Kinetics of cytochrome c and TMPD oxidation by cytochrome c oxidase from the thermophilic bacterium, PS3

Cytochrome caa3 (cytochrome oxidase) from the thermophilic bacterium PS3 can exhibit full catalytic activity in the presence of ascorbate and TMPD or other electron donors and in the absence of added soluble c-type cytochromes. It appears to possess only a low-affinity and not a high-affinity site for the soluble cytochromes. Proteoliposomal cytochrome caa3 develops an effective membrane potential in the presence of ascorbate and TMPD or PMS, in the absence of added soluble cytochrome c. Reduction of the a3 centre is blocked in the presence of cyanide. During reductive titrations of the cyanide-inhibited enzyme, electrons initially equilibrate among three centres, the c haem, the a haem and one of the associated Cu atoms. During steady-state turnover, electrons probably enter the complex via the bound c haem; the a haem and perhaps an associated CuA atom are reduced next. It is concluded that, despite its size and hydrophobic association with the aa3 complex, the haem c-containing subunit can behave in an analogous way to that of mammalian cytochrome c, bound at the high-affinity site of the eucaryotic enzyme.     

Selective removal of subunit VIb increases the activity of cytochrome c oxidase.

Bovine heart cytochrome c oxidase was gel-filtered on Sephacryl S-300 in 0.05% dodecyl maltoside and in the presence or absence of 1 M KCl. The presence of KCl selectively removed subunit VIb from the enzyme complex, resulting in about doubling of enzymatic activity and an increase of the Km for ferrocytochrome c. In contrast, the proton pumping activity of the enzyme was unchanged. The increase of activity is due to removal of subunit VIb and not of lipids, because titration with asolectin or dodecyl maltoside could not abolish the difference in activity between the 12- and 13-subunit enzyme. Attempts to reconstitute cytochrome c oxidase from its separated components were unsuccessful. It is concluded that subunit VIb suppresses the activity of the mammalian enzyme complex by interaction with the active center.     

Characterization of a covalently linked yeast cytochrome c-cytochrome c peroxidase complex: evidence for a single, catalytically active cytochrome c binding site on cytochrome c peroxidase

A covalent complex between recombinant yeast iso-1-cytochrome c and recombinant yeast cytochrome c peroxidase (rCcP), in which the crystallographically defined cytochrome c binding site [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755] is blocked, was synthesized via disulfide bond formation using specifically engineered cysteine residues in both yeast iso-1-cytochrome c and yeast cytochrome c peroxidase [Papa, H. S., and Poulos, T. L. (1995) Biochemistry 34, 6573-6580]. Previous studies on similar covalent complexes, those that block the Pelletier-Kraut crystallographic site, have demonstrated that samples of the covalent complexes have detectable activities that are significantly lower than those of wild-type yCcP, usually in the range of approximately 1-7% of that of the wild-type enzyme. Using gradient elution procedures in the purification of the engineered peroxidase, cytochrome c, and covalent complex, along with activity measurements during the purification steps, we demonstrate that the residual activity associated with the purified covalent complex is due to unreacted CcP that copurifies with the covalent complex. Within experimental error, the covalent complex that blocks the Pelletier-Kraut site has zero catalytic activity in the steady-state oxidation of exogenous yeast iso-1-ferrocytochrome c by hydrogen peroxide, demonstrating that only ferrocytochrome c bound at the Pelletier-Kraut site is oxidized during catalytic turnover.     

Complex-formation between cytochrome c and cytochrome c peroxidase. Kinetic studies

1. The kinetics of ferrocytochrome c peroxidation by yeast peroxidase are described. Kinetic differences between the older and more recent preparations of the enzyme most probably arise from differences in intrinsic turnover rates. 2. The time-courses of cytochrome c peroxidation by the enzyme follow essentially first-order kinetics in phosphate buffer. Deviations from first-order kinetics occur in acetate buffer, and are due to a higher enzymic turnover rate in this medium accompanied by a greater tendency to autocatalytic peroxidation of cytochrome c. 3. The kinetics of ferrocytochrome c peroxidation by yeast peroxidase are interpreted in terms of a mechanism postulating formation of reversible complexes between the peroxidase and both reduced and oxidized cytochrome c. Formation of these complexes is inhibited at high ionic strengths and by polycations. 4. Oxidized cytochrome c can act as a competitive inhibitor of ferrocytochrome c peroxidation by peroxidase. The K(i) for ferricytochrome c is approximately equal to the K(m) for ferrocytochrome c and thus probably accounts for the observed apparent first-order kinetics even at saturating concentrations of ferrocytochrome c. 5. The results are discussed in terms of a possible analogy between the oxidations of cytochrome c catalysed by yeast peroxidase and by mammalian cytochrome oxidase.     

Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.     

Kinetic studies on the reaction between cytochrome c oxidase and ferrocytochrome c.

In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with 'visible' copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the 'visible' copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and 'visible' copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.     

Cloning, overproduction and characterization of cytochrome c peroxidase from the purple phototrophic bacterium Rhodobacter capsulatus.

The bacterial cytochrome c peroxidase (BCCP) from Rhodobacter capsulatus was purified as a recombinant protein from an Escherichia coli clone over-expressing the BCCP structural gene. BCCP from Rb. capsulatus oxidizes the Rhodobacter cytochrome c2 and reduces hydrogen peroxide, probably functioning as a detoxification mechanism. The enzyme binds two haem c groups covalently. The gene encoding BCCP from Rb. capsulatus was cloned through the construction of a 7-kb subgenomic clone. In comparison with the protein sequence, the sequence deduced from the gene has a 21-amino-acid N-terminal extension with the characteristics of a signal peptide. The purified recombinant enzyme showed the same physico-chemical properties as the native enzyme. Spectrophotometric titration established the presence of a high-potential (Em=+270 mV) and a low-potential haem (between -190 mV and -310 mV) as found in other BCCPs. The enzyme was isolated in the fully oxidized but inactive form. It binds calcium tightly and EGTA treatment of the enzyme was necessary to show calcium activation of the mixed valence enzyme. This activation is associated with the formation of a high-spin state at the low-potential haem. BCCP oxidizes horse ferrocytochrome c better than the native electron donor, cytochrome c2; the catalytic activities ('turnover number') are 85 800 min(-1) and 63 600 min(-1), respectively. These activities are the highest ever found for a BCCP.     

The role of subunit III in bovine cytochrome c oxidase. Comparison between native, subunit III-depleted and Paracoccus denitrificans enzymes

In order to obtain information on the role of subunit III in the function and aggregation state of cytochrome c oxidase, the kinetics of ferrocytochrome c oxidation by the bovine cytochrome c oxidase depleted of its subunit III were studied and compared with those of the oxidase isolated from P. denitrificans which contains only two subunits. The aggregation state of both enzymes dispersed in dodecyl maltoside was also compared. The two-subunit oxidase from P. denitrificans gave linear Eadie-Hofstee plots and the enzyme resulted to be monomeric (Mr = 82 000) both, in gel filtration and sucrose gradient centrifugation studies. The bovine heart subunit III depleted enzyme, under conditions when the P. denitrificans cytochrome c oxidase was in the form of monomers, was found to be dimeric by sucrose gradient centrifugation analysis. At lower enzyme concentrations monomers were, however, detected by gel filtration. Depletion of subunit III was accompanied by the loss of small polypeptides (VIa, VIb and VIIa) and of almost all phospholipid (1-2 molecules were left per molecule of enzyme). The electron-transfer activity of the subunit III-depleted enzyme showed a monophasic Eadie-Hofstee plot, which upon addition of phospholipids became non-linear, similar to that of the control bovine cytochrome c oxidase. One of the roles of subunit III may be that of stabilising the dimers of cytochrome c oxidase. Lack of this subunit and loss of phospholipid is accompanied by a change in the kinetics of electron transfer, which might be the consequence of enzyme monomerisation.     

Circular-dichroic properties and secondary structure of Pseudomonas aeruginosa soluble cytochrome c oxidase.

The c.d. spectra of Pseudomonas aeruginosa cytochrome c oxidase in the oxidized state and the reduced state are reported in the visible- and u.v. absorption regions. In the visible region the comparison between the spectra of reduced cytochrome c oxidase and ferrocytochrome c-551 allows the identification of the c.d. bands mainly due to the d1 haem chromophore in cytochrome c oxidase. In the near-u.v. region the assignment of some of the observed peaks to the haem groups and to the aromatic amino acid residues is proposed. A careful analysis of the data in the far-u.v. region leads to the determination of the relative amounts of alpha-helix and beta-sheet in the enzyme, giving for the first time a picture of its secondary structure. A significant difference in this respect between the reduced and the oxidized species is observed and discussed in the light of similar conclusions reported by other workers.     

The electric potential field around cytochrome c and the effect of ionic strength on reaction rates of horse cytochrome c.

