. To examine the cyanide reaction with cytochrome aa3 in situ, we studied the spectral characteristics and the reaction kinetics of cyanide with reduced brain cytochrome aa3 in an isolated perfused rat head preparation. Anaesthetized rats underwent bilateral carotid-arterial cannulation. The head (skull intact, muscle removed) was perfused with a crystalloid solution containing Na2S2O4, and the animal was then decapitated. By means of reflectance spectrophotometry the reaction of cyanide with cytochrome aa3 was continuously monitored with the use of the 590 nm-575 nm, 610 nm-575 nm and 590 nm-610 nm wavelength pairs. We found that: the kinetics of the absorbance change at 590 nm and 610 nm were similar, with almost identical apparent rate constants, suggesting that these spectral changes are the results of the formation of a single complex; the difference spectrum obtained on addition of cyanide to the fully reduced preparation showed a peak at 588 nm and a trough at 610 nm, consistent with spectral characteristics of the cyanide-ferrocytochrome aa3 complex in isolated enzyme and isolated mitochondria in vitro; this observation underscores the accuracy of monitoring the effects of inhibitors of mitochondrial function on cytochrome redox reactions in situ; the half-maximal (K0.5) effect was approx. 50 microM, significantly lower than that in vitro. The lower apparent K0.5 for cyanide in this preparation in situ may be due to a difference in the pH of the two systems. This approach provides the means to study the inhibitors of mitochondrial function in intact brain under a physiological environment.  相似文献   

4.
Cytochrome oxidation-reduction responses to two mitochondrial electron transport inhibitors, carbon monoxide (CO) and cyanide (CN), were studied in the intact brains of fluorocarbon-circulated rats. In vivo reflectance spectrophotometry indicated that cortical b-type cytochromes (564 nm) were highly resistant to reduction by CN in the presence of O2 but showed reduction responses to the administration of 1-5% CO in 90% O2. In contrast, cyanide-sensitive cytochromes aa3 (605 nm) and c + c1 (551 nm) did not increase their reduction levels during exposure to 5% CO in 90% O2. The in vivo CO-mediated b-cytochrome reduction responses did not occur after pretreatment with the cytochrome b inhibitor, antimycin A. Transmission spectrophotometry of superfused hemoglobin-free rat brain slices confirmed cortical b-type cytochromes to be CN-resistant in the presence of O2. Another cytochrome absorbing at 445 nm also was resistant to reduction by 1-mM cyanide in vitro, but it could be reduced anaerobically. The reduced 445-nm cytochrome bound CO in the presence of cyanide. We postulate that this CN-resistant CO binding component might account for in vivo cytochrome aa3-CO interactions and directly or indirectly modulate cytochrome b reduction responses to CO. In any event, the spectral data indicate different primary tissue target sites for CO and CN in living rat brain and also suggest different bioenergetic consequences of exposure to the two agents.  相似文献   

5.
Using the isolated perfused rat hindlimb and the fluorocarbon-transfused rat, we have examined the optical characteristics of the rat skeletal muscle in the near-infrared region. The total contribution of myoglobin and cytochromes to the overall absorbance change was less than 10%. Analyzing transmitted light at 700, 730, and 805 nm, we found linear relationships between the absorbance and the hemoglobin concentrations at hematocrit values from 15 to 50% in the inflowing perfusate. Based on the relationship, we determined the ratio of absorption coefficients at 700, 730, and 805 nm of oxy- and deoxy-hemoglobins of blood in the thigh muscle. The values in thigh muscle were significantly smaller than those in hemoglobin solutions for deoxygenated blood. On the other hand, the values in thigh muscle were larger than those in hemoglobin solutions for oxygenated blood. Solving simultaneous equations by the use of these absorption coefficients, we calculated the changes in the contents of oxy-, deoxy-, and total hemoglobins in the anesthetized rat hindlimb under various conditions. The oxygen saturation of blood determined by our optical method in the thigh muscle was very close to that in the vena cava measured directly with a gas analyzer.  相似文献   

