Circular dichroism and UV-resonance Raman investigation of the temperature dependence of the conformations of linear and cyclic elastin

We used electronic circular dichroism (CD) and UV resonance Raman (UVRR) spectroscopy at 204 nm excitation to examine the temperature dependence of conformational changes in cyclic and linear elastin peptides. We utilize CD spectroscopy to study global conformation changes in elastin peptides, while UVRR is utilized to probe the local conformation and hydrogen bonding of Val and Pro peptide bonds. Our results indicate that at 20 degrees C cyclic elastin predominantly populates distorted beta-strand, beta-type II and beta-type III turn conformations. At 60 degrees C, the beta-type II turn population increases, while the distorted beta-strand population decreases. Linear elastin predominantly adopts distorted beta-strand and beta-type III turn conformations with some beta-type II turn population at 20 degrees C. Increasing temperature to 60 degrees C results in a small increase in the turn population.     

Ubiquinol-cytochrome c oxidoreductase of higher plants. Isolation and characterization of the bc1 complex from potato tuber mitochondria.

A procedure is described for isolation of active ubiquinol-cytochrome c oxidoreductase (bc1 complex) from potato tuber mitochondria using dodecyl maltoside extraction and ion exchange chromatography. The same procedure works well with mitochondria from red beet and sweet potato. The potato complex has at least 10 subunits resolvable by gel electrophoresis in the presence of dodecyl sulfate. The fifth subunit carries covalently bound heme. The two largest ("core") subunits either show heterogeneity or include a third subunit. The purified complex contains about 4 mumol of cytochrome c1, 8 mumol of cytochrome b, and 20 mumol of iron/g of protein. The complex is highly delipidated, with 1-6 mol of phospholipid and about 0.2 mol of ubiquinone/mol of cytochrome c1. Nonetheless it catalyzes electron transfer from a short chain ubiquinol analog to equine cytochrome c with a turnover number of 50-170 mol of cytochrome c reduced per mol of cytochrome c1 per s, as compared with approximately 220 in whole mitochondria. The enzymatic activity is stable for weeks at 4 degrees C in phosphate buffer and for months at -20 degrees C in 50% glycerol. The activity is inhibited by antimycin, myxothiazol, and funiculosin. The complex is more resistant to funiculosin and diuron than the beef heart enzyme. The optical difference spectra of the cytochromes were resolved by analysis of full-spectrum redox titrations. The alpha-band absorption maxima are 552 nm (cytochrome c1), 560 nm (cytochrome b-560), and 557.5 + 565.5 nm (cytochrome b-566, which has a split alpha-band). Extinction coefficients appropriate for the potato cytochromes are estimated. Despite the low lipid and ubiquinone content of the purified complex, the midpoint potentials of the cytochromes (257, 51, and -77 mV for cytochromes c1, b-560, and b-566, respectively) are not very different from values reported for whole mitochondria. EPR spectroscopy shows the presence of a Rieske-type iron sulfur center, and the absence of centers associated with succinate and NADH dehydrogenases. The complex shows characteristics associated with a Q-cycle mechanism of redox-driven proton translocation, including two pathways for reduction of b cytochromes by quinols and oxidant-induced reduction of b cytochromes in the presence of antimycin.     

Electron transport in the membrane of lutoids from the latex of Hevea brasiliensis.

1. An antimycin-insensitive NADH-cytochrome c oxidoreductase (E.C. 1.6.99.3) activity can be demonstrated in the membrane of lutoids isolated from the latex of Hevea brasiliensis. This electron transport system can also use ferricyanide as an electron acceptor, but is unable to oxidize NADPH. 2. Two beta-type cytochromes are present in the membranes. Cytochrome beta563 is partially reduced by NADH and ascorbate, but is not reducible by NADPH. It shows a double peak at 555 and 561 nm at 77 degrees K. A second cytochrome, cytochrome beta561, seems to be reducible by hydrosulfite only. 3. In the reduced state, these cytochromes do not combine with CO. The occurrence of cytochrome P-450 could not be demonstrated. 4. The role of the NADH oxidation system is considered in relation to the biosynthesis of polyisoprene compounds in the latex.     