1. The electric potential fields around tuna ferri- and ferrocytochrome c were calculated assuming that (i) all of the lysines and arginines are protonated, (ii) all of the glutamic and aspartic acids and the terminal carboxylic acid are dissociated, and (iii) the haem has a net charge of +1e in the oxidized form. 2. Near the haem crevice high values for the potential (greater than +2.5 kT/e) are found. Consequently, electron transfer via the haem edge is favored if the oxidant or reductant is negatively charged. 3. The inhomogeneous distribution of charges leads to a dipole moment of 244 and 238 debye for oxidized and reduced tuna cytochrome c, respectively. Horse cytochrome c has dipole moments of 303 (oxidized) and 286 (reduced) debye. 4. A line through the positive and negative charge centres, the dipole axis, crosses the tuna cytochrome c surface at Ala 83 (positive part) and Lys 99 (negative part). The direction of the dipole axis of horse cytochrome c is very similar. Since the centre of the domain on the cytochrome c surface, which is involved in the binding to cytochrome c oxidase, is found at the beta-carbon of the Phe 82 in horse cytochrome c (Ferguson-Miller, S., Brautigan, D.L. and Margoliash, E. (1978) J. Biol. Chem. 253, 149--159) it is suggested that the direction of the dipole is of physiological importance. 5. The activity coefficients of horse ferri- and ferrocytochrome c were calculated as a function of ionic strength using a formula derived by Kirkwood (Kirkwood, J.G. (1934) J. Chem. Phys. 2, 351--361). 6. Due to the high net charge at pH 7.5 the influence of the dipole moments of horse ferri- and ferrocytochrome c on the respective activity coefficients can be neglected at I less than or equal to 50 mM. 7. Using the Br?nsted relation the effect of ionic strength on reaction rates of horse cytochrome c was calculated. Good agreement is found between theory and experimental results reported in the literature.     

Dicyclohexylcarbodiimide binds specifically and covalently to cytochrome c oxidase while inhibiting its H+-translocating activity

We have investigated the covalent binding of dicyclohexylcarbodiimide (DCCD) to cytochrome c oxidase in relation to its inhibition of ferrocytochrome c-induced H+ translocation by the enzyme reconstituted in lipid vesicles. DCCD bound to the reconstituted oxidase in a time- and concentration-dependent manner which appeared to correlate with its inhibition of H+ translocation. In both reconstituted vesicles and intact beef heart mitochondria, the DCCD-binding site was located in subunit III of the oxidase. The apolar nature of DCCD and relatively minor effects of the hydrophilic carbodiimide, 1-ethyl-(3-dimethylaminopropyl)-carbodiimide, on H+ translocation by the oxidase indicate that the site of action of DCCD is hydrophobic. DCCD also bound to isolated cytochrome c oxidase, though in this case subunits III and IV were labeled. The maximal overall stoichiometries of DCCD molecules bound per cytochrome c oxidase molecule were 1 and 1.6 for the reconstituted and isolated enzymes, respectively. These findings point to subunit III of cytochrome c oxidase having an important role in H+ translocation by the enzyme and indicate that DCCD may prove a useful tool in elucidating the mechanism of H+ pumping.     

Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrate-ferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the Km (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the 'association' rate but also by increasing the 'dissociation' rate for bound cytochrome c converting the 'primary' (T) site at high salt concentrations into a site similar kinetically to the 'secondary' (L) site at low ionic strength. A finite Km of 170 microM at very high ionic strength indicates a ratio of K infinity m/K 0 M of about 5000. It is proposed that anions either modify the E10 of cytochrome C bound at the primary (T) site of that they perturb an equilibrium between two forms of bound c in favour of a less active form.     

Electron transfer between soluble and immobilized mammalian cytochrome c. Equilibrium and kinetic studies on immobilized cytochrome c.

Horse heart cytochrome c was covalently bound to Sepharose 4B and its redox properties were measured under various experimental conditions. The equilibrium constant for the electron exchange between the oxidized and the reduced form of cytochrome c when one of the two forms was in the semi-solid state and the other one in solution was close to 1. Matrix-bound ferrocytochrome c is very stable to autoxidation and is not oxidized by O2 even in the presence of mammalian cytochrome oxidase. Oxidation occurs if catalytic amounts of soluble cytochrome c are added to the reaction mixture. The rate of oxidation of matrix-bound ferrocytochrome c in the presence of cytochrome oxidase and catalytic amounts of soluble cytochrome c may be correlated with the rate of electron transfer between soluble and matrix-bound cytochrome c. This rate is more than two orders of magnitude lower than that reported for the homonuclear (between identical species) electron transfer in solution.     

