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The effect of low-level irradiation on the structural and functional organization of the cytochrome part of the respiratory chain in tumor carrier rats' liver is studied. The preliminary low-level irradiation leading to the mitochondrial cytochrome a, b and c content reduction at the latent stage of Guerin's carcinoma is shown. At the same time, the maximal reduction of the content of all liver cytochromes is observed at the terminal stages of oncogenesis. The content of cytochome c undergoes the most significant changes in the liver mitochondrial fracture. The possible mechanism of mitochondrial haem-containing cytochromes content reduction may be associated with the disorder of their formation caused by the heam synthesis inhibition found in our study. Simultaneously, the cytochrome oxydase (key enzyme of the cytochrome part) activity inhibition is observed to be caused by preliminary low-level irradiation at the latent growth stage of Guerin's carcinoma. The determined differences between irradiated and non-irradiated tumor carrier groups allow us to come to the conclusion that low-level irradiation has an impact only at the initial stages of the aftereffect. At the following stages, the state of the cytochrome part of the respiratory chain is defined by growth conditions of tumor.

One of the most puzzling aspects of the biological impact of polycyclic aromatic hydrocarbon compounds is that they elicit an apparently unrelated variety of toxic, teratogenic, and carcinogenic responses in exposed animals and in humans. At the cellular level, these environmental toxicants affect cell cycle regulatory mechanisms and signal transduction pathways in ways that are equally diverse and often contradictory. For example, depending on the particular cell lines studied, exposure to these compounds may lead to cell proliferation, to terminal differentiation, or to apoptosis. These effects are mediated by the aryl hydrocarbon receptor, a ligand-activated transcription factor well known for its regulatory activity on the expression of several phase I detoxification cytochrome P450 genes. Research into the molecular mechanisms of aryl hydrocarbon receptor function has uncovered a novel role for this protein during cell cycle progression. The activated receptor acts as an environmental sensor and cell cycle checkpoint that commits cells exposed to adverse environmental stimuli to arrest before the onset of DNA replication.

Light-absorption spectra and afferent chemoreceptor discharge were simultaneously recorded on superfused rat carotid bodies (CBs) under the influence of cytochrome a3-CuB ligands (O2, CN-, CO) in order to identify the primary mitochondrial cytochrome c oxidase (CCO) oxygen sensor. Spectra could be described on the basis of weighted light-absorption spectra of cytochrome b558 of the NAD(P)H oxidase and mitochondrial cytochromes b and c, CCO, cytochrome a3, and an unusual cytochrome a peaking at 592 nm. Discharge signals were deconvoluted into phasic and tonic activity for comparing different CB responses. The spectral weight of cytochrome a592 decreased significantly starting at high PO2 (100 mm Hg) and low sodium cyanide (CN-, 10 mM) accompanied by increasing phasic peak discharge. Combined CO-hypoxia or CO-CN- application inhibited photolysis of CO-stimulated chemoreceptor discharge, revealing photometrically cytochrome a592 as central in oxygen sensing. Control spectra in tissue from sympathetic and nodose ganglia did not show any cytochrome a592 contribution. According to these results, cytochrome a592 is assumed as a unique component of CB CCO, revealing in contrast to other cytochromes an apparent low PO2 and high CN- affinity, probably due to a shortcut of electron flow within CCO between CuA and cytochrome a3-CuB.

The genes encoding 'cytochrome a1'-like hemoprotein of Magnetospirillum magnetotacticum were identified and sequenced. Three ORFs, mcalI, mcaI and hosA, were included in the sequenced region. The six histidine residues which were predicted to associate with the prosthetic cofactors of heme-copper oxidase superfamily were conserved in the hemoprotein. However, none of the amino acid residues which were proposed to participate in the oxygen-reducing and the coupled proton pumping reactions in cytochrome c oxidase were at all conserved in the hemoprotein.

The electron-transferring flavoprotein (ETF) has been detected in two large soluble-protein complexes partially purified from sonicated porcine liver mitochondria. Size-exclusion chromatography and sucrose-density ultracentrifugation suggested molecular masses in the region of 390 to 420 kDa for the two complexes. Activities of medium-chain acyl-CoA dehydrogenase, sarcosine dehydrogenase and ETF:ubiquinone oxidoreductase were also detected. No evidence of oxidative-phosphorylation properties was obtained. Treatment with antimycin A inhibited the activity of both complexes. Pyridine haemochromogens, prepared from the partially purified species, show the presence of cytochrome proteins. The possible composition of these complexes and their relationship to the electron transport chain are discussed.