Low-temperature spectral properties of the respiratory chain cytochromes of mitochondria from Crithidia fasciculata

Highly purified mitochondrial preparations from the trypanosomatid hemoflagellate, Crithidia fasciculata (A.T.C.C. No.11745), were examined by low-temperature difference spectroscopy. The cytochrome a+a₃ maximum of hypotonically-treated mitochondria reduced with succinate, was shifted from 605 nm at room temperature to 601 nm at 77 °K. The Soret maximum, found at 445 nm at 23 °C, was split at 77 °K into two approximately equally absorbing species with maxima at 438 and 444 nm. A prominent shoulder observed at 590 nm with hypotonically-treated mitochondria was not present in spectra of isotonic controls.

The cytochrome b maxima observed in the presence of succinate plus antimycin A were shifted from the 431 and 561 nm positions observed at 23 °C to 427 and 557 nm at 77 °K. Multiple b cytochromes were not apparent.

Unlike other soluble c-type cytochromes, the maximum of cytochrome c₅₅₅ was not shifted at 77 °K although it was split to give a 551 nm shoulder adjacent to the 555 nm maximum. This lack of a low-temperature blue shift was true for partially purified hemoprotein preparations as well as in situ in the mitochondrial membrane.

Using cytochrome c₅₅₅-depleted mitochondria, a cytochrome c₁ pigment was observed with a maximum at 420 nm and multiple maxima at 551, 556, and 560 nm. After extraction of non-covalently bound heme, the pyridine hemochromogen difference spectrum of cytochrome c₅₅₅-depleted preparations exhibited an maximum at 553 nm at room temperature.

The reduced rate of succinate oxidation by cytochrome c₅₅₅-depleted mitochondria and the ferricyanide requirement for the reoxidation of cytochrome c₁, even in the presence of antimycin, indicated that cytochrome c₅₅₅-mediated electron transfer between cytochromes c₁ and a+a₃ in a manner analagous to that of cytochrome c in mammalian mitochondria.     

A method for in situ characterization of b- and c-type cytochromes in Escherichia coli and in complex III from beef heart mitochondria by combined spectrum deconvolution and potentiometric analysis

An analytical technique for the in situ characterization of b- and c-type cytochromes has been developed. From evaluation of the results of potentiometric measurements and spectrum deconvolutions, it was concluded that an integrated best-fit analysis of potentiometric and spectral data gave the most reliable results. In the total cytochrome b content of cytoplasmic membranes from aerobically grown Escherichia coli, four major components are distinguished with alpha-band maxima at 77 K of 555.7, 556.7, 558.6 and 563.5 nm, and midpoint potentials at pH 7.0 of 46, 174, -75 and 187 mV, respectively. In addition, two very small contributions to the alpha-band spectrum at 547.0 and 560.2 nm, with midpoint potentials of 71 and 169 mV, respectively, have been distinguished. On the basis of their spectral properties they should be designated as a cytochrome c and a cytochrome b, respectively. In Complex III, isolated from beef heart mitochondria, five cytochromes are distinguished: cytochrome c1 (lambda m (25 degrees C) = 553.5 nm; E'0 = 238 mV) and four cytochromes b (lambda m (25 degrees C) = 558.6, 561.2, 562.1, 566.1 nm and E'0 = -83, 26, 85, -60 mV).     

The impact of the thermal sensitivity of cytochrome c oxidase on the respiration rate of Arctic charr red muscle mitochondria