6.
B Chance  C Saronio    J S Leigh  Jr 《The Biochemical journal》1979,177(3):931-941
Compound C2 is a product of the reaction of O2 and the mixed-valence state of cytochrome oxidase. The mixed-valence state of membrane-bound cytochrome oxidase is obtained at -24 degrees C, by using either ferricyanide or yeast peroxidase complex ES as oxidants, and the configurations of oxidized haem a and its associated copper (a3+Cua2+) and of reduced haem a3 and its associated copper (ac3+.CO.Cua3+) are obtained. The mixed-valence-state cytochrome oxidase mixed with O2 at -24 degrees C and flash-photolysed at -60 to -100 degrees C reacts with O2 and initially forms an oxy compound (A2) similar to that formed from the fully reduced state (A1). Thereafter the course of the reaction differs from that obtained in the fully reduced state, and absorbance increases are observed at 740--750 nm and 609 nm and a decrease at 444 nm, with no increase in absorbance at 655 nm. One possible attribution of the absorbance increases is to charge-transfer interaction between the iron of haem a3 and the copper associated with haem a3, Cua3(2+), having properties of a type-I 'blue' copper. A possible attribution of the decrease in absorbance at 444 nm is to liganding of a3(2+). A related explanation is that the 609 nm absorbance involves a charge-transfer interaction of both iron and copper as a mixed-valence binuclear complex, Cua3, having properties of a non-blue copper. Intermediates in addition to Compound C2 are not yet identifiable by chemical or spectroscopic tests. The kinetic and equilibrium properties of Compound C2 are described.  相似文献   

7.
Titration of cyanide-incubated cytochrome c oxidase (ox heart cytochrome aa3) with ferrocytochrome c or with NNN'N'-tetramethyl-p-phenylenediamine initially introduces two reducing equivalents per mol of cytochrome aa3. The first equivalent reduces the cytochrome a haem iron; the second reducing equivalent is not associated with reduction of the 830 nm chromophores (e.p.r.-detectable copper) but is probably required for reduction of the e.p.r.-undetectable copper. Excess reductant introduces a third reducing equivalent into the cyanide complex of cytochrome aa3. During steady-state respiration in the presence of cytochrome c and ascorbate, the 830 nm chromophore is almost completely oxidized. It is reduced more slowly than cytochrome a on anaerobiosis. In the presence of formate or azide, some reduction at 830 nm can be seen in the steady state; in an oxygen-pulsed system, a decrease in steady-state reduction of cytochromes c and a is associated with ab increased reduction of the 830 nm species. In the formate-inhibited system the reduction of a3 on anaerobiosis shows a lag phase, the duration of which corresponds to the time taken for the 830 nm species to be reduced. It is concluded that the e.p.r.-undetectable copper (CuD) is reduced early in the reaction sequence, whereas the detectable copper (CUD) is reduced late. The latter species is probably that responsible for reduction of the cytochrome a3 haem. The magnetic association between undetectable copper and the a3 haem may not imply capability for electron transfer, which occurs more readily between cytochrome a3 and the 830 nm species.  相似文献   

8.
An a-type cytochrome was purified from Halobacterium halobium. The cytochrome showed an absorption spectrum similar to that of cytochrome aa3; it showed absorption peaks at 420 and 598 nm in the resting state, peaks at 441 and 602 nm in the reduced form, and its CO compound showed peaks at 430 and 600 nm. The cytochrome molecule was composed of only one kind of polypeptide with the molecular weight of 40,000. The cytochrome contained two heme a molecules in the molecule but no copper. The cytochrome did not show cytochrome c oxidase activity. Midpoint redox potential at pH 8.0 of the cytochrome was determined to be +0.31 V. The amino acid composition of the cytochrome resembled that of subunit I of mitochondrial cytochrome aa3. While two molecules of heme a were reduced with sodium dithionite, only one of two heme a molecules was reduced with ascorbate plus TMPD. The heme a reduced with ascorbate plus TMPD did not react with molecular oxygen or carbon monoxide, while one of two heme a molecules reduced with sodium dithionite was oxidized by molecular oxygen and combined with carbon monoxide.  相似文献   

