Absence Of Cytochrome Research Articles

Spectroelectrochemical studies of stoichiometry, energetics, and kinetics of heme proteins: cytochrome c and cytochrome c oxidase

Two heme proteins, cytochrome c and cytochrome c oxidase, have been investigated in our laboratory by spectroelectrochemical techniques. In this paper we describe our spectroelectrochemical approach for the evaluation of stoichiometry, energetics and kinetics associated with each of these heme proteins and their mixtures. The spectroelectrochemical method involves the electrochemical generation of a mediator-titrant (either oxidant or reductant) at an optically transparent electrode. The subsequent exchange of electrons between the mediator-titrant and the heme protein is monitored spectrophotometrically by light passing directly through the optically transparent electrode and the solution. The heme protein can be cycled conveniently between its reduced and oxidized forms. An accurate assessment of the number of electrons ( n value) transferred to or from the heme protein can be obtained from the charge required for titration. n values for cytochrome c oxidase, cytochrome c, modified cytochrome c and myoglobin have been measured in this way. U o′ values, for cytochrome c and for the redox components of cytochrome c oxidase have been evaluated from the shape of absorbance-charge curves obtained by the spectroelectrochemical method. Assignments are made on the basis of comparisons between experimental curves and computer-calculated curves. Potentials are reported for cytochrome c oxidase in the presence and absence of cytochrome c. The applicability of spectroelectrochemical methods for the measurement of electron exchange with heme proteins is demonstrated.

Reactivity of cytochrome oxidase with ascorbate

When cytochrome oxidase is solubilized in bile acid, ascorbate alone is capable of reducing the oxidase and induces oxygen uptake in the absence of cytochrome c. Cytochrome oxidase is organized into vesicular structures in the absence of detergent in phosphate buffer. In this, spectral changes are brought about by ascorbate, but there is negligible oxygen uptake in the absence of cytochrome c.

Biochemical and biophysical studies on cytochrome c oxidase. XVIII. Potentiometric titrations of cytochrome c oxidase followed by circular dichroism

1. Potentiometric circular dichroism titrations of cytochrome c oxidase, carried out in the absence of cytochrome c, confirm the potentiometric equivalence of the two heme a groups of cytochrome c oxidase. In the presence of cytochrome c, two different midpoint potentials are found for the two heme a groups of cytochrome c oxidase. 2. Circular dichroism difference spectra (reduced minus oxidized) of the two heme a components of cytochrome c oxidase have been obtained by means of this potentiometric titration. On reduction of the first heme a group a circular dichroism difference spectrum is obtained with peaks at 425, 442 and 602.5 nm; the second heme a group shows difference peaks at 434, 447 and 608 nm. Whereas both heme a groups contribute about equally to the absorbance difference spectrum, the second heme a group reduced contributes about twice as much to the circular dichroism difference spectrum as does the first heme a group. 3. From these spectral and circular dichroism differences it is concluded that, on reduction of or ligand binding to cytochrome c oxidase, conformational changes occur which affect the symmetry of the environments of the heme a groups.

Heme—Heme interaction in cytochrome c oxidase: The cooperativity of the hemes of cytochrome c oxidase as evidenced in the reaction with CO

Isolated and purified cytochrome c oxidase from beef heart muscle mitochondria (Kuboyama et al. (1972) J. Biol. Chem. 247, 6375–6383) is shown to be very similar to the hemoprotein in situ with respect to its EPR absorption properties and the half-reduction potentials of the hemes and copper. The half-reduction potentials of cytochromes a and a 3 in the purified cytochrome c oxidase are 205 mV and 360 mV, respectively, and these values are the same in the presence and absence of cytochrome c. Low-temperature EPR spectra show that the binding of CO to reduced cytochrome a 3 changes the oxidized cytochrome a from high spin ( g 6) to low spin ( g 3). In samples at 5–8 °K the photodissociation of the reduced cytochrome a 3 CO compound shifts the spectrum of the oxidized low-spin cytochrome a to a lower g value and converts approximately 5% of the low-spin form to a high-spin form. The heme-heme interaction demonstrated in this reaction is very fast as evidenced by the fact that even at 5 °K the measured change in oxidized cytochrome is complete within 5 msec.