Objective: To simultaneously determine the effect of propofol on myocardial oxygenation, mitochondrial function, and whole organ function in an isolated heart model, using optical reflectance spectroscopy., Design: Controlled laboratory investigation., Setting: Research laboratory., Subjects: Twenty adult guinea pigs., Interventions: Isolated hearts were perfused alternately with a modified oxygenated Krebs-Henseleit buffer and with buffer containing varied concentrations of propofol. Ninety seconds of ischemia were produced during perfusion with each solution studied., Measurements and Main Results: Myoglobin oxygen saturation, cytochrome c and cytochrome a/a3 redox state, and ventricular pressure were continuously measured from isolated guinea pig hearts during a 2-hr period. Myoglobin oxygen saturation increased and both cytochromes became more oxidized in the presence of propofol. During ischemia, myoglobin desaturation and cytochrome reduction were delayed and less complete in the presence of propofol. The mean ischemic time to 50% myoglobin desaturation was, on average, 14.3 secs with buffer perfusion, and increased to 24.5, 27.9, and 41.8 secs, with 50, 100, and 200 microM propofol perfusion, respectively. Ventricular function decreased linearly with increasing propofol concentration. From baseline buffer perfusion, maximal dP/dt per cardiac cycle decreased on average by 30.4%, 40.9%, and 69.4%, with 50, 100, and 200 microM propofol perfusion, respectively., Conclusions: Propofol impairs either oxygen utilization or inhibits electron flow along the mitochondrial electron transport chain in the guinea pig cardiomyocyte. Propofol also significantly decreases ventricular performance in the isolated perfused heart. These effects are linearly correlated with propofol concentration in the range studied.

The electronic absorption spectrum of solubilized beef heart cytochrome c oxidase was analyzed in the 400-500 nm region to identify the origin of doublet features appearing in the second derivative spectrum associated with ferrocytochrome a. This doublet, centered near 22,600 cm(-1), was observed in the direct absorption spectrum of the a(2+)a(3)(3+).HCOO(-) form of the enzyme at cryogenic temperatures. Since evidence for this doublet at room temperature is obtained only on the basis of the second derivative spectrum, a novel mathematical approach was developed to analyze the resolving power of second derivative spectroscopy as a function of parameterization of spectral data. Within the mathematical limits defined for resolving spectral features, it was demonstrated that the integrated intensity of the doublet feature near 450 nm associated with ferrocytochrome a is independent of the ligand and oxidation state of cytochrome a(3). Furthermore, the doublet features, also observed in cytochrome c oxidase from Paracoccus denitrificans, were similarly associated with the heme A component and were correspondingly independent of the ligand and oxidation state of the heme A(3) chromophore. The doublet features are attributed to lifting of the degeneracy of the x and y polarized components of the B state of the heme A chromophore associated with the Soret transition.

The iron-oxidizing activity of a moderately thermophilic iron-oxidizing bacterium, strain TI-1, was located in the plasma membrane. When the strain was grown in Fe2+ (60 mM)-salts medium containing yeast extract (0.03%), the plasma membrane had iron-oxidizing activity of 0.129 mumol O2 uptake/mg/min. Iron oxidase was solubilized from the plasma membrane with 1.0% n-octyl-beta-D-glucopyranoside (OGL) containing 25% (v/v) glycerol (pH 3.0) and purified 37-fold by a SP Sepharose FF column chromatography. Iron oxidase solubilized from the plasma membrane was stable at pH 3.0, but quite unstable in the buffer with the pH above 6.0 or below 1.0. The optimum pH and temperature for iron oxidation were 3.0 and 55 degrees C, respectively. Solubilized enzyme from the membrane showed absorption peaks characteristic of cytochromes a and b. Cyanide and azide, inhibitors of cytochrome c oxidase, completely inhibited iron-oxidizing activity at 100 microM, but antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and myxothiazol, inhibitors of electron transport systems involved with cytochrome b, did not inhibit enzyme activity at 10 microM. The absorption spectrum of the most active enzyme fraction from SP Sepharose FF column chromatography (4.76 mumol O2 uptake/mg/min) compared with lower active fractions from the chromatography (0.009 and 2.10 mumol O2 uptake/mg/min) showed a large alpha-peak of cytochrome a at 602 nm and a smaller alpha-peak of cytochrome b at 560 nm. The absorption spectrum of pyridine ferrohemochrome prepared from the most highly purified enzyme showed an alpha-peak characteristic of heme a at 587 nm, but not the alpha-peak characteristic of heme c at 550 nm. The cytochrome a, but not cytochrome b, in the most highly purified enzyme fraction was reduced by the addition of ferrous iron at pH 3.0, indicating that electrons from Fe2+ were transported to cytochrome a, but not cytochrome b. These results strongly suggest that cytochrome a, but not cytochromes b and c, is involved in iron oxidation of strain TI-1.