9.
1. Purified mitochondria have been prepared from wild type Paramecium tetraurelia and from the mutant Cl1 which lacks cytochrome aa3. Both mitochondrial preparations are characterized by cyanide insensitivity. Their spectral properties and their redox potentials have been studied. 2. Difference spectra (dithionite reduced minus oxidized) of mitochondria from wild type P. tetraurelia at 77 K revealed the alpha peaks of b-type cytochrome (s) at 553 and 557 nm, of c-type cytochrome at 549 nm and a-type cytochrome at 608 nm. Two alpha peaks at 549 and 545 nm could be distinguished in the isolated cytochrome c at 77 K. After cytochrome c extraction from wild type mitochondria, a new peak at 551 nm was unmasked, probably belonging to cytochdrome c1. The a-type cytochrome was characterized by a split Soret band with maxima at 441 and 450 nm. The mitochondria of the mutant Cl1 in exponential phase of growth differed from the wild type mitochondria in that cytochrome aa3 was absent while twice the quantity of cytochrome b was present. In stationary phase, mitochondria of the mutant were characterized by a new absorption peak at 590 nm. 3. Cytochrome aa3 was present at a concentration of 0.3 nmol/mg protein in wild type mitochondria and ubiquinone at a concentration of 8 nmol/mg protein both in mitochondria of the wild type and the mutant Cl1. Cytochrome aa3 was more susceptible to heat than cytochromes b and c,c1.  相似文献   

10.
Compartmentation of ATP within renal proximal tubular cells   总被引:2,自引:0,他引:2  
Temperature-dependent spin changes of the heme iron atom on cytochrome P-450scc were studied by optical absorption and circular dichroism measurements. The optical absorption and circular dichroism spectra of cholesterol-free cytochrome P-450scc did not change between 10 and 26 degrees C. In contrast, the absorbance at 390 nm and the ellipticity at 330 nm of cholesterol-bound cytochrome P-450scc decreased upon temperature elevation, and the absorbance at 424 nm correspondingly increased. These spectral changes were reversible in respect of temperature. The far-ultraviolet circular dichroism spectra of both cholesterol-bound and -free cytochrome P-450scc were not affected by temperature. In addition, bound cholesterol molecule is not released from the cytochrome molecule by increasing temperature. From these results, we propose that temperature modulates specific interactions between the heme protein and bound cholesterol rather than the gross secondary structural changes of the protein.  相似文献   

11.
Singular value decomposition (SVD) was used to deconvolute the spectral changes occurring in the near infrared region during potentiometric titrations of cytochrome aa3. Overall oxidized minus reduced difference spectra revealed a broad absorbance feature centered near 830 nm with an apparent Em near 250 mV. However, SVD did not isolate any spectral species with an absorbance centered near 830 nm. It was found that the spectral changes occurring in the wavelength region from 650 to 950 nm were associated mainly with cytochromes a and a3. It was concluded that the absorbance at 830 nm should not be used as an independent measure of the concentration of CuA in cytochrome aa3.  相似文献   

12.
The visible and near infrared magnetic circular dichroism (MCD) spectra of equilibrium high-spin ferrous derivatives of myoglobin, hemoglobin, horseradish peroxidase and mitochondrial cytochrome c oxidase at 15 K are compared with those of the corresponding proteins in nonequilibrium conformations produced by low-temperature photodissociation of CO-complexes of these proteins as well as of O2-complexes of myoglobin and hemoglobin. Over all the spectral region (450-800 nm) the intensities of MCD bands of hemoproteins studied in equilibrium conformation are shown to be strongly temperature-dependent, including a negative band at ca. 630 nm and positive bands at ca. 690 nm and at ca. 760 nm. In contrast to the absorption spectra, the low-temperature MCD spectra of high-spin ferrous hemoproteins differ significantly, reflecting the peculiarities in the heme iron coordination sphere which are created by a protein conformation. The MCD spectra reveal clearly the structural changes in the heme environment which occur on ligand binding. On the basis of assignment of d leads to d and charge-transfer transitions in the near infrared region the correlation is suggested between the wavelength position of the MCD band at approx. 690 nm and the value of iron out-of-plane displacement as well as between the location of the band at approx. 760 nm and the Fe-N epsilon (proximal histidine) bond strength (length) in equilibrium and nonequilibrium conformations of the hemoproteins studied. The high sensitivity of low-temperature MCD spectra to geometry at heme iron is discussed.  相似文献   