Rhizobium leguminosarum cytochromes (Vicia faba)

1. By difference spectrophotometry the cytochrome pattern of two strains of Rhizobium leguminosarum — effective and partly-effective — was studied in pure culture and in bacteroids isolated from legume root nodules1. 2. In pure culture both strains contained cytochromes c, b and a-a3 as well as cytochrome a2 which appeared under semi-anaerobic conditions, especially in the effective strain. In Rhizobia cytochrome a2 was detected for the first time. 3. Bacteroids of the effective strain contained cytochromes c, b and some CO-reactive pigments which remained incompletely identified. Bacteroids of the partly-effective strain contained cytochrome a (a = peak at 597 nm at − 196°) which was absent from bacteroids of the effective strain. 4. The data obtained show that the low O2 concentration within nodules does not seem to be the only factor responsible for the absence of cytochromes a from N2-fixing bacteroids.

Evaluation of the energetics of cytochrome C oxidase in the absence of cytochrome C

Evaluation of the energetics of cytochrome C oxidase in the absence of cytochrome C

The mitochondrial derivative of the spermatozoon of Drosophila before and after fertilization

The ultrastructure of the paracrystalline mitochondrial derivative of the Drosophila spermatozoon has been investigated in sperms stored for different intervals after mating within the genital apparatus of inseminated females and in sperms which penetrated the egg. The paracrystalline lattice remains structurally intact even after the most prolonged storage. Only after fertilization does the derivative exhibit structural modifications which following a characteristic pattern lead to the formation of lamellar bodies present up to the onset of the cellular blastoderm stage of the egg development. The behaviour of the mitochondrial derivative during the sperm storage, its fate at fertilization, and the absence of cytochrome c oxidase activity from its crystalline texture, as demonstrated by cytochemical tests, suggest that the typical biochemical activity of mitochondria is not a major function of this highly differentiated sperm component. The functional significance of the derivative is discussed in relation to a possible role at fertilization and to sperm metabolism.

Biochemical and biophysical studies on cytochrome c oxidase. X. Spectral and potentiometric properties of the hemes and coppers

1. 1. Potentiometric titrations of highly purified cytochrome c oxidase, carried out in the absence of cytochrome c at the wavelengths 410, 424, 445 and 605 nm, show that both heme a groups act as independent one-electron acceptors with the same midpoint potential ( E 0′ = 280 mV, n = 1.0). The titration of the copper atoms at 830 nm suggests an equilibrium between one- and two-electron acceptors ( E 0′ = 280 mV, n = 1.6). 2. 2. When cytochrome c is present, the copper atoms titrate as single-electron acceptors ( E 0′ = 280 mV, n = 1.0). Two heme a groups with different midpoint potentials ( E 0′ = 370 mV, n = 1.0 and E 0′ = 230 mV, n = 1.0) contribute about equally to the absorbance differences on reduction at 410, 424, 445 and 605 nm. 3. 3. Both in the absence and presence of cytochrome c the difference spectrum (reduced minus oxidized) of the heme a group that is reduced at high oxidation-reduction potentials peaks at 603.5 and 444 nm, whilst that of the heme a group that is reduced at low redox potentials peaks at 607 and 446 nm. This indicates that the difference spectrum of one of the heme a groups depends on the redox state of the other. 4. 4. No evidence is found for Lemberg's ( Physiol. Rev. 49 (1969) 48–121) suggestion that cytochrome a 3 absorbs maximally at 411 nm and cytochrome a at 423 nm. 5. 5. It is concluded that the heme a groups in cytochrome c oxidase, in the absence of inhibitors such as CO, azide and cyanide, are formally equivalent.

Biochemical and biophysical studies on cytochrome aa3. VII. The effect of cytochrome c on the oxidation-reduction potential of isolated cytochrome aa3

1. 1. In the absence of cytochrome c, the haem groups of cytochrome aa 3 titrate as indistinguishable identities, each having E′ o = 280 mV and n = 1. 2. 2. In the presence of cytochrome c, 45% of the absorbance change on reduction at 605 nm is due to a high-potential component with E′ o = 335–360 mV, n = 1.2 and 55% to a low-potential component with E′ o = 200–250 mV, n = 1.0. At 445 nm both components contribute equally to the difference spectrum (reduced minus oxidized).

