B.F. Van Gelder

On cytochrome c oxidase I. The extinction coefficients of cytochrome a and cytochrome a3

Summary 1. In agreement with Gibson, a shoulder at 420–424 m μ is present in the absorption spectra of preparations of cytochrome c oxidase (ferrocytochromec: oxygen oxidoreductase, EC 1.9.3.1) reduced with Na 2 S 2 O 4 . An examination of the spectra in the presence and absence of cyanide showed that this is due to the presence of equal amounts of non-reducible cytochrome a and non-reducible cytochrome a 3 . 2. Cytochrome c oxidase was titrated with NADH and phenazine metho-sulphate. It was found that each mole of NADH reduced 1 mole of haem a and 1 atom of copper. 3. Functionally inactive copper present in the enzyme preparation, added Cu(II) and Fe(III) salts and mitochrome, a modification product of cytochrome c oxidase, are not reduced by phenazine methosulphate, under the conditions of the titration. 4. The values of Δem M (reduced minus oxidized) at 605 m μ , and 445 m μ , respectively, were: cytochrome a , 19.4 and 82; cytochrome a 3 , 4.6 and 82; cytochrome aa 3 , 24.0 and 164. 5. Concentrations of cytochrome a, calculated on the basis of the widely used value of 16.5 for Δem M ( A 605 m μ A 630 m μ ), are 150% too high.

Demonstration by EPR spectroscopy of the functional role of iron in soybean lipoxygenase-1

1. The EPR spectrum at 15 degrees K of soybean lipoxygenase-1 in borate buffer pH 9.0 has been studied in relation to the presence of substrate (linoleic acid), product (13-L-hydroperoxylinoleic acid) and oxygen. 2. The addition of 13-L-hydroperoxylinoleic acid to lipoxygenase-1 at pH 9.0 gives rise to the appearance of EPR lines at g equals 7.5, 6.2, 5.9 and 2.0, and an increased signal at g equals 4.3. 3. In view of the effect of the end product on both the kinetic lag period of the aerobic reaction and the fluorescence of the enzyme, it is concluded that 13-L-hydroperoxylinoleic acid is required for the activation of soybean lipoxygenase-1. Thus it is proposed that the enzyme with iron in the ferric state is the active species. 4. A reaction scheme is presented in which the enzyme alternatingly exists in the ferric and ferrous states for both the aerobic and anaerobic reaction.

Generation of superoxide radicals during the autoxidation of mammalian oxyhemoglobin

Abstract It is shown that during autoxidation of mammalian oxyhemoglobin to methemoglobin superoxide anions (O2- are generated. We suggest that the physiological role of superoxide dismutase in erythrocytes is to prevent the deleterious action of the superoxide anion.

Isolation procedure and some properties of myeloperoxidase from human leucocytes

1. A rapid isolation procedure with a high yield for pure myeloperoxidase (donor:H2O2 oxidoreductase, EC 1.11.1.7) from normal human leucocytes is described. The enzyme was solubilized from leucocytes with the detergent, cetyltrimethylammonium bromide, and purified to apparent homogeneity. The yield of the enzyme was 17% with an absorbance ratio A430nm/A280nm = 0.85. 2. The purified enzyme showed three isoenzyme bands after polyacrylamide gel electrophoresis; ultracentrifuge studies indicated one homogeneous band with a molecular weight of 144 000. After reduction of myeloperoxidase, sodium dodecyl sulfate gel electrophoresis resolved an intense band (63 000 daltons) and a weak band (81 000 daltons). 3. The carbohydrate content of the enzyme was at least 2.5%. Mannose, glucose and N-acetylglucosamine were present. The amino acid composition is reported. 4. The EPR spectrum exhibited a high-spin heme signal with rhombic symmetry (gx = 6.92, gy = 5.07 and gz = 1.95). Upon acidification this signal was converted into a signal with more axial symmetry (g perpendicular = 5.89). At high pH (9.5) the EPR spectrum of the enzyme only shows low-spin ferric heme resonances. The circular dichroism spectra of ferric and ferrous myeloperoxidase in the visible and ultraviolet region show maxima and minima in ellipticity.

