Resonance Raman studies of CuA-modified cytochrome c oxidase

Modification of the CuA site in mammalian cytochrome c oxidase has been used to elucidate the functional role of this center in the catalytic cycle of the enzyme. Both heat treatment in detergents and chemical modification by p-(hydroxymercuri)benzoate (pHMB) convert CuA to a lower potential type II center and effectively remove the site from the electron-transfer pathway during turnover. In this study, resonance Raman spectroscopy has been employed to investigate the effects of these CuA modifications on the heme active sites. The Raman data indicate some environmental perturbation of the heme a3 chromophore in the modified derivatives. Only pHMB modification and SB-12 heat treatment produced significant effects in the Raman spectra of the fully reduced enzyme. These perturbations are much less evident in the spectra obtained within 10 ns of CO photolysis from the fully reduced species of the modified enzymes. Transient Raman studies further indicate that the half-time for CO religation in the modified enzymes is quite similar to that of the native protein.     

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.     

Resonance raman studies of oxo intermediates in the reaction of pulsed cytochrome bo with hydrogen peroxide

Cytochrome bo from Escherichia coli, a member of the heme-copper terminal oxidase superfamily, physiologically catalyzes reduction of O(2) by quinols and simultaneously translocates protons across the cytoplasmic membrane. The reaction of its ferric pulsed form with hydrogen peroxide was investigated with steady-state resonance Raman spectroscopy using a homemade microcirculating system. Three oxygen-isotope-sensitive Raman bands were observed at 805/X, 783/753, and (767)/730 cm(-)(1) for intermediates derived from H(2)(16)O(2)/H(2)(18)O(2). The experiments using H(2)(16)O(18)O yielded no new bands, indicating that all the bands arose from the Fe=O stretching (nu(Fe)(=)(O)) mode. Among them, the intensity of the 805/X cm(-)(1) pair increased at higher pH, and the species giving rise to this band seemed to correspond to the P intermediate of bovine cytochrome c oxidase (CcO) on the basis of the reported fact that the P intermediate of cytochrome bo appeared prior to the formation of the F species at higher pH. For this intermediate, a Raman band assignable to the C-O stretching mode of a tyrosyl radical was deduced at 1489 cm(-)(1) from difference spectra. This suggests that the P intermediate of cytochrome bo contains an Fe(IV)=O heme and a tyrosyl radical like compound I of prostaglandin H synthase. The 783/753 cm(-)(1) pair, which was dominant at neutral pH and close to the nu(Fe)(=)(O) frequency of the oxoferryl intermediate of CcO, presumably arises from the F intermediate. On the contrary, the (767)/730 cm(-)(1) species has no counterpart in CcO. Its presence may support the branched reaction scheme proposed previously for O(2) reduction by cytochrome bo.     

Structural comparison of cytochrome c2and cytochrome c6

Photosynthesis Research - This review compares the three-dimensional structures of the solublec-type cytochromes that functionally link membrane-bound energy transducingcomplexes in algal,...     

Complex-formation between cytochrome c and cytochrome c peroxidase. Equilibrium and titration studies

1. Physical studies of complex-formation between cytochrome c and yeast peroxidase are consistent with kinetic predictions that these complexes participate in the catalytic activity of yeast peroxidase towards ferrocytochrome c. Enzyme-ferricytochrome c complexes have been detected both by the analytical ultracentrifuge and by column chromatography, whereas an enzyme-ferrocytochrome c complex was demonstrated by column chromatography. Estimated binding constants obtained from chromatographic experiments were similar to the measured kinetic values. 2. The physicochemical study of the enzyme-ferricytochrome c complex, and an analysis of its spectrum and reactivity, suggest that the conformation and reactivity of neither cytochrome c nor yeast peroxidase are grossly modified in the complex. 3. The peroxide compound of yeast cytochrome c peroxidase was found to have two oxidizing equivalents accessible to cytochrome c but only one readily accessible to ferrocyanide. Several types of peroxide compound, differing in available oxidizing equivalents and in reactivity with cytochrome c, seem to be formed by stoicheiometric amounts of hydrogen peroxide. 4. Fluoride combines not only with free yeast peroxidase but also with peroxidase-peroxide and accelerates the decomposition of the latter compound. The ligand-catalysed decomposition provides evidence for one-electron reduction pathways in yeast peroxidase, and the reversible binding of fluoride casts doubt upon the concept that the peroxidase-peroxide intermediate is any form of peroxide complex. 5. A mechanism for cytochrome c oxidation is proposed involving the successive reaction of two reversibly bound molecules of cytochrome c with oxidizing equivalents associated with the enzyme protein.     