Insoluble hexokinase in the brush border region of rat intestinal epithelial cells

In several reports it has been shown that a considerable amount of the total hexokinase activity in rat intestinal epithelial cells is particle-bound [l-4] . Srivastava et al. [4] determined the intracellular distribution of hexokinase activity in the mucosa of both rat and guinea pig small intestine. They found that in rat 60% of the hexokinase activity was associated with the particulate fraction of homogenates. Further subfractionation and characterization of the subfractions by means of marker enzymes, led the authors to conclude that the particle-bound hexokinase was predominantly recovered in the mitochondrial fraction [4]. During our studies on energy metabolism of intestinal epithelium it was found that ATP contributed less than 15% to the total amount of adenine nucleotides [5]. This low phosphorylation potential led us to investigate the energy metabolism in a part of the cell where mitochondria are absent, the brush border region. Preparations of brush borders were made essentially according to Harrison and Webster [6]. After removal of viscous material contaminating the everted intestine by 1 min vibration (100 Hz) in cold saline, brush borders were released by further vibration for 20 min in 2.5 mM EDTA, containing 1 mM dithiothreitol (pH 7.4). As described by Harrison and Webster [6], the brush borders were harvested by centrifugation (2 min at 700 g) after treatment with glass fibers to remove nuclear aggregates. The precipitates were washed in cold isolation medium 3-5 times. The material obtained did not contain mitochondrial material, as judged by the absence of cytochrome c oxidase. However, the washed brush border did contain hexokinase activity. The Michaelis constants for this hexokinase are lob4 M for glucose (compare also

The Utility of Superoxide Dismutase in Studying Free Radical Reactions: II. THE MECHANISM OF THE MEDIATION OF CYTOCHROME c REDUCTION BY A VARIETY OF ELECTRON CARRIERS

Abstract The rate of reduction of cytochrome c by the ferredoxin-TPN reductase of spinach or by chicken liver xanthine dehydrogenase is markedly augmented by a variety of electron carriers. Menadione, flavin mononucleotide, flavin adenine dinucleotide, N-methylphenazinium methosulfate, methylene blue, and 2,6-dichlorobenzenone indophenol were all effective. When cytochrome c reduction was mediated aerobically by menadione, flavin mononucleotide, or flavin adenine dinucleotide, superoxide dismutase was an inhibitor. When cytochrome c reduction was mediated by these same electron carriers in the absence of molecular oxygen, superoxide dismutase was without effect. When cytochrome c reduction was mediated by N-methylphenazinium methosulfate, methylene blue, or 2,6-dichlorobenzenone indophenol, superoxide dismutase was without effect whether or not oxygen was present. These results indicate that of these electron carriers only the reduced forms of menadione and of the flavins preferentially reduce O2 to the superoxide radical in the presence of cytochrome c. In the absence of cytochrome c, it was shown that reduced methylene blue is likewise capable of the univalent reduction of oxygen. In this case O-2 was detected by its ability to oxidize epinephrine to adrenochrome.

Kinetic studies on the interaction of TMPD with cytochrome c and cytochrome c oxidase

The kinetics of the reduction of cytochrome c by ascorbate and ascorbate plus TMPD 3 3 Abbreviation used: TMPD = N: N: N′: N′ tetramethyl p-phenylenediamine. have been examined. TMPD increases the reduction rate of cytochrome c by a factor of 30 relative to ascorbate alone. It is concluded that this is the principal reason why TMPD increases the activity of cytochrome c oxidase at a fixed concentration of cytochrome c and markedly diminishes the apparent K m value for cytochrome c. The reduction of cytochrome oxidase by ferrocytochrome c is identical in the presence or absence of TMPD (3.5 × 10 7 m −1 sec −1). The oxidation of TMPD in the absence of cytochrome c shows an average maximal turnover of two electrons (heme a) −1 sec −1 when cytochrome a measured at 605 mμ is over 60% reduced. This identifies a slow rate of electron transfer between a and a 3. It is suggested that in the presence of cytochrome c the reaction pathway may be directly from c to a 3.