Nitric oxide reductase (NOR) is a key enzyme in denitrification, reforming the N-N bond (making N2O from two NO molecules) in the nitrogen cycle. It is a cytochrome bc complex which has apparently only two subunits, NorB and NorC. It contains two low-spin cytochromes (c and b), and a high-spin cytochrome b which forms a binuclear center with a non-heme iron. NorC contains the c-type heme and NorB can be predicted to bind the other metal centers. NorB is homologous to the major subunit of the heme/copper cytochrome oxidases, and NOR thus belongs to the superfamily, although it has an Fe/Fe active site rather than an Fe/Cu binuclear center and a different catalytic activity. Current evidence suggests that NOR is not a proton pump, and that the protons consumed in NO reduction are not taken from the cytoplasmic side of the membrane. Therefore, the comparison between structural and functional properties of NOR and cytochrome c- and quinol-oxidizing enzymes which function as proton pumps may help us to understand the mechanism of the latter. This review is a brief summary of the current knowledge on molecular biology, structure, and bioenergetics of NOR as a member of the oxidase superfamily.

Background: The Bcl-2 family of proteins plays a key role in the regulation of apoptosis. Some family members prevent apoptosis induced by a variety of stimuli, whereas others promote apoptosis. Competitive dimerisation between family members is thought to regulate their function. Homologous domains within individual proteins are necessary for interactions with other family members and for activity, although the specific mechanisms might differ between the pro-apoptotic and anti-apoptotic proteins., Results: Using a cell-free system based on extracts of Xenopus eggs, we have investigated the role of the Bcl-2 homology domain 3 (BH3) from different members of the Bcl-2 family. BH3 domains from the pro-apoptotic proteins Bax and Bak, but not the BH3 domain of the anti-apoptotic protein Bcl-2, induced apoptosis in this system, as determined by the rapid activation of specific apoptotic proteases (caspases) and by DNA fragmentation. The apoptosis-inducing activity of the BH3 domains requires both membrane and cytosolic fractions of cytoplasm, involves the release of cytochrome c from mitochondria and is antagonistic to Bcl-2 function. Short peptides, corresponding to the minimal sequence of BH3 domains required to bind anti-apoptotic Bcl-2 family proteins, also trigger apoptosis in this system., Conclusions: The BH3 domains of pro-apoptotic proteins are sufficient to trigger cytochrome c release, caspase activation and apoptosis. These results support a model in which pro-apoptotic proteins, such as Bax and Bak, bind to Bcl-2 via their BH3 domains, inactivating the normal ability of Bcl-2 to suppress apoptosis. The ability of synthetic peptides to reproduce the effect of pro-apoptotic BH3 domains suggests that such peptides may provide the basis for engineering reagents to control the initiation of apoptosis.

The characteristics of mitochondria of yeast cells expressing the pro-apoptotic gene Bax or coexpressing Bax and the anti-apoptotic gene Bcl-xL have been investigated in whole cells, isolated mitochondria and permeabilized spheroplasts. It is found that Bax-induced growth arrest of yeast cells is related to two defects in the respiratory chain: (i) a decrease in the amount of cytochrome c oxidase, the terminal enzyme of the respiratory chain, and (ii) a dramatic increase in the release of cytochrome c to the cytosol. Other components of the inner mitochondrial membrane (bc1 complex and F0F1-ATPase) are unaffected. Coexpression of Bcl-xL almost fully prevented the effect of Bax. Surprisingly, these results obtained in yeast parallel similar observations reported in mammalian cells.

