Mammalian heart cytochrome oxidase - Oregon State BB ... Title MAMMALIAN HEART CYTOCHROME OXIDASE

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Text of Mammalian heart cytochrome oxidase - Oregon State BB ... Title MAMMALIAN HEART CYTOCHROME OXIDASE

  • AN ABSTRACT OF THE THESIS OF

    Mario Kuboyama for the Doctor of Philosophy in Biochemistry (Name) (Degree) (Major)

    Date thesis is presented October 26, 1964

    Title MAMMALIAN HEART CYTOCHROME OXIDASE

    Abstract approved

    Preparation of a cytochrome £-cytochrome oxidase complex

    was achieved, using crystalline beef cytochrome£ and purified

    cytochrome oxidase from beef heart muscle, or from the

    Keilin-Hartree preparation. Succinate oxidase was reconstitu­

    ted with the complex.

    A mixture of cytochrome oxidase, solubilized with non-

    ionic detergent, and excess cytochrome£ was exposed to sonic

    irradiation. The cytochrome £-cytochrome oxidase complex was

    isolated using gel filtration of the mixture. The ratio of

    the concentration of cytochrome £ to cytochrome oxidase in

    the complex was increased with the length of sonic treatment.

    The complex with the maximum ratio of one was separated after

    a 45-60 minute period of sonic irradiation.

    Differential sedimentation and chromatography on Sephadex

    showed the complex to be an integrated entity of two components.

  • The complex could be £armed with cytochrome oxidase and either

    intact cytochrome £ or guanidin~ted cytochrome £· However~

    both acetylated cytochrome £ and succinylated cytochrome £

    completely £ailed to £orm any kind o£ complex with cytochrome

    oxidase These results indicate that the interacting £orceo

    between the two components is mainly electrostatic.

    Absolute and di££erence absorption spectra of the carbon

    monoxide compound o£ the comple~ showed a unique, distinct

    maximum at 415 mM. On the other nand, a "£ree mixture" of

    both components exhibited no absorption peak at the same wave

    length in the presence of CO. The £ormation o£ this CO com~

    pound was prevented by sodium cholate. A£ter separation by

    means o£ cation exchange resin, neither component o£ the

    complex showed the absorption ~aximum at 415 mM in the pre­

    sence of CO. From these and other observations, the possi­

    bility o£ a con£ormational change in the protein moiety o£ the

    cytochromes as a result of the interaction was hypothesi~ed in

    re£erence to the electron transport mechanism.

    In£rared spectrum of the c~mplex showed no superposition

    o£ absorption bands o£ the two components but showed a unique

    -1 band at 950 and at 1050 em •

    The physiological activity of the complex was veri£ied

    by the £unctional reconstitution o£ succinate oxidase with

    soluble succinate dehydrogenase, the cytochrome £-£ 1

    particle 1

    and the complex. The reconstituted succinate oxidase was

  • inhibited by the usual respiratory inhibitors in the same

    manner as the Keilin-Hartree heart muscle preparation. The

    carbon monoxide inhibition of the reconstituted succinate

    oxidase was reversed by light. The behavior of the complex

    in the oxidation of ascorbate was the same as that of the

    heart muscle preparation. Addition of a catalytic amount of

    tetramethyl-p-phenylenediamine dramatically stimulated

    ascorbate oxidation.

    The cytochrome oxidase (cytochrome ~plus cytochrome ~3 ) was functionally and structurally reconstituted from cytochrome

    a and intact heme a. About sixty percent of the original

    cytochrome oxidase activ~ty was recovered from an incubation

    mixture of cytochrome ~' ascorbate-EDTA, cytochrome £ and

    hematin a. Hematin a was prepared frpm cytochrome oxidase pr

    directly from heart muscle mince by means of metalation of

    purified porphyrin ~· After removal of excessive hematin ~

    by Sephadex chromatography, the reconstituted oxidase showed

    spectral properties almost identical to those of the intact

    preparation; a slight difference was found at the a-band.

  • MAMMALIAN HEART CYTOCHROME OXIDASE

    by

    MORIO KUBOYAMA

    A THESIS

    submitted to

    OREGON STATE UNIVERSITY

    in partial fulfillment of the requirements for the

    degree of

    DOCTOR OF PHILOSOPHY

    Jtme 1965

  • APPROVED:

    Pro£eesor o£ Chemistry

    In Charge o£ Major

    Chairman o£ Department o£ Chemistry

    /

    Dean o£ Graduate School

    Dat~ thesis ~s presented October 26, 1964

    Typed by Clistie Stoddard

  • ACKNOWLEDGEMENT

    The author wishes to express his deep appreciation to

    Professor Tsoo E. King £or his most careful guidance and

    continued interest. Without his penetrating insight and

    generous patience, none o£ this work would have been doneo

    The author is also grateful to Dean Vernon Ho Cheldelin

    £or his kind interest and generosity.

