Biochemical and biophysical studies on cytochrome aa3. VI. Reaction of cyanide with oxidized and reduced enzyme

1.

1. The reaction of cyanide with cytochrome aa₃ in the fully oxidized and reduced states has been studied. In both cases a single molecule of cyanide is bound reversibly per molecule of aa₃ (that is, 1 mole cyanide per 2 equivalents of haem a).     

Ionic strength effects on cytochrome aa3 kinetics

1. The occurrence of an optimal ionic strength for the steady-state activity of isolated cytochrome aa₃ can be attributed to two opposite effects: upon lowering of the ionic strength the affinity between cytochrome c and cytochrome aa₃ increases, whereas in the lower ionic strength region the formation of a less active cytochrome c-aa₃ complex limits the ferrocytochrome c association to the low affinity site.2. At low ionic strength, the reduction of cytochrome c-aa₃ complex by ferrocytochrome c₁ proceeds via non-complex-bound cytochrome c. Under these conditions the positively charged cytochrome c provides the electron transfer between the negatively charged cytochromes c₁ and aa₃.3. Polylysine is found to stimulate the release of tightly bound cytochrome c from the cytochrome c-aa₃ complex. This property points to the existence of negative cooperativity between the two binding sites. We suggest that the stimulation is not restricted to polylysine, but also occurs with cytochrome c.4. Dissociation rates of both high and low affinity sites on cytochrome aa₃ were determined indirectly. The dissociation constants, calculated on the basis of pre-steady-state reaction rates at an ionic strength of 8.8 mM, were estimated to be 0.6 nM and 20 μM for the high and low affinity site, respectively.     

Occurrence of cytochrome aa3 in Anacystis nidulans

The cytochrome content of membrane fragments prepared from the bluegreen alga (cyanobacterium) Anacystis nidulans was examined by difference spectrophotometry. Two b-type cytochromes and a hitherto unknown cytochrome a could be characterized. In the reduced-minus-oxidised difference spectra the a-type cytochrome showed an α-band at 605 nm and a γ-band at 445 nm. These bands shifted to 590 and 430 nm, respectively, in CO difference spectra. NADPH, NADH and ascorbate reduced the cytochrome through added horse heart cytochrome c as electron mediator. In presence of KCN the reduced-minus-oxidised spectrum showed a peak at 600 nm and a trough at 604 nm. Photoaction spectra of O₂ uptake and of horse heart cytochrome c oxidation by CO-inhibited membranes showed peaks at 590 and 430 nm. These findings are consistent with cytochrome aa₃ being the predominant respiratory cytochrome c oxidase in Anacystis nidulans.     

Biochemical and biophysical studies on cytochrome aa3 II. Conformations of oxidized cytochrome aa3

1.

1. The spectral properties of ‘oxygenated’ cytochrome c oxidase, prepared by passing air through the dithionite-reduced enzyme solution, were compared with those of the ferric enzyme.     

The pre-steady state reaction of ferrocytochrome c with the cytochrome c-cytochrome aa3 complex

1. Using stopped-flow technique we have investigated the electron transfer form cytochrome c to cytochrome aa₃ and to the (porphyrin) cytochrome c-cytochromeaa₃ complex.2. In a low ionic strength medium, the pre-steady state reaction occurs in a biphasic way with rate constants of at least 2 · 10⁸ M^?1 · s^?1 and about 10⁷ M^?1 · s^?1 (I = 8.8 mM, pH 7.0, 10° C), respectively.3. A comparison of the rate constants, determined in the presence of an excess of cytochrome c with those found in the presence of an excess of cytochrome aa₃ reveals the existence of two slower reacting sites on the functional unit (2 hemes and 2 coppers) of cytochrome aa₃. On basis of these results we discuss various models. If no site-site interactions are assumed (non-cooperative model) cytochrome aa₃ has 2 high and 2 low affinity sites available for the reaction with ferrocytochrome c. If negative cooperativity occurs, cytochrome aa₃ has 2 high affinity sites which change into 2 low affinity sites upon binding of one cytochrome c molecule. The latter model is favoured.     