Structure of an electron transfer complex. I. Covalent cross-linking of cytochrome c peroxidase and cytochrome c

Cytochrome c peroxidase and cytochrome c form a noncovalent electron transfer complex in the course of the peroxidase-catalyzed reduction of H2O2. The two hemoproteins were cross-linked in 40% yield to a covalent 1:1 complex with the aid of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex was found to be a valid model of the noncovalent electron transfer complex for the following reasons. The covalent complex had only 5% residual peroxidase activity toward exogeneous ferrocytochrome c indicating that the cross-linked cytochrome c covers the electron-accepting site of cytochrome c peroxidase. The residual peroxidase activity was almost independent of ionic strength indicating that the electron-accepting site is much less accessible even when ionic bonds between the two cross-linked hemoproteins are severed. The rate of reduction of heme c by ascorbate is 15 times slower in the covalent complex than in free cytochrome c and is independent of ionic strength. Although the covalent complex may not have been entirely pure with respect to the number and location of the cross-links, two major cross-links could be localized to within a few residues. One is from Lys 13 of cytochrome c to an acidic residue in positions 32, 33, 34, 35, or 37 of cytochrome c peroxidase, the other from Lys 86 of cytochrome c to a carboxyl group in the same cluster of acidic residues. The result stresses the importance of a peculiar stretch of acidic residues of cytochrome c peroxidase and of Lys 13 and 86 of cytochrome c.     

The reduction of porphyrin cytochrome c by hydrated electrons and the subsequent electron transfer reaction from reduced porphyrin cytochrome c to ferricytochrome c.

1. Hydrated electrons, produced by pulse radiolysis react with porphyrin cytochrome c with a bimolecular rate constant of 3-10(10) M-1 S-1 at 21 degrees C and pH 7.4. 2. After the reduction step an absorbance change with a half-life of 5 microns is observed with the spectral range of 430-470 nm. A relatively stable intermediate then decays with a half-life of 15 s. 3. The spectrum of the intermediate observed 50 microns after the generation of hydrated electrons shows a broad absorption band between 600 and 700 nm and a peak at 408 nm. The spectrum is attributed to the protonated form of an initially produced porphyrin anion radical. 4. Reduced porphyrin cytochrome c reacts with ferricytochrome c with a bimolecular constant of 2-10(5) M-1- S-1 in 2 mM phosphate pH 7.4, at 21 degrees C and of 2 - 10(6) M-1-S-1 under the same conditions but at 1 M ionic strength. It is proposed that electron transfer in an analogous exchange reaction between ferrocytochrome c and ferricytochrome c occurs via the exposed part of the haem.     

Titration and steady-state behaviour of the 830 nm chromophore in cytochrome c oxidase.

Titration of cyanide-incubated cytochrome c oxidase (ox heart cytochrome aa3) with ferrocytochrome c or with NNN'N'-tetramethyl-p-phenylenediamine initially introduces two reducing equivalents per mol of cytochrome aa3. The first equivalent reduces the cytochrome a haem iron; the second reducing equivalent is not associated with reduction of the 830 nm chromophores (e.p.r.-detectable copper) but is probably required for reduction of the e.p.r.-undetectable copper. Excess reductant introduces a third reducing equivalent into the cyanide complex of cytochrome aa3. During steady-state respiration in the presence of cytochrome c and ascorbate, the 830 nm chromophore is almost completely oxidized. It is reduced more slowly than cytochrome a on anaerobiosis. In the presence of formate or azide, some reduction at 830 nm can be seen in the steady state; in an oxygen-pulsed system, a decrease in steady-state reduction of cytochromes c and a is associated with ab increased reduction of the 830 nm species. In the formate-inhibited system the reduction of a3 on anaerobiosis shows a lag phase, the duration of which corresponds to the time taken for the 830 nm species to be reduced. It is concluded that the e.p.r.-undetectable copper (CuD) is reduced early in the reaction sequence, whereas the detectable copper (CUD) is reduced late. The latter species is probably that responsible for reduction of the cytochrome a3 haem. The magnetic association between undetectable copper and the a3 haem may not imply capability for electron transfer, which occurs more readily between cytochrome a3 and the 830 nm species.     