    Acknowledgement is due colleagues at Science Research

    Institute £or their advice, especially Dr. Pat W. Camerino

    and Mrs. Carole M. Staneslow £or their help in the prepara­

    tion o£ the thesis.

    The author is indebted to the U. S. Educational

    Commission in Japan and the U. S. Public Health Service £or

    financial support during his study at Oregon State University.

  • 1

    TABLE OF CONTENTS

    Page

    GENERAL INTRODUCTION-.~~-- .. -~--------------------------=--

    1. CYTOCHROME c-CYTOCHROME OXIDASE COMPLEX

    EXP~RIMENTAL PROCEDURES

    Materials----------------=--------------~--------- Methods------------------------------------------­

    8 10

    RESULTS

    Preparation of Cytochrome c-Cytochrome Oxidase Complexes and Electronic Absorption Specta of the Complex----------------~------------------- Infrared Spectra of the Complex and Individual Components--------------------~-~----------------- Integrity of tne Cytochrome £~Cytochrome Oxidase Complex; Dissociation of the Complex-------------­ Complex Formation of Chemically Modified Cytochrome c with Cytochrome Oxidase-------------­ CO-compoun~and eN-compound of the Complex-------- Ascorbate Oxidation with Cytochrome £-Cytochrome Oxidase Complex and TM~D Stimulation-------------­

    15

    28

    35

    40 41

    53

    DISCUSSION-----------~~--------~--~-----~----------- 56

    2. RECONSTITUTION QF SUCCINATE CYTOCHROME OXIDASE COMPLEX

    OXIDASE WITH CYTOCHROME £­

    INTRODUCTION---------------------------------------­ 64

    EXPERIMENTAL PROCEDURES

    Materials----------------------------------------­ 65

    RESULTS

    Reconstitution of Succinate Oxidase with Cytochrome £-Cytochrome Oxidase Complex, Solu­ ble Succinate Dehydrogenase and Cytochrome k-£

    1 Particle; Turnover Number------------------------­ 66

  • LIST OF FIGURES

    Fig~r~ Page

    l Sephadexcolumnchromatography of the sonically irradiated mixture of cytochrome £ and cyto­ chrome oxidase----------------------------------­ 18

    2 Elution pattern of the cytochrome £-cytochrome oxidase complex of molar ratio l from a Sephadex column--------------------------------­ 20

    JA Absorption spectra of cytochrome £-cytoch~ome oxidase complex--------------------------------­ ~~

    JB The absolute absorption spectra of the complex (1:1) in the ultraviolet region-----------------~ 25

    ~ Difference spectra of the cytochrome £­ cytochrome oxidase complex---------------------­ 27

    S Infrared spectrum of cytochrome £­ cytochrome oxidase complex---------~------------ 30

    6 Infrared spectrum of cytochrome c--------------­ 32

    7 Infrared spectrum of cytochrome oxidase--------­

    8 Dissociation of oxidase complex

    cytochrome £-cytochrome at different ionic strengths-~~- 39

    9 CO difference spectrum of cytochrome £­ cytochrome oxidase complex and effect of cholate

    10 Absorption spectra of cytochrome oxidase after SO minutes of sonic irradiation----------------­ ~6

    ll Absorption spectra of cytochrome £ separated from the cytochrome £-cytochrome oxidase complex----------------------------------------­ ~8

    12 CO spectra: (A) The cytochrome c-cytqchrome oxidase complex, (B) Reconstituted succinate oxidase, (C) The Keilin-Hartree heart muscle preparation------------------------------------­ so

    lJ CN compound of the cytochrome £-cytochrome oxidase complex--------------------------------­ 52

  • LIST OF FIGURES (continued)

    Figure Page

    14: Ascorbate oxidation with the cytochrome E.­ cytochrome oxidase complex and with the Keilin­ Hartree heart muscle preparation~~~--------------- 55

    15 Replotted CO difference spectrum of Ball's "electron transmitter t;!ystem"""----------.-------·-­ 59

    16 Reconstitution of succinate oxidase with succinate cytochrome £ reductase and cytochrqme £-cytochrome oxidase complex---------------~-----~ 69

    17 Effect of the concentration of the cytochrome £• cytochrome oxidase complex on the activity of the reconstituted succinate oxidase------------------­ 71

    18 Photo-irradiation effect on the carbon monoxide inhibited reconstituted succinate oxidase and the Keilin-Hartree heart muscle preparation------- 76

    19 Optimal pH for succinate oxidase activity of the reconstituted succinate oxidase·------~------- 81

    20 Optimal pH for succina