ADP-dependent palmitoylcarnitine sensitivity of mitochondria isolated from the perfused rabbit heart.

The function of mitochondria as influenced by the depletion of oxidizable substances in the myocardium was examined during perfusion of the isolated rabbit heart. Mitochondria were isolated using proteinase and KCl–albumin medium, and their respiration was measured polarographically at 25 °C.(i) Mitochondria of nonperfused hearts (control) responded to repeated additions of ADP with repeated and reproducible "state 4 to state 3 transitions" in the presence of a variety of oxidizable substrates (glutamate, oxoglutarate, acetate, pyruvate, β-hydroxybutyrate, succinate, and palmitoyicamitine).(ii) The heart of a fed animal was perfused almost to exhaustion in a substrate-free medium and mitochondria were isolated and tested. The following findings were obtained (iii–viii).(iii) The respiratory control was gradually lost in a response to repeated additions of ADP, when palmitoylcamitine was used as the substrate. A larger quantity of mitochondria in a given assay partially counteracted this gradual loss of the respiratory control.(iv) The respiratory control and P/O ratios of mitochondria obtained from these hearts were slightly depressed as compared to those of the control, when measured using glutamate or oxoglutarate. Malate addition was required for pyruvate oxidation by mitochondria from perfused hearts.(v) The state 3 respiratory rate of the mitochondria obtained from an exhausted heart was about one-quarter that of the control mitochondria regardless of substrates used. The values were independent of the amount of mitochondria present in the assay system.(vi) With paimitoyicamitine as the substrate, the respiratory control ratio calculated from the first state 3 to state 4 transition was influenced by the amount of mitochondria present. The ratio was depressed at low concentrations of mitochondria.(vii) The rate of exogenous NADH oxidation in the absence of cytochrome c and the optical density of the mitochondrial suspension at 520 mμ, were similar with mitochondria prepared from nonperfused and perfused hearts. An addition of cytochrome c accelerated the rate of NADH oxidation by the latter more than the control.(viii) Unstimulated ATPase activity was high in mitochondria of the perfused heart; the enzyme was stimulated by DNP to a lesser degree and by a smaller quantity of paimitoyicarnitine than the control.(ix) The mitochondria prepared from the perfused heart of a fasted animal and from the fed rabbit's heart perfused in the presence of glucose, albumin, or 30 mM KCl (cardiac arrest) showed a normal respiratory function.(x) The mitochondria of the heart in anoxic arrest showed uncoupling of oxidative phosphorylation and a low respiratory rate regardless of the hydrogen donors used.(xi) It is concluded that a prolonged lack of hydrogen sources in perfused exhausted rabbit hearts results in the mitochondrial dysfunction.

Respiratory activity of isolated mammalian nuclei: III. The reduced nicotinamide adenine dinucleotide oxidase of rat liver nuclei and nucleoli

Rat liver nuclei and nucleoli have been shown to catalyze the reduction of neotetrazolium or oxygen with NADH as a substrate. Maximal NADH oxidase activity of nuclei requires the presence of exogenous cytochrome c; nucleoli require added polyethylenesulfonate as well as cytochrome c for demonstrable activity. Nuclei can also oxidize NADPH in the presence of NAD, neither nuclei nor nucleoli can oxidize succinic acid or a variety of other potential substrates, either with or without cytochrome c. Chemical and enzymic analysis as well as analysis by light and electron microscopy and low-temperature spectroscopy indicate the complete absence of contaminating organelles which could conceivably catalyze the observed activities. Isolated nuclei were consistently found to contain a complement of cytochrome c, in the absence of cytochromes a + a 3. Nucleoli which were active in NADH oxidation when supplemented with cytochrome c were found to be essentially devoid of hemoproteins.

ACRIFLAVINE INDUCTION OF DIFFERENT KINDS OF "PETITE" MITOCHONDRIAL POPULATIONS IN SACCHAROMYCES CEREVISIAE.