Objective: Mitochondrial cytochrome a,a3 redox shifts can be determined by near-infrared wavelength reflection. Since near-infrared wavelengths penetrate skin and bone, a potential exists to noninvasively measure mitochondrial oxidation using this phenomenon. The purpose of this study was to compare conventional parameters of resuscitation with regional measurements of spectroscopically derived cytochrome redox state in a hemorrhagic shock model., Design: Prospective, controlled laboratory investigation., Setting: Animal research laboratory of a university medical center., Subjects: New Zealand white rabbits (n = 23), weighing 2 to 3 kg., Interventions: After anesthesia and instrumentation, the subjects underwent laparotomy with placement of near-infrared spectroscopy probes on the stomach, liver, kidney, and hamstring muscle. Baseline measurements were obtained, and phlebotomy was used to reduce cardiac output by 60% for 30 mins. Animals were resuscitated with shed autologous blood and crystalloid to reach baseline cardiac output (0.9%), and were monitored for an additional 60 mins., Measurements and Main Results: Significant correlations between mitochondrial cytochrome a,a3 redox state, cardiac output, and oxygen delivery were observed throughout shock and resuscitation. However, gastric cytochrome oxidation did not recover after shock, despite systemic evidence of adequate resuscitation (p < .05)., Conclusions: Resuscitation from severe hemorrhagic shock may not uniformly restore cellular oxygenation, despite normalization of traditional parameters of resuscitation. Direct monitoring of cytochrome oxidation may be useful in identifying regional areas of dysoxia.

The effects of chronic, around the clock, low-frequency electrostimulation on the respiratory chain activity and cytochrome content of freshly isolated mitochondria were evaluated in rabbit skeletal muscle before and after 30 days of continuous or cyclical electrostimulation using a totally implantable system and a training programme now used in humans. The respiratory activity measured in state III increased strongly after electrostimulation. The efficiency of the respiratory chain increased significantly after electrostimulation but the activity of complex [(reduced nicotinamide adenine dinucleotide dehydrogenase) did not increase. The amount of cytochromes a and a3, b562, and c and c1 increased clearly after electrostimulation. The respiratory activity rate of mitochondria obtained after continuous electrostimulation was apparently higher than after cyclical electrostimulation. Chronic uninterrupted low-frequency electrostimulation, using a clinical training programme, induces an increase in mitochondrial respiratory chain activity in purified mitochondria of skeletal muscle. These changes are the basis of induced resistance to fatigue in fast-to-slow muscle conversion by chronic electrostimulation.

A microbial cyanide sensor was prepared, consisting of immobilized Saccharomyces cerevisiae and an oxygen electrode. When the electrode was inserted into a solution containing glucose, the respiration activity of the microorganisms increased. The change in the respiration activity is monitored with the oxygen electrode. When cyanide is added to the sample solution, the electron transport chain reaction of the respiration system in the mitochondria is inhibited, resulting in a decrease in respiration. The inhibition is caused by cyanide binding with respiration enzymes such as the cytochrome oxidase complex in the mitochondrial inner membrane. Therefore, the cyanide concentration can be measured from the change in the respiration rate. When the sensor was applied to a batch system at pH 8.0 and 30 degrees C, the cyanide calibration curve showed linearity in the concentration range between 0.3 microM and 150 microM CN-.

The heme axial ligands of bd-type ubiquinol oxidase of Escherichia coli were studied by EPR and optical spectroscopies using nitric oxide (NO) as a monitoring probe. We found that NO bound to ferrous heme d of the air-oxidized and fully reduced enzymes with very high affinity and to ferrous heme b595 of the fully reduced enzyme with low affinity. EPR spectrum of the 14NO complex of the reduced enzyme exhibited an axially symmetric signal with g-values at g = 2.041 and g = 1.993 and a clear triplet of triplet (or a triplet of doublet for the 15NO complex) superhyperfine structure originating from a nitrogenous proximal ligand trans to NO was observed. This EPR species was assigned to the ferrous heme d-NO complex. This suggests that the proximal axial ligand of heme d is a histidine residue in an anomalous condition or other nitrogenous amino acid residue. Furthermore, the EPR line shape of the ferrous heme d-NO was slightly influenced by the oxidation state of the heme b595. This indicates that heme d exists in close proximity to heme b595 forming a binuclear center. Another axially symmetric EPR signal with g-values at g(parallel) = 2.108 and g(perpendicular) = 2.020 appeared after prolonged incubation of the reduced enzyme with NO and was attributed to the ferrous heme b595-NO complex.