13.
Cytochrome aa3 from Nitrosomonas europaea   总被引:3,自引:0,他引:3  
Cytochrome c oxidase has been purified from the ammonia oxidizing chemoautotroph Nitrosomonas europaea by ion-exchange chromatography in the presence of Triton X-100. The enzyme has absorption maxima at 420 and 592 nm in the resting state and at 444 and 598 nm in the dithionite-reduced form; optical extinction coefficient (598 nm minus 640 nm) = 21.9 cm-1 nM-1. The enzyme has approximately 11 nmol of heme a and approximately 11 nmol of copper per mg of protein (Lowry procedure). There appear to be three subunits (approximate molecular weights 50,800, 38,400, and 35,500), two heme groups (a and a3), and two copper atoms per minimal unit. The EPR spectra of the resting and partially reduced enzyme are remarkably similar to the corresponding spectra of the mitochondrial cytochrome aa3-type oxidase. Although the enzyme had been previously classified as "cytochrome a1" on the basis of its ferrous alpha absorption maximum (598 nm), its metal content and EPR spectral properties clearly show that it is better classified as a cytochrome aa3. Neither the data reported here nor a review of the literature supports the existence of cytochrome a1 as an entity discrete from cytochrome aa3. The purified enzyme is reduced rapidly by ferrous horse heart cytochrome c or cytochrome c-554 from N. europaea, but not with cytochrome c-552 from N. europaea. The identity of the natural electron donor is as yet unestablished. With horse heart cytochrome c as electron donor, the purified enzyme could account for a significant portion of the terminal oxidase activity in vivo.  相似文献   

14.
Heme-copper relationship of cytochrome oxidase in rat brain in situ   总被引:1,自引:0,他引:1  
The role of copper aa3 in relation to heme aa3 of cytochrome oxidase in electron transfer and oxygen utilization is poorly understood in vitro. In an attempt to study this in situ, we have simultaneously monitored the steady state redox changes of heme aa3 and copper aa3 in an isolated perfused rat head model (skull intact, muscle removed). By means of reflectance spectrophotometry the redox reactions of heme aa3 and copper aa3 were continuously monitored using 605-625 nm and 815-920 nm wavelength pairs, respectively. The reaction kinetics of these components in response to transient perfusion interruption in energized and de-energized preparations were then examined. We found that in response to perfusion interruption, soon after full reduction, the copper signal begins to change toward oxidation despite continuation of anoxic insult and progressive reduction of heme aa3. This phenomenon disappeared by pretreatment of the preparation with 2,4-dinitrophenol. A schematic sequence of electron transport in situ is proposed which emphasizes an active role for Cua in this sequence.  相似文献   

15.
In three fully crossed, three-way, two-by-two-by-four experiments, male weanling Long-Evans rats were fed a basal diet supplemented with vanadium (ammonium metavanadate)-at 0 and 1 μg/g, cystine at 3.0 and 8.5 mg/g, and iron (ferric sulfate) at 0 (Expts. 1 and 2) or 5 (Expt. 3), 15, 100, and 500 μg/g. After 6 wk, a relationship between vanadium and iron that was influenced by dietary cystine was found. The interaction among vanadium, iron, and cystine was demonstrated best by the hematocrit and hemoglobin findings, which were similar. In all Expts., hematocrits were depressed in rats fed the two lower levels of iron. In Expts. 2 and 3, vanadium deprivation exacerbated the depression of hematocrits in rats fed 15 μg iron and 3.0 mg cystine/g diet. In Expt. 1, the effect was similar, but less marked. On the other hand, in Expts. 1 and 3 when supplemental cystine was 8.5 mg/g, vanadium deprivation did not exacerbate, but tended to alleviate the depression of hematocrits in rats fed 15 μg iron/g diet. When dietary iron was 15 μg/g in Expt. 2, the exacerbation of the depression of hematocrits by vanadium deprivation was much less in rats fed 8.5 rather than 3.0 mg cystine/g diet. Dietary vanadium had little effect on depressed hematopoiesis in severely iron-deficient rats. The findings indicated that vanadium neither substitutes for iron at some metabolic site, nor stimulates iron absorption; but has a positive influence on the utilization of iron after absorption.  相似文献   