To assess if cytochrome c oxidase could determine the response of mitochondrial respiration to changes in environmental temperature in ectotherms, we performed KCN titration of the respiration rate and cytochrome c oxidase activity in mitochondria from Arctic charr (Salvelinusfontinalis) muscle at four different temperatures (1 degrees C, 6 degrees C, 12 degrees C, and 18 degrees C). Our data showed an excess of cytochrome c oxidase activity over the mitochondrial state 3 respiration rate. Mitochondrial oxygen consumption rates reached approximately 12% of the cytochrome c oxidase maximal capacity at every temperature. Also, following titration, the mitochondrial respiration rate significantly decreased when KCN reached concentrations that inhibit almost 90% of the cytochrome c oxidase activity. This strongly supports the idea that the thermal sensitivity of the maximal mitochondrial respiration rate cannot be dictated by the effect of temperature on cytochrome c oxidase catalytic capacity. Furthermore, the strong similarity of the Q10s of mitochondrial respiration and cytochrome c oxidase activity suggests a functional or structural link between the two. The functional link could be coevolution of parts of the mitochondrial system to maintain optimal functions in most of the temperature range encountered by organisms.     

Multiple light-induced reactions of cytochromes b and c in Rhodopseudomonas spheroides

Illumination of chromatophore preparations from Rhodopseudomonas spheroides causes the oxidation of a cytochrome c and a slight oxidation of a cytochrome b with a maximum at 560nm. When illuminated in the presence of antimycin A the oxidation of cytochrome c was more pronounced and cytochrome b(560) was reduced; the dark oxidation of cytochrome b(560) was biphasic in the presence of succinate, but not in the presence of NADH, a less effective reductant. Split-beam spectroscopy showed that, in addition to the reduction of cytochrome b(560), another pigment with maxima at 565 and 537nm. was reduced and was more rapidly oxidized in the dark than cytochrome b(560). This pigment, tentatively identified as cytochrome b(565), was also detected in spectra at 77 degrees k, after brief illumination at room temperature; the maxima at 77 degrees k were at 562 and 536nm. In the absence of antimycin A, light caused a transient reduction of cytochrome b(565) and an oxidation of cytochrome b(560). Dark oxidation of b(565) was rapid, even in the presence of antimycin A and succinate. Difference spectra, at 77 degrees k, of ascorbate-reduced minus succinate-reduced chromatophores or of anaerobic succinate-reduced minus aerobic succinate-reduced chromatophores suggested that two cytochromes c were present, with maxima at 547 and 549nm. When chromatophores frozen at 77 degrees k were illuminated both these cytochromes c were oxidized, indicating a close association with the photochemical reaction centre. A scheme involving two reaction centres is proposed to explain these results.     

Quinol-cytochrome c oxidoreductase from the thermophilic bacterium PS3. Purification and properties of a cytochrome bc1(b6f) complex

A quinol-cytochrome c oxidoreductase (cytochrome bc1 complex) has been purified from plasma membranes of a thermophilic Bacillus, PS3, by ion-exchange chromatography in the presence of Triton X-100. The purified enzyme shows absorption bands at 561-562 nm and 553 nm at room temperature, and 560, 551, and 547 nm at 80 K upon reduction, and gives an ESR signal similar to that of a Rieske-type iron sulfur center. Its contents of protohemes, heme c, and non-heme iron are about 23, 10, and 21 nmol/mg of protein, respectively. The enzyme consists of four polypeptides with molecular masses of 29, 23, 21, and 14 kDa judging from their electrophoretic mobilities in the presence of sodium lauryl sulfate. Since the staining intensities of the respective bands are almost proportional to their molecular masses, the monomer complex (87 kDa) of the subunits probably consists of a cytochrome b having two protohemes, a cytochrome c1 and an Fe2-S2-type iron sulfur center. The 29 and 21 kDa subunits were identified as cytochromes c1 and b, respectively, and the 23-kDa subunit is probably an iron-sulfur protein, since the 14-kDa polypeptide can be removed with 3 M urea without reducing the content of non-heme iron. Several characteristics of the subunits and chromophores indicate that the PS3 enzyme is rather similar to cytochrome b6f (a bc1 complex equivalent) of chloroplasts and Cyanobacteria. The PS3 complex catalyzes reduction of cytochrome c with various quinol compounds in the presence of P-lipids and menaquinone. The turnover number at pH 6.8 was about 5 s-1 at 40 degrees C and 50 s-1 at 60 degrees C. The enzyme is heat-stable up to 65 degrees C.     