Cytochrome bd oxidase is a bacterial terminal oxidase that contains three cofactors: a low-spin heme (b558), a high-spin heme (b595), and a chlorin d. The center of dioxygen reduction has been proposed to be a binuclear b595/d site, whereas b558 is mainly involved in transferring electrons from ubiquinol to the oxidase. Information on the nature of the axial ligands of the three heme centers has come from site-directed mutagenesis and spectroscopy, which have implicated a His/Met coordination for b558 (Spinner, F., Cheesman, M. R., Thomson, A. J., Kaysser, T., Gennis, R. B., Peng, Q., & Peterson, J. (1995) Biochem. J. 308, 641-644; Kaysser, T. M., Ghaim, J. B., Georgiou, C., & Gennis, R. B. (1995) Biochemistry 34, 13491-13501), but the ligands to b595 and d are not known with certainty. In this work, the three heme chromophores of the fully reduced cytochrome bd oxidase are studied individually by selective enhancement of their resonance Raman (rR) spectra at particular excitation wavelengths. The rR spectrum obtained with 413.1-nm excitation is dominated by the bands of the 5cHS b595(2+) cofactor. Excitation close to 560 nm yields a rR spectrum dominated by the 6cLS b558(2+) heme. Wavelengths between these values enhance contributions from both b595(2+) and b558(2+) chromophores. The rR bands of the ferrous chlorin become the major features with red laser excitation (595-650 nm). The rR data indicate that d2+ is a 5cHS system whose axial ligand is either a weakly coordinating protein donor or a water molecule. In the low-frequency region of the 441.6-nm spectrum, we assign a rR band at 225 cm-1 to the (b595)Fe(II)-N(His) stretching vibration, based on its 1.2-cm(-1) upshift in the 54Fe-labeled enzyme. This observation provides the first physical evidence that the proximal ligand of b595 is a histidine. Site-directed mutagenesis had suggested that His 19 is associated with either b595 or d (Fang, H., Lin, R. -J., & Gennis, R. B. (1989) J. Biol. Chem. 264, 8026-8032). On the basis of the present study, we propose that the proximal ligand of b595 is His 19. We have also studied the reaction of cyanide with the fully reduced cytochrome bd oxidase. In approximately 700-fold excess cyanide (approximately 35 mM), the 629-nm UV/vis band of d2+ is blue-shifted to 625 nm and diminished in intensity. However, the rR spectra at each of three different gamma(0) (413.1, 514.5, and 647.1 nm) are identical with or without cyanide, thus indicating that both b595 and d remain as 5cHS species in the presence of CN-. This observation leads to the proposal that a native ligand of ferrous chlorin d is replaced by CN- to form the 5cHS d2+ cyano adduct. These findings corroborate our companion study of the "as-isolated" enzyme in which we proposed a 5cHS d3+ cyano adduct (Sun, J., Osborne, J. P., Kahlow, M. A., Kaysser, T. M., Hill, J. J., Gennis, R. B., & Loehr, T. M. (1995) Biochemistry 34, 12144-12151). To further characterize the unusual and unexpected nature of these proposed high-spin cyanide adducts, we have obtained EPR spectral evidence that binding of cyanide to fully oxidized cytochrome bd oxidase perturbs a spin-state equilibrium in the chlorin d3+ to yield entirely the high-spin form of the cofactor.

The terminal segment of the aerobic respiratory chain of the thermoacidophilic archaeon Sulfolobus sp. strain 7 is an unusual caldariellaquinol oxidase supercomplex, which contains at least one b-type and three spectroscopically distinguishable a-type cytochromes, one copper, and a Rieske-type FeS center. In this paper, we report the purification and characterization of two different forms of the archaeal a-type cytochromes, namely, a three-subunit cytochrome a583-aa3 subcomplex and a single-subunit cytochrome aa3 derived from the cytochrome subcomplex, in order to facilitate further studies on the terminal oxidase segment of Sulfolobus. The optical and EPR spectroscopic analyses suggest the presence of two different low-spin heme centers and one high-spin heme center in the purified cytochrome a583-aa3 subcomplex, and one low-spin and one high-spin hemes in cytochrome aa3, respectively. The Rieske-type FeS center detected in the purified cytochrome supercomplex was absent in two forms of the a-type cytochrome oxidase, indicating its association with cytochrome b562. The crystal field parameters of the lowspin heme a583 center indicate that its axial ligands may be similar to those of cytochromes c, rather than conventional bis-histidine ligation. In spite of the absence of any c-type cytochrome, a ferrocytochrome c oxidase activity was detected in the archaeal purified cytochrome a583-aa3 subcomplex with no quinol oxidase activity, but not in the purified cytochrome oxidase supercomplex, which has been tentatively interpreted as a representative of electron transfer from the Rieske FeS center to cytochrome a583 in vivo. Thus, our results indicate the following scheme for the intramolecular electron transfer of the terminal oxidase supercomplex from Sulfolobus sp. strain 7: [caldariellaquinol-->] b562-->Rieske FeS center-->a583 aa3-->molecular oxygen.

