Bob F. Van Gelder

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). 2. 2. The difference spectrum of fully formed ferric aa3-cyanide complex minus oxidizid enzyme with maxima at 432, 540 and 585 nm resembles that of other cyanide ferrichaemoproteins; it appears to be a low-spin complex of cytochrome a3. 3. 3. The equilibrium constant for complex formation with ferric aa3 (KD) was approximately 1 μM; equilibration at 4° took several days as the rate constant for cyanide binding at low cyanide concentrations is only 1.8 M−1·sec−1 (pH 7·4, 21°). At high cyanide concentrations, the rate of spectroscopic complex formation becomes independent of cyanide with a first order constant of 0.018 sec−1. 4. 4. The difference spectrum obtained on addition of cyanide to ferrous aa3 shows a peak at 587 nm but only a small diminution of absorbance at 445 nm. The equilibrium constant for complex formation with the ferrous enzyme was 100 μM; the rate constant for cyanide binding was 150 M−1·sec−1, apparently representing a simple bimolecular step. 5. 5. The cytochrome aa3-azide complex reacts more rapidly with cyanide than does the free ferric enzyme; the rate constant for cyanide displacement of azide is 25 M−1·sec−1. The rate limiting step at high cyanide concentrations has the same velocity in the precence as in the absence of azide. 6. 6. The reaction of ferric enzyme with cyanide is interpreted in terms of a two-step reaction involving an initial weak binding to the protein (K′α of 10 mM) followed by binding to the a3 haem. A conformational change accompanying azide binding is proposed that facilitates the subsequent initial weak binding of cyanide (K′α (inpresenceofN3−) = 0.7 mM).

Biochemical and biophysical studies on cytochrome aa3: VIII. Effect of cyanide on the catalytic activity

Abstract 1. 1. Cyanide inhibits the catalytic activity of cytochrome aa 3 in both polarographic and spectrophotometric assay systems with an apparent velocity constant of 4·10 3 M −1 ·s −1 and a K i that varies from 0.1 to 1.0 μM at 22 °C, pH 7·3. 2. 2. When cyanide is added to the ascorbate-cytochrome c -cytochrome aa 3 −O 2 system a biphasic reduction of cytochrome c occurs corresponding to an initial K i of 0.8 μM and a final K i of about 0.1 μM for the cytochrome aa 3 −cyanide reaction. 3. 3. The inhibited species ( a 2 + a 3 3+ HCN) is formed when a 2+ a 3 3+ reacts with HCN, when a 2+ a 3 2+ HCN reacts with oxygen, or when a 3+ a 3 3+ HCN (cyano-cytochrome aa 3 ) is reduced. Cyanide dissociates from a 2+ a 3 3+ HCN at a rate of 2·10 −3 s −1 at 22 °C, pH 7.3. 4. 4. The results are interpreted in terms of a scheme in which one mole of cyanide binds more tightly and more rapidly to a 2+ a 3 3+ than to a 3+ a 3 3+ .

Biochemical and biophysical studies on cytochrome aa3. V. Binding of cyanide to cytochrome aa3

Abstract 1. 1. The effect of cyanide on the enzymic activity of cytochrome aa 3 shows that 1 mole cyanide is tightly bound to 1 mole cytochrome aa 3 . This is confirmed by the isolation of this complex (cyano-cytochrome aa 3 ). 2. 2. The rate constant for cyanide binding is 2 M −1 · sec −1 (pH 8.0, 0). From the K D of 7 · 10 −7 M (obtained from equilibrium dialyses), a dissociation rate constant of 1.4 · 10 −6 sec −1 is calculated. 3. 3. The time needed for equilibration of cyanide and cytochrome aa 3 depends on the redox state of the enzyme. 4. 4. Under conditions of reducing preincubation with ascorbate and cytochrome c the inhibition is noncompetitive towards cytochrome c with a K i of 8 · 10 −8 –9 × 10 −8 M. 5. 5. In the presence of reducing equivalents cyanide dissociates readily from cyano-cytochrome aa 3 to form enzymically active cytochrome aa 3 . The dissociation rate constant is 2 · 10 −3 sec −1 (pH 6.0, 25°). 6. 6. It is suggested, that the cavity in which the haem of cytochrome aa 3 is buried is more closed in the oxidized than in the reduced form and that the conformation is determined by the redox state of cytochrome a .