Photoreductive titration of the resonance Raman spectra of cytochrome oxidase in whole mitochondria.

A photoreductive titration of the resonance Raman (RR) spectra of cytochrome c oxidase in whole mitochondria was recorded by exploiting the preferential enhancement of the Raman signals of reduced cytochrome oxidase excited at 441.6 nm. When the sample was cooled to about--10 degrees C, it was possible to slow down the photoreductive effect of the laser and to record RR spectra at various states of reduction. Compared to the earliest recorded scan (most oxidized), the dithionite-reduced sample shows the appearance of new bands at 216, 363, 560, and 1665 cm-1. At intermediate stages of photoreduction, the 216- and 560-cm-1 bands appear before the 363- and 1665-cm-1 bands; photoreduction induces full intensity in the former bands, whereas the latter bands are photoreduced to 50% of the dithionite-reduced intensity. The relative intensities of a doublet at 1609--1623 cm-1 are affected by reduction: the band at 1609 cm-1 is weaker in the earlier scans; in later scans this band has grown to equal intensity with the 1623-cm-1 band. We conclude that this reductive titration of the RR spectrum of cytochrome c oxidase reflects three states in its reduction. The behavior of the doublet at 1609--1623 cm-1 suggests that the two hemes are nonequivalent but interacting. The band at 216 cm-1 may be indicative of an iron-copper interaction that is affected by the presence of external ligands.     

The reaction of cytochrome omicron in Escherichia coli with oxygen. Low-temperature kinetic and spectral studies.

1. The reactions of cytochrome omicron in intact cells of aerobically grown Escherichia coli with O2 and CO have been studied at low temperature. 2. Flash photolysis of CO-liganded cells in the presence of O2 and at temperatures between -79 and -102 degrees C results in the oxidation of kinetically heterogeneous beta-type cytochromes (including cytochrome omicron), but not of cytochrome d. 3. The reaction of reduced cytochrome omicron with O2 involves O2 binding to give intermediate(s) with spectral characteristics similar to that of the reduced oxidase-CO complex. Observation in the alpha-region suggests that unexplained ligand dissociation accompanies the initial O2 binding. 4. At temperatures below -98 degrees C, an 'end point' in the reaction is reached; further reaction and oxidation of cytochrome omicron occurs on raising the temperature. 5. There is a linear relationship between the rate of formation of the oxygen compound and the O2 concentration up to 0.5 mM. The second-order constant for its formation (k+1) is 0.91 M-1.S-1 at -101 degrees C. The reaction is not readily reversible, the value of k-1 being 1.4 X 10(-5) S-1 and the kd 1.5 X 10(-5) M. 6. The energy of activation for this reaction at low temperatures is 29.9kJ (7.1 kcal)/mol. 7. The reaction with O2 is distinguished from that with CO by the markedly lower velocity and high photolytic reversibility of the latter. 8. Comparisons are drawn between the intermediate(s) in the O2 reaction of cytochrome omicron in E. coli and those identified in other bacteria and in the reaction of cytochrome aa3 with O2.     

Spectral evidence for the existence of a second cytochrome o in whole cells of Vitreoscilla

Cytochrome o, a protoheme IX pigment, has been proposed as the terminal oxidase of the filamentous bacterium, Vitreoscilla. Aerobic and anaerobic photolysis of CO-liganded whole cells demonstrated the presence of a second CO-reactive pigment, cytochrome o'. At temperatures lower than -100 degrees C, anaerobic photolysis dissociated only about 25% of the total CO-liganded components to reveal the unliganded cytochrome o'. At these temperatures, the photolysis of cytochrome o could not be demonstrated. At warmer temperatures, recombination of CO with the reduced cytochrome o' occurred with an apparent energy of activation of 5.8 kcal/mol. Aerobic photolysis of whole cells demonstrated two oxygen-bound intermediates. At temperatures lower than -95 degrees C, a spectrally distinct compound with absorption maxima at 428, 534, and 564 nm appeared (form I'); the apparent second order rate constant (k+1) for the formation of this intermediate was found to be 9.1 M-1 s-1, the reverse rate (k-1) was 9.9 X 10(-5) s-1, and the equilibrium constant (Kd) was 1.1 X 10(-5) M. This oxygen intermediate of cytochrome o' is spectrally and kinetically similar to the oxygen intermediate of cytochrome o seen in Escherichia coli. At temperatures warmer than -90 degrees C, photolysis of aerobic samples resulted in the immediate formation of a second oxygen-bound intermediate (form I) with absorption maxima at 422, 534, and 564 nm. This second intermediate results from the binding of oxygen to the cytochrome o (oxygenated cytochrome o). These data support the proposal that whole cells of Vitreoscilla contain two alternative pathways of electron transport, one terminating with cytochrome o and the other with cytochrome o'.     

