Biochemical and biophysical studies on cytochrome c oxidase XV. Reaction with fluoride

1. Fluoride is a mixed-type inhibitor of the cytochrome c oxidase activity with a K_i for the free enzyme of 10 mM and a K_i for the cytochrome c-complexed enzyme of 35 mM.

2. Fluoride shifts the γ-band of the enzyme from 423 to 421 nm and the -band from 597 to 598 nm. The difference spectrum (oxidized enzyme in the presence of fluoride minus oxidized enzyme) has peaks at 400, 453, 482, 605 and 638 nm and troughs at 430, 520, 552 and 674 nm. The changes in absorbance are small (about 3% at absorbance maxima) with respect to those of other hemoproteins.

3. On addition of fluoride to isolated cytochrome c oxidase 3 reactions can be distinguished: (I) a bimolecular binding reaction (K_on = 4 M⁻¹ · s⁻¹ and k_off = 2.9 · 10⁻²s⁻¹ at 25 °C, pH 7.4) contributing at 638 nm and 430 nm; (II) a first-order reaction (k = 2.4 · 10⁻²) s⁻¹ at 22 °C, pH 7.2) visible mainly at 430 nm and (III) a very slow reaction with a half-time in the order of 10 min.

4. The spectroscopic dissociation constants for the fluoride binding, determined from Hill plots using the absorbance changes at 638 and 430 nm, are similar (7 and 10 mM, respectively, at 22 °C, pH 7.2).

5. A mechanism for the reaction is discussed in which the bimolecular binding reaction is followed by a conformational change of the enzyme-fluoride complex.     

Biochemical and biophysical properties of purified phospholipid vesicles containing bovine heart cytochrome c oxidase

Liposomes containing bovine heart cytochrome c oxidase (COV) prepared by the cholate dialysis technique were purified from those devoid of the enzyme using discontinuous sucrose density ultra centrifugation to eliminate interference in proton-pumping assays. This technique was also used to purify liposomes containing cytochrome c oxidase depleted in subunit III (COV-III), a COX enzyme preparation with altered subunit structure, to assess if the technique could be applied to COX enzymes in which structural and functional changes have occurred. Upon discontinuous sucrose density ultra gradient ultracentrifugation, either COV or COV-III were separated into two bands. Liposomes devoid of enzyme sedimented into the 12% sucrose layer, whereas enzyme-containing liposomes (pCOV or pCOV-III) were found in the 13% sucrose layer. The yield of both pCOV or pCOV-III was greater than 60% (based on heme aa(3) content), suggesting a similar distribution of cytochrome c oxidase (COX) and subunit III-depleted enzyme (COX-III) in the purified liposomes. The number of COX or COX-III molecules per phospholipid vesicle in purified fractions was estimated to be two. Removal of subunit III (M(r)=29,918) from COX resulted in a 30% decrease in electron transfer activity (either in COV-III or pCOV-III) when compared with COV and pCOV, respectively. Both pCOV and pCOV-III exhibited low endogenous proton permeability, as assessed by possessing high respiratory control ratios (14 and greater) and by having similar valinomycin concentration dependencies for stimulation of electron transfer activity in the presence of saturating amounts of CCCP. COV-III and pCOV-III exhibited a 39-44% decrease in proton-pumping activity when compared with COV and pCOV. These results showed that the separation of COX containing liposomes from those lacking enzyme by sucrose density gradient centrifugation can be used to characterize the biophysical properties of these liposomes.     

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.     

Biochemical and biophysical studies on cytochrome c oxidase XVI. Circular dichroic study of cytochrome c oxidase and its ligand complexes

1. CD spectra of cytochrome c oxidase have been determined both in the absence and presence of the extrinsic ligands CO, NO, cyanide and azide.2. CO and NO affect the CD spectrum of cytochrome c oxidase in a similar way.3. Cyanide and azide also affect the CD spectrum of cytochrome c oxidase in a similar way, but distinctly different from CO and NO.4. From the CD spectra of the oxidized and reduced enzyme, in the presence and absence of extrinsic ligands, CD difference spectra (reduced minus oxidized) are calculated for the so-called cytochrome a and cytochrome a₃ moieties of the enzyme.5. These spectra are largely dependent on the extrinsic ligand used. It is therefore concluded that these spectra do not represent independent cytochrome a and cytochrome a₃ difference spectra, but that heme-heme interactions occur within the cytochrome c oxidase molecule, in such a way that binding of a ligand to one of the heme a groups of cytochrome c oxidase affects the spectral properties of the other heme a group.6. As a consequence, ligand-binding studies cannot give information as to the pre-existence of separate cytochrome a and cytochrome a₃ moieties in the absence of extrinsic ligands.     

Biochemical and biophysical studies on cytochrome c oxidase XIV. The reaction with cytochrome c as studied by pulse radiolysis

1. The reduction of cytochrome c oxidase by hydrated electrons was studied in the absence and presence of cytochrome c.

2. Hydrated electrons do not readily reduce the heme of cytochrome c oxidase. This observation supports our previous conclusion that heme a is not directly exposed to the solvent.

3. In a mixture of cytochrome c and cytochrome c oxidase, cytochrome c is first reduced by hydrated electrons (k = 4 · 10¹⁰ M⁻¹ · s⁻¹ at 22 °C and pH 7.2) after which it transfers electrons to cytochrome c oxidase with a rate constant of 6 · 10⁷ M⁻¹ · s⁻¹ at 22 °C and pH 7.2.

4. It was found that two equivalents of cytochrome c are oxidized initially per equivalent of heme a reduced, showing that one electron is accepted by a second electron acceptor, probably one of the copper atoms of cytochrome c oxidase.

5. After the initial reduction, redistribution of electrons takes place until an equilibrium is reached similar to that found in redox experiments of Tiesjema, R. H., Muijsers, A. O. and Van Gelder, B. F. (1973) Biochim. Biophys. Acta 305, 19–28.     

Interactions of sulphide and other ligands with cytochrome c oxidase. An electron-paramagnetic-resonance study.

The effect of sulphide on resting oxidized cytochrome c oxidase was studied by both e.p.r. and optical-absorption spectroscopy. Excess sulphide causes some reduction of cytochrome a, CuA and CuB, and the formation of the cytochrome a3-SH complex after about 1 min. After several hours in the presence of excess sulphide only the e.p.r. signals due to low-spin ferricytochrome a3-SH persist, giving a partially reduced species. Re-oxidation of this partially reduced sulphide-bound enzyme by ferricyanide makes all of the metal centres except CuB detectable by e.p.r. We conclude that sulphide has reduced and binds to CuB as well as to ferricytochrome a3. Sulphide binding to cuprous CuB may raise its mid-point potential and make re-oxidation difficult. Addition of reductant (ascorbate + NNN'N'-tetramethyl-p-phenylenediamine) and sulphide together to the oxidized resting enzyme produces a species in which cytochrome a and CuA are nearly completely reduced and cytochrome a3 is e.p.r.-detectable as approx. 80% of one haem in the low-spin sulphide-bound complex. The g = 12 signal of this partially reduced derivative is almost unchanged in magnitude relative to that of the resting enzyme; this suggests that the g = 12 signal may arise from less than 20% of the enzyme and that it may be relatively unreactive to both ligation and reduction. Such a reactivity pattern of the g = 12 form of the oxidase is also demonstrated with the ligands F- and NO, which are thought to bind to cytochrome a3 and CuB respectively.