16.
In the presence of micromolar concentrations of H2O2, ferric cytochrome c oxidase forms a stable complex characterized by an increased absorption intensity at 606-607 nm with a weaker absorption band in the 560-580 nm region. Higher (millimolar) concentrations of H2O2 result in an enzyme exhibiting a Soret band at 427 nm and an alpha-band of increased intensity in the 589-610 nm region. Addition of H2O2 to ferric cytochrome c oxidase in the presence of cyanide results in absorbance increases at 444nm and 605nm. These changes are not seen if H2O2 is added to the cyanide complex of the ferric enzyme. The results support the idea that direct reaction of H2O2 with ferric cytochrome a 3 produces a 'peroxy' intermediate that is susceptible to further reduction by H2O2 at higher peroxide concentrations. Electron flow through cytochrome a is not involved, and the final product of the reaction is the so-called 'pulsed' or 'oxygenated' ferric form of the enzyme.  相似文献   

17.
1. Techniques and experiments are described concerned with the millisecond kinetics of EPT-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O2 or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy. 2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (less than 50 ms) approx. 0.5 electron equivalent per heme a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (less than 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a. 3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O2 in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O2 side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates. 4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms., whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO. 5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C.R., Hansen, R.E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477-2481). Both the low-spin (g=o; 2.2; 1.5) and slowly appearing high-spin (g=6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a3, when it is present in a state of interaction with EPR-undectectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.  相似文献   

18.
Temperature coefficients of both cat and toad brain have been calculated for the active metabolic state induced by electrical stimulation. Values are higher than most of the values previously reported for "rest" metabolism, whether calculated from Arrhenius plots or from linear graphs. Relative rates of oxidative metabolism were obtained by measuring the time course of the transient changes in NADH fluorescence and cytochrome aa3 absorption by reflectance techniques directly from the surface of the exposed cat cerebral cortex in vivo and from the isolated intact toad brain mounted in a cuvet. These findings demonstrate that such optical methods accurately record metabolic processes.  相似文献   

19.
Reflectance spectrophotometry through the skull was used to investigate carbon monoxide (CO) binding by tissue hemoproteins in the brains of barbiturate-anesthetized Sprague-Dawley rats. After splenectomy and extensive perfluorotributylamine exchange transfusion, steady-state spectral scans were obtained in Soret and visible wave-length regions during O2 ventilation, during subsequent exposure to O2-enriched gases containing 1, 3, or 5% CO, and finally after N2 anoxia. These CO exposures were well-tolerated and electroencephalograph (EEG) activity continued to be present. Initial difference spectra were influenced by CO binding to residual hemoglobin, but spectral evidence of CO-mediated b-type cytochrome reduction was obtained in the visible region as CO concentration was increased to 3 or 5%. This was associated with Soret spectra compatible with formation of the reduced cytochrome a3-CO complex. Reduction of cytochrome a at 605 nm and cytochrome c + c1 at 550 nm was absent. These findings may indicate respiratory chain branching through b cytochromes, either to a separate a3-like oxidase independent of the classical cytochrome aa3 or to an unidentified alternative CO-sensitive oxidase.  相似文献   

20.
The findings in the current studies with pure cytochrome aa3 confirm the findings in an accompanying paper pertaining to cytochrome aa3 in mitochondria (Reddy et al., 1985). In both cases, three Nernstian titrations are seen with Em values near 200, 260, 340 mV with n values of 2, 2, and 1. Similarly, the alpha absorption features of the difference spectra in both cases were centered near 602, 605, and 607 mn. The component with Em approximately 200 mV was identified as heme a3 on the basis of experiments conducted in an atmosphere of carbon monoxide, and in both cases, the carbon monoxide-liganded species did not display an elevated Em. In the current studies, unique Soret absorbance features are added to the difference spectra for the three Nernstian transitions. Specifically, absorption peaks at 429, 446, and 448 nm go with the alpha peaks seen respectively at 602, 605, and 607 nm. Evidence was presented to support the hypothesis that the redox state of heme alpha may control the redox potential of heme a3.  相似文献   

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