Analysis of deamidation of small, acid-soluble spore proteins from Bacillus subtilis in vitro and in vivo.

Deamidation of one specific asparagine residue in an alpha/beta-type small, acid-soluble spore protein (SASP) of Bacillus subtilis took place readily in vitro (time for 50% deamidation [t(1/2)], approximately 1 h at 70 degrees C), and the deamidated SASP no longer bound to DNA effectively. However, DNA binding protected against this deamidation in vitro. A mutant alpha/beta-type SASP in which the reactive asparagine was changed to aspartate also failed to bind to DNA in vitro, and this protein did not restore UV radiation and heat resistance to spores lacking the majority of their alpha/beta-type SASP. When expressed in Escherichia coli, where it is bound to DNA, the alpha/beta-type SASP deamidated with a t(1/2) of 2 to 3 h at 95 degrees C. However, the alpha/beta-type SASP was extremely resistant to deamidation within spores (t(1/2), >50 h at 95 degrees C). A gamma-type SASP of B. subtilis also deamidated readily in vitro (t(1/2) for one net deamidation, approximately 1 h at 70 degrees C), but this protein (which is not associated with DNA) deamidated fairly readily in spores (t(1/2), approximately 1 h at 95 degrees C). Total spore core protein also deamidated in vivo, although the rate was two- to threefold slower than that of deamidation of total protein in heated vegetative cells. These data indicate that protein deamidation is slowed significantly in spores, presumably due to the spore's environment. However, alpha/beta-type SASP are even more strongly protected against deamidation in vivo, presumably by their binding to spore DNA. Thus, not only do alpha/beta-type SASP protect spore DNA from damage; DNA also protects alpha/beta-type SASP.     

The electron-transport system of mitochondria from the slime mould Physarum polycephalum

A method is described for the preparation of mitochondria from the slime mould Physarum polycephalum; the mitochondria were not coupled. P. polycephalum mitochondria oxidized added NADH via a rotenone-insensitive pathway, but the oxidation of malate plus glutamate was rotenone sensitive; both of these substrates reduced much less cytochrome b than did succinate, in both aerobic and anaerobic steady states. Spectroscopy at 77 degrees K separated three absorption maxima in the alpha-band region, at 560nm, 553nm and one at 547nm due to cytochrome c. The absorption at 553nm was increased in the aerobic steady state by the addition of 2-heptyl-4-hydroxyquinoline N-oxide, suggesting that it was due to a b-type cytochrome. All three absorption maxima appeared in the aerobic steady state after the addition of a range of substrates. The respiratory activity with different substrates and the response to inhibitors of respiration were similar to those previously described for fungus mitochondria (Weiss et al., 1970; Erickson & Ashworth, 1969). When grown under conditions of haem limitation the mitochondria contained a lower concentration of cytochromes than normal.     