Avers, Charlotte J. (Rutgers, The State University, New Brunswick, N. J.), Cynthia R. Pfeffer, and Martha W. Rancourt. Acriflavine induction of different kinds of "petite" mitochondrial populations in Saccharomyces cerevisiae. J. Bacteriol. 90:481-494. 1965.-Mutant frequencies induced by 1 or 2 hr in 16 and 64 mug/ml of acriflavine were significantly higher during acceleration and log-phase exposures than during lag or stationary phases. From these induced petites, 59 colonies were selected at random and established in pure culture. All strains were analyzed histochemically for mitochondrial cytochrome oxidase and succinic dehydrogenase (SDH) reactions. On the basis of counts of stained mitochondria per cell obtained by light microscopy, four different cell phenotypes were recognized among the mutant strains: (i) reduced cytochrome oxidase, wild-type SDH; (ii) reduced cytochrome oxidase, high SDH; (iii) absent cytochrome oxidase, high SDH; and (iv) absent cytochrome oxidase, wild-type SDH. The last group was the most common, characterizing 43 of the 59 strains. Electron microscopy showed differences in mitochondrial ultrastructure for the various cell phenotype classes. Electron histochemical localizations showed cytochrome oxidase reaction product only on mitochondrial membranes of respiration-competent cells. Both reactive and unreactive mitochondria occurred in the same cell in mutants with partial respiratory competence. Different mitochondrial subpopulation mixtures characterized the mutant strains, many of which had at least two kinds of respiratory-competent types per chondriome. The diverse chondriomes comprised a stable feature of the mutants, since they have been maintained unchanged during serial transfer for more than 1 year in culture. Together with earlier reports of at least two kinds of mitochondria in wild-type cells, the evidence indicated that mitochondria were capable of regulating some portion of their phenotype. The recognition of mitochondrial phenotypes was proposed as an initial step in a formal analysis of organelle heredity.

The fine structure of mitochondria in the frog nephron correlated with cytochrome oxidase activity

The high cytochrome oxidase activity of the proximal tubule of the nephron of Rana pipiens markedly decreases under certain environmental conditions. Proximal tubular cells with high cytochrome oxidase activity have mitochondria with transverse cristae and numerous dense mitochondrial granules in the matrix. Proximal tubular cells with low or absent cytochrome oxidase activity have a heterogeneous population of mitochondria, some having transverse cristae, others having longitudinally oriented internal membranes. Mitochondrial granules are virtually absent from the latter forms. “Ring”, crescentic, and oval forms with parallel, concentric internal membranes are also seen in the absence of cytochrome oxidase activity. Distal tubular cytochrome oxidase activity does not vary significantly, and the mitochondria uniformly have transverse cristae and dense mitochondrial granules in the matrix. The relationship of the modified mitochondria to similar forms reported previously in other situations under physiological and pathological conditions, and to mitochondria in tissues known to have low oxidative capacity, is discussed. It is concluded that no similar mitochondrial forms have previously been associated with low cytochrome oxidase activity, and, conversely, no mitochondria of low oxidative capacity have previously been reported to have structural transformations similar to those described. A possible relationship to defective oxidative phosphorylation is discussed. It is suggested that some of the internal lamellar membranes of “myelin” figures may be remnants of mitochondrial internal membranes, but no strong or conclusive evidence can be offered. It is thought that both the enzymatic and the structural changes in mitochondria reflect a specific adaptive response of the proximal tubular cell. The modified mitochondria may well have acquired specific metabolic and functional properties, but the possibility of their being degenerative in nature has not been excluded.

Reconstruction of the reaction mechanism of cytochrome oxidase.

PREVIOUSLY it has been generally believed that the substrates of the cytochrome oxidase system, such as p-phenylene diamine and hydroquinone, are only oxidized by cytochrome oxidase through cytochrome c. However, since p-phenylene diamine is fairly well oxidized by particulate cytochrome oxidase preparations in the absence of cytochrome c, whereas the other substrates, such as hydroquinone are not, Okunuki1 has suggested that the reductant of the so-called Nadi-reaction can reduce not only cytochrome c but also cytochrome oxidase. This was confirmed spectrophotometrically by Smith2.