The reaction of cytochrome aa3 with (porphyrin) cytochrome c as studied by pulse radiolysis

(1) Using the pulse-radiolysis and stopped-flow techniques, the reactions of iron-free (porphyrin) cytochrome c and native cytochrome c with cytochrome aa₃ were investigated. The porphyrin cytochrome c anion radical (generated by reduction of porphyrin cytochrome c by the hydrated electron) can transfer its electron to cytochrome aa₃. The bimolecular rate constant for this reaction is 2·10⁷ M^?1·s^?1 (5 mM potassium phosphate, 0.5% Tween 20, pH 7.0, 20°C). (2) The ionic strength dependence of the cytochrome c-cytochromeaa₃ interaction was measured in the ionic strength range between 40 and 120 mM. At ionic strengths below 30 mM, a cytochrome c-cytochrome aa₃ complex is formed in which cytochrome c is no longer reducible by the hydrated electron. A method is described by which the contributions of electrostatic forces to the reaction rate can be determined. (3) Using the stopped-flow technique, the effect of the dielectric constant (?) of the reaction medium on the reaction of cytochrome c with cytochrome aa₃ was investigated. With increasing ? the second-order rate constant decreased.     

Control of respiration in proteoliposomes containing cytochrome aa3. I. Stimulation by valinomycin and uncoupler

1. Both valinomycin and p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) are required for full release of respiration by cytochrome c oxidase-containing proteoliposomes (prepared by sonicating beef heart cytochrome aa₃ in salt solution with 4 parts phosphatidylcholine, 4 parts phosphatidylethanolamine and 2 parts cardiolipin) in the presence of external ascorbate and cytochrome c. In the absence of valinomycin the response to FCCP is rather sluggish, as reported by Wrigglesworth et al. (1976) (Abstracts, 10th Int. Congr. Biochem., No. 06-6-230).2. The K_m for cytochrome c in 67 mM, pH 7.4, phosphate buffer with ascorbate as substrate, was 9 μM in both absence and presence of valinomycin and FCCP. Energization thus acts non-competitively towards cytochrome c oxidation.3. The apparent K_m for oxygen is greater in the energized than in the deenergized state; double reciprocal plots of respiration rate versus oxygen concentration are concave downward in the absence of uncouplers, as found with intact mitochondria. Energization thus acts “competitively” towards oxygen.4. Despite the lack of a functional ATPase system, all the kinetic features of energization found in intact mitochondria can be mimicked in the reconstituted liposomes. This supports the chemiosmotic idea that electrical and perhaps H⁺ gradients modify the oxidase activity in reconstituted vesicles.     

Superoxide oxidase and reductase activity of cytochrome b559 in photosystem II

This study provides evidence for the superoxide oxidase and the superoxide reductase activity of cytochrome b₅₅₉ (cyt b₅₅₉) in PSII. It is reported that in Tris-treated PSII membranes upon illumination, both the intermediate potential (IP) and the reduced high potential (HP^red) forms of cyt b₅₅₉ exhibit superoxide scavenging activity and interconversion between IP and HP^red form. When Tris-treated PSII membranes were illuminated in the presence of spin trap EMPO, the formation of superoxide anion radical (O₂⁻) was observed, as confirmed by EPR spin-trapping spectroscopy. The observations that the addition of enzymatic (superoxide dismutase) and non-enzymatic (cytochrome c, α-tocopherol and Trolox) O₂⁻ scavengers prevented the light-induced conversion of IP ↔ HP^red cyt b₅₅₉ confirmed that IP and HP^red cyt b₅₅₉ are reduced and oxidized by O₂⁻, respectively. Redox changes in cyt b₅₅₉ by an exogenous source of O₂⁻ reconfirmed the superoxide oxidase and reductase activity of cyt b₅₅₉. Furthermore, the light-induced conversion of IP to HP^red form of cyt b₅₅₉ was completely inhibited at pH > 8 and by chemical modification of the imidazole ring of histidine residues using diethyl pyrocarbonate. We proposed that a change in the environment around the heme iron, induced by the protonation and deprotonation of His²² residue generates a favorable condition for the oxidation and reduction of O₂⁻, respectively.     

Time-resolved single-turnover of ba3 oxidase from Thermus thermophilus

The kinetics of the oxidation of fully-reduced ba₃ cytochrome c oxidase from Thermus thermophilus by oxygen were followed by time-resolved optical spectroscopy and electrometry. Four catalytic intermediates were resolved during this reaction. The chemical nature and the spectral properties of three intermediates (compounds A, P and O) reproduce the general features of aa₃-type oxidases. However the F intermediate in ba₃ oxidase has a spectrum identical to the P state. This indicates that the proton taken up during the P → F transition does not reside in the binuclear site but is rather transferred to the covalently cross-linked tyrosine near that site. The total charge translocation associated with the F → O transition in ba₃ oxidase is close to that observed during the F → O transition in the aa₃ oxidases. However, the P_R → F transition is characterized by significantly lower charge translocation, which probably reflects the overall lower measured pumping efficiency during multiple turnovers.     