Bovine cytochrome c oxidases, purified from heart, skeletal muscle, liver and kidney, differ in the small subunits but show the same reaction kinetics with cytochrome c

(1) Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate of purified cytochrome c oxidase preparations revealed that bovine kidney, skeletal muscle and heart contain different cytochrome c oxidase isoenzymes, which show differences in mobility of the subunits encoded by the nuclear genome. No differences in subunit pattern were observed between the oxidase preparations isolated from kidney and liver. (2) The kinetics of the steady-state reactions between bovine ferrocytochrome c and the four types of bovine cytochrome c oxidase preparation were compared under conditions of both high- and low-ionic strength. Also the pre-steady-state kinetics were studied. Only minor differences were observed in the electron-transfer activity of the isoenzymes. Thus, our experiments do not support the notion that the subunits encoded by the nuclear genome act as modulators conferring different activities to the isoenzymes of cytochrome c oxidase. (3) The cytochrome c oxidase preparation from bovine skeletal muscle was found to consist mainly of dimers, whereas the enzymes isolated from bovine kidney, liver and heart were monomeric.

An EPR study of the photodissociation reactions of oxidised cytochrome c oxidase-nitric oxide complexes

Complexes of oxidised cytochrome c oxidase with NO in the absence and presence of ligands such as formate, fluoride and cyanide are photodissociable. After photodissociation at 10 K the EPR spectrum of the high-spin cytochrome a3+3 in the absence of ligands or in the presence of fluoride or formate disappears - as does the EPR spectrum of the low-spin cytochrome a3+3 in the presence of cyanide. The action spectra of the photodissociation reaction of these complexes show slight differences but all have maxima at 640-660 nm and below 400 nm, and are assigned to a diamagnetic Cu+B-NO+ complex. The differences in the action spectra in the presence of various ligands are due to binding of these anions to the cytochrome (a3-CuB) couple. The disappearance of the cytochrome a3 signal upon photodissociation of the Cu+B-NO+ complex is explained by a magnetic interaction between cytochrome a3+3 and Cu2+B in the photodissociated complex. The temperature at which NO recombines with Cu2+B is about 30 K and slightly affected by the presence of added ligands. It is suggested that in the oxidised ligand-cytochrome c oxidase complexes the coupling ligand between cytochrome a3+3 and Cu2+B is cyanide, fluoride and formate. The observation that two ligands may bind simultaneously to the cytochrome a3-CuB couple leads to further support for the notion that during turnover of cytochrome c oxidase both metal ions are involved in binding and reduction of oxygen.

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-cytochrome aa3 complex. 2. In a low ionic strength medium, the pre-steady state reaction occurs in a biphasic way with rate constants of at least 2.10(8) M-1.s-1 and about 10(7) M-1.S-1 (I=8.8 mM, pH 7.0, 10 degrees 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 cytochrome c oxidase-azide-nitric oxide complex as a model for the oxygen-binding site.

The complex of cytochrome c oxidase with NO and azide has been studied by EPR at 9.2 and 35 GHz. This complex which shows delta ms = 2 EPR triplet and strong anisotropic signals, due to the interaction of cytochrome a2+3 X NO (S = 1/2) and Cu2+B (S = 1/2), is photodissociable . Its action spectrum is similar to that of cytochrome a2+3 X NO with bands at 430, 560 and 595 nm, but shows an additional band in the near ultraviolet region. The quantum yield of the photodissociation process of cytochrome a2+3 X NO in the metal pair appears to depend on the redox state of CuB. When the photolysed sample was warmed to 77 K, a complex was observed with the EPR parameters of cytochrome a3+3 - N-3 - Cu1 +B (S = 1/2). This process of electron and ligand transfer can be reversed by heating the sample to 220 K. It is suggested that in the triplet species azide is bound to Cu2+B whereas NO is bridged between Cu2+B and the haem iron of the cytochrome a2+3. The complex has a triplet ground state and a singlet excited state with an exchange interaction J = -7.1 cm-1 between both spins. The anisotropy in the EPR spectra is mainly due to a magnetic dipole-dipole interaction between cytochrome a2+3 X NO and Cu2+B. From simulations of the triplet EPR spectra obtained at 9 and 35 GHz, a value for the distance between the nitroxide radical and Cu2+B of 0.33 nm was found. A model of the NO binding in the cytochrome a3-Cu pair shows a distance between the haem iron of cytochrome a3 and CuB of 0.45 nm. It is concluded that the cytochrome a3-CuB pair forms a cage in which the dioxygen molecule is bidentate coordinated to the two metals during the catalytic reaction.