Effect of growth at low temperature on the alternative pathway respiration in Acanthamoeba castellanii mitochondria

Mitochondria of amoeba Acanthamoeba castellanii in addition to the conventional cytochrome pathway possess, like plant mitochondria, a cyanide-resistant alternative quinol oxidase. In mitochondria isolated from amoeba batch culture grown temporarily at low temperature (6 degrees C), higher respiration was accompanied by lower coupling parameters as compared to control culture (grown at 28 degrees C). In the presence of benzohydroxamate, respiratory rates and coupling parameters were similar in both types of mitochondria indicating that growth in cold conditions did not disturb the cytochrome pathway. Increased contribution of alternative oxidase in total mitochondrial respiration in low-temperature-grown amoeba cells was confirmed by calculation of its contribution using ADP/O measurements. Furthermore, in mitochondria from low-temperature- grown cells the content of the alternative oxidase was increased and correlated with the increase in the unstimulated and GMP-stimulated cyanide-resistant respiratory activity. A possible physiological role of higher activity of alternative oxidase as response to growth at a low temperature in unicellular organisms, such as amoeba, is discussed.     

Presence of three B-type cytochromes in swine cerebral microsomes

In swine cerebral microsomes purified with sucrose density gradient and glycerol-cholate gradient centrifugations, it was observed that a new b-type cytochrome which had alpha-peak at 560 nm and Soret peak at 428 nm at 23 degrees C was reduced preferentially by anaerobic NADPH in the presence of cyanide. The b5-type cytochromes were reduced completely by both NADH and NADPH anaerobically. Three b-type cytochromes were partially purified into two b-type, spectroscopically distinct from each other, and the new b-type (b560-5) cytochromes.     

The reaction with oxygen of cytochrome oxidase (cytochrome d) in Escherichia coli K12: optical studies of intermediate species and cytochrome b oxidation at sub-zero temperatures

Optical changes in d- and b-type cytochromes, following initiation of the reaction of cytochrome oxidase d with O2, have been studied in cells and derived membrane particles from oxygen-limited cultures of Escherichia coli K12. At successively higher temperatures between -132 and -88 degrees C, the first scan after photolysis of the Co-liganded, reduced oxidase in the presence of O2 and a slow increase in absorbance at 675 to 680 nm due to an unidentified chromophore. A similar sequence occurs when a single sample is scanned repetitively at -91 degrees C. At higher temperatures, oxidation of at least two spectrally distinct cytochromes b occurs. Selective photolysis of the cytochrome d-CO complex with a He-Ne laser shows that neither of these cytochromes is the CO-binding cytochrome o436. In all oxidation states examined, no absorbance in the 720 to 860 nm region was observed; it is concluded that both cytochromes d and o436 lack redox-active copper that has an environment similar to the copper(s) in mitochondrial cytochrome c oxidase. The amount of cytochrome d650 (but not the amount of reduced cytochrome o436) formed after photolysis is directly proportional to the oxygen concentration in the sample at the time of freeze trapping. The results are discussed in relation to the composition and mechanism of action of cytochrome d.     

Effect of heat treatment on oxidase activity and proton-pumping capability of proteoliposome-incorporated beef heart cytochrome aa3

By incubating beef heart cytochrome c oxidase at 43-45 degrees C, selective inactivation of the H+-pumping function is possible without affecting cytochrome c oxidase activity; proteoliposomes reconstituted with heated enzyme (43.5 degrees C for 60 min at pH 7.0) showed an apparent H+/e- ratio of only 0.3 and a turnover with cytochrome c plus ferrocyanide as substrate of 20 s-1, while those with the intact enzyme showed an apparent H+/e- ratio somewhat greater than 1.0 and a turnover of 19 s-1. This decrease in the H+/e- ratio could not be attributed to a stimulation of H+ permeability upon heating, since the respiratory control ratio and the magnitude of membrane potential formation remained almost the same in the two cases. A pH-dependent Em (midpoint redox potential) change of cytochrome a in the presence of cyanide was still observed after the heat treatment. Heating induced a small spectral shift in the Soret region of the oxidized (resting) enzyme; the peak of the heated enzyme was at 421 nm, while that of the intact enzyme was at 419 nm. The spectral shift obtained by pulsing the enzyme with oxygen under turnover conditions is also altered.     

Catalytic activity of cytochromes c and c1 in mitochondria and submitochondrial particles.