The aerobic respiratory system of the thermoacidophilic archaeon, Sulfolobus sp. strain 7, is unusual in that it consists of only a- and b-type cytochromes but no c-type cytochromes. In previous studies, a novel cytochrome oxidase a583-aa3 subcomplex has been purified, which showed a ferrocytochrome c oxidase but no caldariellaquinol oxidase activity (Wakagi, T., Yamauchi, T., Oshima, T., Müller, M., Azzi, A., and Sone, N. (1989) Biochem. Biophys. Res. Commun. 165, 1110-1114). We show here that the cytochrome subcomplex could be copurified with a non-CO-reactive cytochrome b562 as a novel terminal oxidase "supercomplex," which also contained a Rieske-type FeS cluster at gy = 1.89. It contained one copper and all four heme centers detectable in the archaeal membranes by the low temperature spectrophotometry and the potentiometric titration: cytochromes b562 (+146 mV), a583 (+270 mV), and aa3 (+117 and +325 mV). The presence of one copper atom indicates that it contains the conventional heme a3-CuB binuclear center for reducing molecular oxygen. In conjunction with the presence of a Rieske-type FeS center, inhibitor studies suggest that the terminal oxidase segment of the respiratory chain of Sulfolobus sp. strain 7 is a functional fusion of respiratory complexes III and IV, where cytochrome b562 and the Rieske-type FeS center probably play a central role in the oxidation of caldariellaquinol. This archaeal terminal oxidase supercomplex reconstitutes the in vitro succinate oxidase respiratory chain for the first time together with caldariellaquinone and the purified cognate succinate:caldariellaquinone oxidoreductase complex. The reconstitution system requires caldariellaquinone for the activity, and is highly sensitive to cyanide and 2-heptyl-4-hydroxy-quinoline-N-oxide. These results are also discussed in terms of the evolutionary considerations.

Wild-type and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides were characterized by EPR spectroscopy. A pH-induced g12 signal, seen previously in mammalian cytochrome oxidase and assigned to the presence of a bridging carboxyl ligand in the bimetallic cytochrome a3-CuB site, is found also in the bacterial enzyme. Mutation of glutamate-286 to glutamine inactivates the enzyme but does not affect this signal, demonstrating that the carboxyl group of this residue is not the bridging ligand. Three mutants, M106Q, located one helix turn below a histidine ligand to cytochrome a, and T352A as well as F391Q, located close to the bimetallic center, are shown to affect dramatically the low-spin heme signal of cytochrome a. These mutants are essentially inactive, suggesting that these three mutations result in alterations to cytochrome a that render the oxidase non-functional.

Biochemical, physiological and histological data have established that 55-65% of retinal mitochondria are located in the photoreceptor inner segments and suggested that photoreceptors have at least a two-fold greater oxygen consumption (QO2) than the remaining inner retina. QO2 in isolated whole rat retina (QWR), outer retina (QOR) and inner retina (QIR) was measured during dark and rod-saturating light adaptation. The effects of function-specific chemical agents on QWR, QOR and QIR during dark and light adaptation were determined. In addition, the oxidation-reduction (redox) potential of cytochrome a3 of whole, outer and inner retina was measured during dark and light adaptation. During dark adaptation, the mean QWR was 1.62 mumol O2 (mg dry wt)-1 hr-1 and whole retinal level of reduced cytochrome a3 was 19%. They decreased by 24% and 37% during light adaptation, respectively. To determine QOR and QIR during dark and light adaptation, the outer retina was pharmacologically-isolated from inner retina using L-2-amino-4-phosphonobutyric acid plus kynurenic acid (APB/Kyn). Experiments in the presence or absence of APB/Kyn revealed that: (i) QOR, but not QIR, of the dark-adapted retina was decreased 37% during light adaptation, (ii) the outer and inner retina consumed 65% and 35% of the QWR during dark adaptation, respectively, and 54% and 46% of the QWR during light adaptation, respectively, (iii) the level of reduced retinal cytochrome a3 in the outer, but not inner, retina was decreased 34% during light adaptation, (iv) during light adaptation, the rate of QO2 was equal in the outer and inner retina, and (v) the effects of APB/Kyn were reversible. These results establish that the mean rate of QIR and retinal cytochrome a3 are unchanged during dark or light adaptation. In addition, they suggest that QOR:QIR in the rat may be modeled using a 65%:35% model during DA and a 55%:45% model during LA. All the function-specific agents--IBMX, lead, diltiazem, ouabain, CO2+ plus Mg2+ and verapamil--significantly decreased QWR during dark and light adaptation. A more detailed analysis revealed that IBMX and lead each selectively reduced (> or = 90%) QOR during dark adaptation whereas CO2+ plus Mg2+ and verapamil each selectively reduced (> or = 93%) QIR during dark and light adaptation. These results are consistent with the known pharmacological sites and mechanisms of these agents. Additional experiments determined that the IBMX- and lead-induced inhibition of QOR during dark adaptation resulted, either wholly or partially, from the influx of extracellular Ca2+. During dark adaptation in Ca(2+)-free medium: (i) QWR and QOR increased while QIR was unchanged, (ii) QOR was not decreased in the presence of IBMX and (iii) QOR was only partially decreased in the presence of lead.(ABSTRACT TRUNCATED AT 400 WORDS)