Ferrocyanide as electron donor to cytochrome aa3. Cytochrome c requirement for oxygen uptake

1. In the absence of cytochrome c, ferrocyanide or ferrous sulphate reduces cytochrome c oxidase (EC 1.9.3.1), but no continuous oxygen uptake ensues, as it does with N,N,N′,N′-Tetramethyl-p-phenylenediamine or reduced phenazine methosulphate as reductants, unless a substoichiometric amount of cytochrome c or an excess of clupein is present. Cytochrome c cannot be replaced by porphyrin cytochrome c.2. Cytochrome c, porphyrin cytochrome c and clupein all stimulate the reduction of cytochrome aa₃ by ferrocyanide.3. A model is proposed to explain these findings in which a high-affinity site for cytochrome c on the oxidase regulates the access of hydrophilic electron donors to a low-affinity site, and reduction via the high-affinity site is required for continuous oxygen uptake.4. Furthermore, it is shown that upon reaction of oxidase with ferrocyanide, cyano-oxidase is formed.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b₅ has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b₅vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b₅ upon the interaction with its redox partners is discussed.     

The effect of pH and ionic strength on the pre-steady-state reaction of cytochrome c and cytochrome aa3

(1) In the pH range between 5.0 and 8.0, the rate constants for the reaction of ferrocytochrome c with both the high- and low-affinity sites on cytochrome aa₃ increase by a factor of approx. 2 per pH unit. (2) The pre-steady-state reaction between ferrocytochrome c and cytochrome aa₃ did not cause a change in the pH of an unbuffered medium. Furthermore, it was found that this reaction and the steady-state reaction are equally fast in H₂O and ²H₂O. From these results it was concluded that no protons are directly involved in a rate-determining reaction step. (3) Arrhenius plots show that the reaction between ferrocytochrome c and cytochrome aa₃ requires a higher enthalpy of activation at temperatures below 20°C (15–16 kcal/mol) as compared to that at higher temperature (9 kcal/mol). We found no effect of ionic strength on the activation enthalpy of the pre-steady-state reaction, nor on that of the steady-state reaction. This suggests that ionic strength does not change the character of these reactions, but merely affects the electrostatic interaction between both cytochromes.     

Control of respiration in proteoliposomes containing cytochrome aa3 II. Inhibition by carbon monoxide and azide

1. Carbon monoxide (CO) acts competitively towards oxygen when the latter is taken up in respiration by cytochrome aa₃-containing proteoliposomes, both in the presence of p-trifluoromethoxy carbonyl cyanide phenylhydrazone and valinomycin (deenergized state) and in their absence (energized state). At high levels of CO, the double reciprocal plots ($\text{1}\text{v}$ vs. $\text{1}\text{[}\text{O}{}_2\text{]}$) in the energized and deenergized states are parallel, i.e. energization acts “anti-competitively” towards oxygen, and the “respiratory control ratio” decreases as the oxygen concentration decreases.2. Azide acts non-competitively towards cytochrome c when the latter is oxidized by cytochrome aa₃-containing proteoliposomes both in the energized and deenergized (plus p-trifluoromethoxy carbonyl cyanide phenylhydrazone and valinomycin) conditions. At low azide concentrations the apparent K_i for azide is unaffected by energization, but at high azide levels the K_i increases in energized liposomes, i.e. the “respiratory control ratio” decreases as the azide concentration increases.3. It is concluded that the inhibitor experiments are consistent with but do not prove the concept that the oxidase molecules in a single vesicle are responding to a single “energization state” or set of electrochemical gradients. This and other models are discussed.     

The reaction between cytochrome C1 and cytochrome C

The kinetics of electron transfer between the isolated enzymes of cytochrome c₁ and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c₁ and ferricytochrome c is fast; the second-order rate constant (k₁) is 3.0 · 10⁷ M^?1 · s^?1 at low ionic strength (I = 223 mM, 10°C). The value of this rate constant decreases to 1.8 · 10⁵ M^?1 · s^?1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c₁ and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c₁ and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c₁ and ferricytochrome c and the reverse reaction are k₁ = 2.4 · 10⁵ M^?1 · s^?1 and k_?1 = 3.3 · 10⁵ M^?1 · s^?1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10°C). The ‘equilibrium’ constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c²⁺₁ + cytochrome c³⁺ai cytochrome c³⁺₁ + cytochrome c²⁺.