1. Beef heart mitochondria have a cytochrome c1:c:aa3 ratio of 0.65:1.0:1.0 as isolated; Keilin-Hartree submitochondrial particles ahve a ratio of 0.65:0.4:1.0. More than 50% of the submitochondrial particle membrane is in the 'inverted' configuration, shielding the catalytically active cytochrome c. The 'endogenous' cytochrome c of particles turns over at a maximal rate between 450 and 550 s-1 during the oxidation of succinate or ascorbate plus TMPD; the maximal turnover rate for cytochrome c in mitochondria is 300-400 s-1, at 28 degrees-30 degrees C, pH 7.4. 2. Ascorbate plus N,N,N',N'-tetramethyl-p-phenylene diamine added to antimycin-treated particles induces anomalous absorption increases between 555 and 565 nm during the aerobic steady state, which disappear upon anaerobiosis; succinate addition abolishes this cycle and permits the partial resolution of cytochrome c1 and cytochrome c steady states at 552.5-547 nm and 550-556.5 nm, respectively. 3. Cytochrome c1 is rather more reduced than cytochrome c during the oxidation of succinate and of ascorbate + N,N,N',N'-tetramethyl-p-phenylene diamine in both mitochondria and submitochondrial particles; a near equilibrium condition exists between cytochromes c1 and c in the aerobic steady state, with a rate constant for the c1 leads to c reduction step greater than 10(3) s-1. 4. The greater apparent response of the c/aa3 electron transfer step to salts, the hyperbolic inhibition of succinate oxidation by azide and cyanide, and the kinetic behaviour of the succinate-cytochrome c reductase system, are all explicable in terms of a near-equilibrium condition prevailing at the c1/c step. Endogenous cytochrome c of mitochondria and submitochondrial particles is apparently largely bound to cytochrome aa3 units in situ. Cytochrome c1 can either reduce the cytochrome c-cytochrome aa3 complex directly, or requires only a small extra amount of cytochrome c to carry the full electron transfer flux.     

Nuclear and mitochondrial suppression of a mitochondrially inherited cold-sensitive mutation in Aspergillus nidulans

Partial suppressors of a mitochondrially inherited mutation, [cs-67], conferring cold-sensitivity at 20 degrees C were identified. These mapped at one mitochondrial and four unlinked nuclear loci. Most suppressors partially restored the cytochrome aa3 deficiency of the cold-sensitive strain at 20 degrees C. Strains carrying two or more suppressors and [cs-67] showed considerably impaired growth. This effect was temperature-dependent, being more severe at 37 degrees C, and was not expressed in the presence of the [cs-67+] allele. The cytochrome oxidase activity of one of these strains was no more heat-sensitive than that of the wild-type implying that these mutations did not directly modify cytochrome oxidase. The wild-type strain grown in the presence of chloramphenicol and the cold-sensitive strain grown at 20 degrees C had similar cytochrome spectra and mitochondrial membrane protein profiles on sodium dodecyl sulphate gradient acrylamide gels. [cs-67] conferred pleiotropically a low level of resistance to paramomycin at 37 degrees C. It is suggested that [cs-67] and the suppressors act at the level of the mitochondrial ribosome.     

Conformations of cytochrome oxidase: thermodynamic evaluation of the interconversion of the 418- and 428-nm forms

Oxidized cytochrome c oxidase exists in two reasonably well-defined conformations, a high-spin conformation with maximal absorption at 418 nm and a low-spin conformation with maximal absorption at 428 nm. The equilibrium between these two conformations has been studied as a function of pH, pressure, and temperature. pH effects the equilibrium between the two conformations, the maximum fraction of the 418-nm form being found at about pH 6.8. Increasing pressure displaced the equilibrium toward the 428-nm form; the molar volume changes found are independent of pH but strongly dependent on temperature. Increasing temperature over the range -20 to 25 degrees C displaces the equilibrium toward the 428-nm form; the van't Hoff plots that result show a discontinuity at about 10 degrees C. Above 10 degrees C, delta H is relatively constant as a function of pH; below 10 degrees C, delta H is strongly pH dependent. Delta G, delta H, delta S, and delta V have been evaluated for the equilibrium.