The second derivative electronic absorption spectrum of cytochrome c oxidase in the Soret region.
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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. (+info)
Involvement of cytochrome a in iron oxidation of a moderately thermophilic iron-oxidizing bacterium, strain TI-1.
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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. (+info)
Preliminary evidence for the existence of specific functional assemblies between enzymes of the beta-oxidation pathway and the respiratory chain.
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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. (+info)
Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: A di-heme active site?
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Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecond multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b(595) was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the alpha-band of heme b(595). The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d. In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b(595) within a few ps, pointing to a direct interaction between hemes b(595) and d. Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b(595) initially oxidized, a significant part of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps. This caging effect may indicate that ferrous heme b(595) provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein. Taken together, the data indicate physical proximity of the hemes d and b(595) and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd. (+info)
Unusual cytochrome a592 with low PO2 affinity correlates as putative oxygen sensor with rat carotid body chemoreceptor discharge.
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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. (+info)
The oxygen dependence of the mitochondrial respiration rate in ascites tumor cells.
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The effect of the oxygen concentration on the rate of oxygen consumption by 786 and TA3 ascites tumor cell lines has been determined under steady-flow conditions with a membraneless fast-responding O2 electrode and using ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine as electron donors. The reaction was initiated by rapid injection of O2 into anaerobically incubated test system. The time-dependence of the intact cell respiration showed three distinct phases; an early very fast but short duration phase, a subsequent slow phase that prevailed for most of the reaction period and a third phase which preceded the reestablishment of anaerobiosis. Kinetic analysis of the reaction indicated a linkage between the catalytic efficiency and the transmembrane electrochemical potential. The rates of O2 uptake, obtained in the presence of both protonophores and ionophores, were monotonic and pseudo-first order over 90% of the course of O2 consumption. Extrapolation of the observed rates to zero time, at which zero delta mu H+ and thus constant flow prevails, was used to calculate the oxygen concentration for the half-maximal respiratory rate, which was found to be in the range 1.55-2.10 microM O2. No noticeable variation in the value of this kinetic parameter was found between the two cell lines used. Possible reasons for discrepancies in published reports on the oxygen dependence of the cytochrome c oxidase activity in various mitochondrial and reconstituted systems are discussed. (+info)
Homology in the structure and the prosthetic groups between two different terminal ubiquinol oxidases, cytochrome a1 and cytochrome o, of Acetobacter aceti.
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Acetobacter aceti produces two different terminal oxidases dependent on the culture conditions, shaking and static cultures. Cells grown on shaking culture contain cytochrome a1, while cytochrome o is present in cells grown on static culture. Cytochrome a1 and cytochrome o of A. aceti were compared especially with respect to the protein structure and the prosthetic groups. Cytochrome a1 exhibited lower CN sensitivity and higher affinity for O2 than cytochrome o. Both terminal oxidases consisted of four nonidentical polypeptides of which the molecular sizes were identical between both enzymes. Cytochrome a1 cross-reacted with an antibody raised against cytochrome o at the same level as cytochrome o did, and an antibody elicited against cytochrome a1 cross-reacted with both cytochrome o and cytochrome a1 at the same intensity, which indicates that both oxidases are indistinguishable immunochemically. Furthermore, almost the same peptide mapping pattern with chymotrypsin was observed in subunit I and in subunit II between both terminal oxidases, and the amino-terminal sequences in the subunit II of both oxidases were identical at least in their 10 amino acids. As for the prosthetic groups, both oxidases were shown to contain two heme-irons and one copper atom. Further, high performance liquid chromatography analysis of the heme moieties extracted from both the purified enzymes indicated that cytochrome a1 contains hemes b and a at a ratio of 1 to 1, whereas cytochrome o contains the same amounts of hemes b and o. Thus, data indicate that cytochrome a1 and cytochrome o of A. aceti are cytochrome ba and cytochrome bo ubiquinol oxidases, respectively, and that both oxidases have a closely similar protein structure and prosthetic groups, in which only heme a in the heme/copper binuclear center of cytochrome a1 is replaced by heme o in that of cytochrome o. (+info)
Conformational switching at cytochrome a during steady-state turnover of cytochrome c oxidase.
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As an electron transfer-driven proton pump, cytochrome c oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.1) must alternate between two conformations in each valence state of the redox element associated with ion translocation. Using second derivative absorption spectroscopy, the conformation of the cytochrome a cofactor has been investigated during steady-state turnover of this enzyme. Resting cytochrome c oxidase displays a transition for ferric cytochrome a at 430 nm. During aerobic steady-state turnover, this band is replaced by a ferrous cytochrome a transition at 450 nm. When anaerobicity is achieved, the transition occurs at 444 nm. The 450-nm-absorbing species is thus the dominant form during turnover, suggesting that conformational transitions of cytochrome a direct electron transfer during catalysis and may direct as well proton translocation in the last step of the respiratory electron transfer chain. (+info)