The mechanism of reduction of cytochrome c as studied by pulse radiolysis

1. The reaction of hydrated electrons with ferricytochrome c was studied using the pulse-radiolysis technique. 2. In 3.3 mM phosphate-buffer (pH 7.2), 100 mM methanol and at a concentration of cytochrome c of less than 20 muM the reduction kinetics of ferricytochrome c by hydrated electrons is a bimolecular process with a rate constant of 4.5-10-10 M-1-S-1 (21 degrees C). 3. At a concentration of cytochrome c of more than 20 muM the apparent order of the reaction of hydrated electrons with ferricytochrome c measured at 650 nm decreases due to the occurrence of a rate-determining first-order process with an estimated rate constant of 5-10-6s-1 (pH 7.2, 21 degrees C). 4. At high concentration of cytochrome c the reaction-time courses measured at 580 and 695 nm appear to be biphasic. A rapid initial phase (75% and 30% of total absorbance change at 580 and 695 nm, respectively), corresponding to the reduction reaction, is followed by a first-order change in absorbance with a rate constant of 1.3-10-5 S-1 (pH 7.2, 21 degrees C). 5. The results are interpreted in a scheme in which first a transient complex between cytochrome c and the hydrated electron is formed, after which the heme iron is reduced and followed by relaxation of the protein from its oxidized to its reduced conformation. 6. It is calculated that one of each three encounters of the hydrated electron and ferricytochrome c results in a reduction of the heme iron. This high reaction probability is discussed in terms of charge and solvent interactions. 7. A reduction mechanism for cytochrome c is favored in which the reduction equivalent from the hydrated electron is transmitted through a specific pathway from the surface of the molecule to the heme iron.

Separation of enzymically active bovine cytochrome c oxidase monomers and dimers by high performance liquid chromatography

The aggregation state of two types of bovine heart cytochrome c oxidase preparations in the presence of laurylmaltoside was investigated by high performance liquid chromatography in two buffers of ionic strengths of 388 mM and 45 mM, respectively. At high ionic strength, it was found that the Fowler cytochrome c oxidase preparation was monomeric (Mr = 2 X 10(5)), while monomers and dimers (2 X aa3, Mr = 4 X 10(5)) could be isolated from the Yonetani preparation. Under these conditions there was no rapid equilibrium between the two forms. Covalent cytochrome c oxidase-cytochrome c complexes were largely dimeric, and addition of ascorbate and cytochrome c to the oxidase also promoted dimerization. At low ionic strength (I = 45 mM) in the presence of laurylmaltoside the oxidase and the covalent complex with cytochrome c were largely monomeric. In the steady-state oxidation of ferrous horse heart cytochrome c, the monomeric enzyme displayed biphasic kinetics at I = 45 mM. This suggests that the presence of high- and low-affinity reactions is an intrinsic property of the cytochrome c oxidase monomer.

Photodissociation of Cytochrome c Oxidase—Nitric Oxide Complexesa

The dissociation of cytochrome c oxidase-nitric oxide complexes was studied by optical spectroscopy at cryogenic temperatures (15 degrees K). With the reduced cytochrome c oxidase-nitric oxide complex, the observations that were reported by Yoshida et al. were confirmed. Photodissociation of the oxidized cytochrome c oxidase-nitric oxide complex did not induce any significant absorbance changes between 350 and 875 nm. With the azide-nitrosyl-cytochrome c oxidase complex, the illumination caused the dissociation of the a2+(3).NO complex to the unligated state a2+(3). Increasing the temperature to 77 degrees K led to the formation of a new complex, probably a3+(3).N3-. The N3(-)-NO-cytochrome c oxidase complex was the only compound for which appreciable photodissociation was achieved by continuous illumination at room temperature (20 degrees C). The effect of illumination was biphasic. In the first phase the a2+(3).NO complex is dissociated and cytochrome a3 oxidized by an electron transfer to CuB. In the second phase nitric oxide, which is still bound to CuB after the first phase, is expelled from the complex by azide, with a concomitant electron transfer from CuB to cytochrome a.