The electron-transfer kinetics of cytochrome c oxidase were probed by measuring the reduction levels of bound cytochrome c, cytochrome a, and cytochrome a3 during steady-state turnover. Our experimental approach was to measure these reduction levels as a function of (1) the rate of electron input into tightly bound cytochrome c by varying the concentration of TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) and/or cytochrome c and (2) the rate of electron efflux out of cytochrome a (true Kcat) by changing the detergent surrounding cytochrome c oxidase. In most detergent environments, the rate of electron input into cytochrome c is not faster than the rate of electron efflux from cytochrome a. The relatively slow rate of electron input results in incomplete reduction of both cytochrome a and cytochrome c bound a the high-affinity site unless Kcat is very slow. When the high-affinity site is saturated with cytochrome c, the steady-state reduction level of cytochrome a defines Vmax,1, which is the maximum velocity of the high-affinity phase. The remaining fractional oxidation level of cytochrome a determines Vmax,2, the maximum velocity of the low-affinity phase. Therefore, it is the sum Vmax,1 + Vmax,2 which defines the maximum rate of electron transfer between cytochrome a and the bimetallic center, i.e., Kcat. We also were able to evaluate the true Kcat of cytochrome c oxidase in each detergent environment directly from the steady-state reduction levels without any of the complications introduced by the analysis of the polarographic kinetic data.(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the reaction of oxidized "pulsed" cytochrome c oxidase with reduced cytochrome c and with ruthenium(II) hexaammine using stopped-flow mixing. The rate of reduction of Fea3 (the oxygen-binding heme) is not a linear function of the population of reduced Fea (the low-spin heme), as would be expected if electron transfer between these sites is rate-limiting. Instead, the rate can be increased significantly by increasing the driving force of the reductant (lowering of Eh) even after Fea is almost completely reduced. Reduction of Fea3 becomes slower as the pH is raised, and consumption of protons can be seen simultaneously with electron entry into Fea3. Both the reduction of Fea3 and the proton uptake are biphasic. To explain these findings, we propose a model in which (1) intramolecular heme-heme electron transfer is fast, and has an essentially constant rate; (2) when reduction begins, the midpoint potentials of Fea3 and CuB are initially low, and only a small fraction of these centers become reduced; and (3) this reduced population is then stabilized by the uptake of protons. Thus, net reduction of Fea3 and CuB is controlled by the amount of the low-potential population which becomes reduced together with the rate of proton uptake by this reduced low-potential species. Important consequences of this mechanism for the function of the enzyme and for the respiratory chain as a whole are discussed.

Apparent oxygen affinities for Escherichia coli cells and membranes containing a terminal oxidase with only one type of ligand-binding heme, cytochrome o', were measured with oxyleghemoglobin and oxymyoglobin as sensitive oxygen reporters. Two Km values (0.15 to 0.35 microM and 0.016 to 0.085 microM) were detected, well below values determined for the purified oxidase by insensitive electrode methods.