Reactions of mercaptans with cytochrome c oxidase and cytochrome c

1. The steady-state oxidation of ferrocytochrome c by dioxygen catalyzed by cytochrome c oxidase, is inhibited non-competitively towards cytochrome c by methanethiol, ethanethiol, 1-propanethiol and 1-butanethiol with Ki values of 4.5, 91, 200 and 330 microM, respectively. 2. The inhibition constant Ki of ethanethiol is found to be constant between pH 5 and 8, which suggests that only the neutral form of the thiol inhibits the enzyme. 3. The absorption spectrum of oxidized cytochrome c oxidase in the Soret region shows rapid absorbance changes upon addition of ethanethiol to the enzyme. This process is followed by a very slow reduction of the enzyme. The fast reaction, which represents a binding reaction of ethanethiol to cytochrome c oxidase, has a k1 of 33 M-1 . s-1 and a dissociation constant Kd of 3.9 mM. 4. Ethanethiol induces fast spectral changes in the absorption spectrum of cytochrome c, which are followed by a very slow reduction of the heme. The rate constant for the fast ethanethiol reaction representing a bimolecular binding step is 50 M-1 . s-1 and the dissociation constant is about 2 mM. Addition of up to 25 mM ethanethiol to ferrocytochrome c does not cause spectral changes. 5. EPR (electron paramagnetic resonance) spectra of cytochrome c oxidase, incubated with methanethiol or ethanethiol in the presence of cytochrome c and ascorbate, show the formation of low-spin cytochrome alpha 3-mercaptide compounds with g values of 2.39, 2.23, 1.93 and of 2.43, 2.24, 1.91, respectively.     

A covalent complex between horse heart cytochrome c and yeast cytochrome c peroxidase: kinetic properties

The kinetic properties of a 1:1 covalent complex between horse-heart cytochrome c and yeast cytochrome c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) have been investigated by transient-state and steady-state kinetic techniques. Evidence for heterogeneity in the complex is presented. About 50% of the complex reacts with hydrogen peroxide with a rate 20–40% faster than that of native enzyme; 20% of the complex exists in a conformation which does not react with hydrogen peroxide but converts to the reactive form at a rate of 20 ± 5 s⁻¹; 30% of the complex does not react with hydrogen peroxide to form the oxidized enzyme intermediate, cytochrome c peroxidase Compound I. Intramolecular electron transfer between covalently bound ferrocytochrome c and an oxidized site in cytochrome c peroxidase Compound I is too fast to measure, but a lower limit of 600 s⁻¹ can be estimated at 5°C in a 10 mM potassium phosphate buffer at pH 7.5. Free ferrocytochrome c reduces cytochrome c peroxidase Compound I covalently bound to ferricytochrome c at a rate 10⁻⁴ to 10⁻⁵-times slower than for free Compound I. The transient-state ferrocytochrome c reduction rates of Compound I covalently linked to ferricytochrome c are about 70-times too slow to account for the steady-state catalytic properties of the 1:! covalent complex. This indicates that hydrogen peroxide can interact with the 1:1 complex at sites other than the heme of cytochrome c peroxidase, generating additional species capable of oxidizing free ferrocytochrome c.     

Nitric oxide and cytochrome oxidase: substrate,inhibitor or effector?

Endogenously produced nitric oxide (NO) controls oxygen consumption by inhibiting cytochrome c oxidase, the terminal electron acceptor of the mitochondrial electron transport chain. The oxygen-binding site of the enzyme is an iron/copper (haem a3/CuB) binuclear centre. At high substrate (ferrocytochrome c) concentrations, NO binds reversibly to the reduced iron in competition with oxygen. At low substrate concentrations, NO binds to the oxidized copper. Inhibition at the haem iron site is relieved by dissociation of the NO from the reduced iron. Inhibition at the copper site is relieved by oxidation of the bound NO and subsequent dissociation of nitrite from the enzyme. Therefore, NO can be a substrate, inhibitor or effector of cytochrome oxidase, depending on cellular conditions.     

Zinc cytochrome c fluorescence as a probe for conformational changes in cytochrome c oxidase.

Zinc cytochrome c forms tight 1:1 complexes with a variety of derivatives of cytochrome c oxidase. On complex-formation the fluorescence of zinc cytochrome c is diminished. Titrations of zinc cytochrome c with cytochrome c oxidase, followed through the fluorescence emission of the former, have yielded both binding constants (K approximately 7 x 10(6) M-1 for the fully oxidized and 2 x 10(7) M-1 for the fully reduced enzyme) and distance information. Comparison of steady-state measurements obtained by absorbance and fluorescence spectroscopy in the presence and in the absence of cyanide show that it is the reduction of cytochrome a and/or CuA that triggers a conformational change: this increases the zinc cytochrome c to acceptor (most probably cytochrome a itself) distance by some 0.5 nm. Ligand binding to the fully oxidized or fully reduced enzyme leaves the extent of fluorescence quenching unchanged, whereas binding of cyanide to the half-reduced enzyme (a2+CuA+CuB2+-CN(-)-a3(3+)) enhances fluorescence emission relative to that for the fully reduced enzyme, implying further relative movement of donor and acceptor.