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

Abstract 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 Lembergs ( 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 III. The EPR spectrum of NO-ferrocytochrome a3

Abstract 1. 1. The reaction of hydroxylamine with oxidized cytochrome aa 3 results in the formation of NO-ferrocytochrome a 3 . 2. 2. NO-ferrocytochrome a 3 has an EPR spectrum with g x = 2.09, g y = 2.0, g z = 2.005. g z is split into nine lines, due to interaction with two nitrogen nuclei: A N 1 = 21.1 gauss; A N 2 = 6.8 gauss. 3. 3. It is suggested from this spectrum that histidine is the fifth ligand of the iron in cytochrome a 3 .

On cytochrome c oxidase. II. The ratio of cytochrome a to cytochrome a3.

Summary 1. Cytochrome c oxidase was titrated, under anaerobic conditions, with NADH and phenazine methosulphate in the presence of chelating agents. Three different effects of chelating agents on the titration were found from which it can be concluded that cyanide and azide prevent the reduction of half the haem a and half the copper, EDTA and salicylaldoxime block the reduction of half the copper, and fluoride prevents the reduction of half the haem a. 2. It is concluded that the ratio of cytochrome a: cytochrome a3 is 1. 3. Two different kinds of functionally active copper atoms are present in equal amounts. One is unreactive towards chelating agents such as cyanide, azide, EDTA and salicylaldoxime, whereas, under the conditions of the titration, the reduction of the other can be prevented. 4. Chemical copper determinations confirm the site of action of metal-combining compounds under the conditions of the titration. 5. The cyanide-sensitive copper contributes about one-half of the difference spectrum (oxidized minus reduced) at 830 mμ, whereas the contribution of the other half is due either to haem a of cytochrome a or to the cyanide-insensitive copper or to both. The haem a of cytochrome a3 does not contribute to the difference spectrum at 830 mμ.

Electron transfer after flash photolysis of mixed-valence carboxycytochrome c oxidase

The light-induced difference spectra of the fully reduced (a2+ a23+-CO) complex and the mixed-valence carboxycytochrome c oxidase (a3+ a23+-CO) during steady-state illumination and after flash photolysis showed marked differences. The differences appear to be due to electron transfer between the redox centres in the enzyme. The product of the absorbance coefficient and the quantum yield was found to be equal in both enzyme species, both when determined from the rates of photolysis and from the values of the dissociation constants of the cytochrome a23+-CO complex. This would confirm that the spectral properties of cytochrome a3 are not affected by the redox state of cytochrome a and CuA. When the absorbance changes after photolysis of cytochrome a23+-CO with a laser flash were followed on a time scale from 1 mus to 1 s in the fully reduced carboxycytochrome c oxidase, only the CO recombination reaction was observed. However, in the mixed-valence enzyme an additional fast absorbance change (k = 7 X 10(3) s-1) was detected. The kinetic difference spectrum of this fast change showed a peak at 415 nm and a trough at 445 nm, corresponding to oxidation of cytochrome a3. Concomitantly, a decrease of the 830 nm band was observed due to reduction of CuA. This demonstrates that in the partially reduced enzyme a pathway is present between CuA and the cytochrome a3-CuB pair, via which electrons are transferred rapidly.

The effect of pH and ionic strength on the steady-state activity of isolated cytochrome c oxidase

1. The turnover number and apparent Km of isolated beef-heart cytochrome c oxidase were found to increase continuously when the pH was lowered from 8.6 to 4.6 (turnover number 32-630 s-1). In this pH range neither irreversible denaturation of the enzyme nor an optimum for the turnover number was observed. 2. The turnover number of cytochrome c oxidase was found to be independent of ionic strength. It was concluded that the dependence of the activity of cytochrome c oxidase on ionic strength is caused by a change in the value of Km for cytochrome c. 3. The pH dependence of the turnover number of cytochrome c oxidase can be described by a simple model in which at least three sites on the complex of cytochrome c oxidase with cytochrome c (pKa 8.0, 6.5 and 4.8) can take up a proton.

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

Abstract 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 · 10 10 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 · 10 7 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.