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

Three preparations of cytochrome c oxidase, the preparation as traditionally prepared in our laboratory as described by Van Buuren (1992; PhD Thesis, University of Amsterdam), a preparation according to Volpe and Caughey (Biochem. Biophys. Res. Commun. 61 (1974) 502-509) and a preparation of 'fast' cytochrome c oxidase (Brandt, U., Schägger, H. and Von Jagow, G. (1989) Eur. J. Biochem. 182, 705-711), are compared in their reaction with cyanide and carbon monoxide. The reaction with cyanide is nearly as fast for the Van Buuren preparation as for the 'fast' preparation, but much slower for the Volpe-Caughey preparation. Mixed-valence cytochrome c oxidase (cytochrome a3 and CuB reduced with carbon monoxide bound and cytochrome a and CuA oxidized) is prepared by anaerobic incubation with carbon monoxide. With the Van Buuren preparation complete formation of the species takes 4 h, whereas with the Volpe-Caughey preparation it takes 20 h. Longer incubation under CO results in partial reduction of cytochrome a and CuA. With the 'fast' preparation mixed-valence cytochrome c oxidase is formed after more than one day of incubation with CO, but it is stable for at least 3 days. The presence of oxidized cytochrome c did enhance the reactivity towards cyanide and towards carbon monoxide in cytochrome c oxidase of all three preparations. Furthermore, optical and EPR spectra of the preparations of cytochrome c oxidase are compared. The Volpe-Caughey preparation has an intense g' = 12 EPR-signal, the Van Buuren preparation has hardly any g' = 12 signal and the 'fast' preparation has no g' = 12 signal. In the 'fast' preparation the low-spin heme signal is shifted (from g = 3.00 to g = 2.97). The absorbance spectra of the three preparations in the Soret region are similar with a maximum at 424 nm. Only the 'fast' preparation as isolated was completely oxidized, whereas the other preparations were partially reduced. It was concluded that differences in the reaction of cytochrome c oxidase with ligands are determined by the internal or external ligand bound to the cytochrome a3-CuB couple.

Single-turnover studies of the reaction of reduced cytochrome oxidase with oxygen has led to a mechanistic model that specifies a linear sequence of electron transfer from cytochrome c to O2 via the four redox active metals of mitochondrial cytochrome oxidase. Fully reduced oxidase initially forms a dioxygen adduct within which two electrons are transferred at a rate of 6 x 10(4) s-1 to form a peroxy adduct. This two-electron step results in single-electron oxidation of cytochrome alpha 3 and cytochrome alpha. Cytochrome alpha is then re-reduced by CuA in an intramolecular reaction. Subsequent reoxidation of cytochrome alpha occurs at a rate nearly 100-fold slower than its initial oxidation. Oxidation of CuB in this sequence has been the most difficult to determine and here is specified as the third electron transfer step. This reaction sequence is unchanged in the presence of tightly bound cytochrome c, although cytochrome c is rapidly oxidized via CuA and cytochrome alpha. The linear model advanced here has CuA as the acceptor of electrons from cytochrome c, with cytochrome alpha serving as a bridge to deliver electrons from CuA (and cytochrome c) to the binuclear center, cytochrome alpha 3-CuB.

A CO-reactive hemoprotein was purified from the mitochondrial membrane fraction of Tetrahymena pyriformis. It showed absorption peaks at 615 and 455 nm in the reduced form and an alpha peak at 565 nm in the pyridine ferrohemochrome spectrum. Although the spectral properties were apparently similar to those of 'cytochrome a620' which was previously proposed as a mitochondrial terminal oxidase in T. pyriformis, it did not contain any molecules of heme a or copper atoms. Further, it showed neither cytochrome c oxidase nor cytochrome c peroxidase activity. These results suggest that 'cytochrome a620' may not be the terminal oxidase in the mitochondrial respiratory chain of T. pyriformis.

The relationships among mitochondrial O2 availability, O2 delivery, and lactate formation in exercising skeletal muscle remain unclear. Some data suggest that muscle O2 provision is sufficient at maximal O2 consumption (VO2max) to challenge the concept of a mitochondrial O2 limitation at VO2max. The relationships among VO2, mitochondrial O2 availability, and net lactate production were studied over a wide range of exercise intensities. Using near-infrared spectroscopy, the oxidation-reduction state of cytochrome a,a3 was monitored in the canine gracilis in vivo. Twenty adult dogs were anesthetized with alpha-chloralose, intubated, and mechanically ventilated on room air. Five-minute stimulation periods at rates of 2, 3, 4, 5, 7, 8, 10, or 12 stimuli/s were performed. VO2max generally was achieved at a stimulation rate of 8 stimuli/s; mean VO2max was 0.12 +/- 0.09 (SE) ml.min-1 x g-1. The concentration of oxidized mitochondrial cytochrome a,a3 decreased at all work loads relative to resting state and demonstrated a near-linear relationship with muscle VO2 (r2 = 0.99). Muscle lactate efflux and the lactate-pyruvate ratio also were correlated positively with cytochrome a,a3 reduction, suggesting a common regulatory mechanism coupling the processes of aerobic glycolysis and oxidative phosphorylation. At VO2max, the corresponding cytochrome oxidation was not significantly different from that observed at death. Thus, in the gracilis maximal exercise leads to near-complete reduction of cytochrome a,a3 secondary to deficient O2 provision. We conclude that VO2max is limited primarily by O2 delivery to this muscle and not by other factors limiting mitochondrial ATP production or substrate oxidation.