Kinetic studies on mammalian cytochrome c modified with 2-hydroxy-5-hydroxy-5-nitrobenzyl bromide.

The reduction of 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c by the chromous ion was studied by stopped-flow techniques. At pH6.5 the reduction of 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c is complex, showing the presence of three distinct phases. Two chromium concentration-dependent phases are observed (1.1 X 10(5) M-1-S-1, phase 1; 1.25 X 10(4)M-1-S-1, phase 2) and one slow first-order process (0.25S-1, phase 3). A comparison of the static and kinetic difference spectra, along with the data from the reduction of the reoxidized reduced protein, suggests that the slow chromium concentration-independent phase is due to a slow conformational event after fast reduction of the NO2 group. The rates of the chromium concentration-dependent phases show a marked variation with pH above 7.5. The activation energies for the three processes were also measured at 33.2, 38.6 and 69.7 kJ-mol-1 for phases 1, 2 and 3 respectively. The reaction of reduced 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c with CO was foollowed by means of both stopped-flow and flash photolysis. The combination with CO at pH 6.8 as measured in stopped-flow experiments showed two phases, one CO-dependent phase (phase 2, 2.4 X 10(2)M-1-S-1) and one CO-independent phase (phase 1, 0.015S-1). Investigation of the pH-dependence of the phases showed both the rates and amounts of each phase to be pH-invariant. CO recombination, after photolytic removal, was found to be biphasic; a CO-dependent phase (phase 2, 2.4 X 10(2)M-1-S-1) and a CO-independent phase (phase 1, 1.0s-1) were observed. A tentative model which can accommodate these observations is proposed.     

Some properties of mammalian cardiac cytochrome c1.

Investigations into the nature of the axial heme ligands, the strength of the heme crevice, the reactivity with cyanide, and the ascorbate reducibility of cytochrome c1 were performed to explore structure-function relationships of cytochrome c1. The existence of an absorbance band at 690 nm, which was quenched by raising the pH with a pK of 9.2 corresponding to a low spin-low transition, suggested that a methionine residue probably functioned as one of the axial heme iron ligands in this cytochrome. Spectral titrations of cytochrome c1 in the low pH range showed a markedly elevated pK for the low spin-high spin transition relative to cytochrome c. Denaturation studies with urea, the absence of any reaction with cyanide, and the evidence from other lines would appear to indicate that the heme group of cytochrome c1 was reduced by ascorbate at approximately 5% of the rate of reduction of cytochrome c but this rate dramatically increased with increasing pH concomitant with the disappearance of the 690 nm absorbance band. Circular dichroic spectra substantiated that elevated pH produced conformational changes localized to the heme crevice and probably also the regions containing aromatic residues. The enhanced rate of ascorbate reduction was perhaps a consequence of the increased accessibility of the heme iron to ascorbate. Major unfolding of the protein in 8 M urea, however, completely abolished the ascorbate reducibility of cytochrome c1. The buried nature of the heme group of cytochrome c1 would probably preclude transfer of an electron from cytochrome c1 to cytochrome c through a direct Fe-Fe or a heme-heme interaction. This poses an important question concerning the mechanism of this electron transfer between these two cytochromes not only in mitochondria but also in solution.     

Deuterium isotope effect of proton pumping in cytochrome c oxidase

In mitochondria and many aerobic bacteria cytochrome c oxidase is the terminal enzyme of the respiratory chain where it catalyses the reduction of oxygen to water. The free energy released in this process is used to translocate (pump) protons across the membrane such that each electron transfer to the catalytic site is accompanied by proton pumping. To investigate the mechanism of electron-proton coupling in cytochrome c oxidase we have studied the pH-dependence of the kinetic deuterium isotope effect of specific reaction steps associated with proton transfer in wild-type and structural variants of cytochrome c oxidases in which amino-acid residues in proton-transfer pathways have been modified. In addition, we have solved the structure of one of these mutant enzymes, where a key component of the proton-transfer machinery, Glu286, was modified to an Asp. The results indicate that the P3-->F3 transition rate is determined by a direct proton-transfer event to the catalytic site. In contrast, the rate of the F3-->O4 transition, which involves simultaneous electron transfer to the catalytic site and is characteristic of any transition during CytcO turnover, is determined by two events with similar rates and different kinetic isotope effects. These reaction steps involve transfer of protons, that are pumped, via a segment of the protein including Glu286 and Arg481.     

The role of plastidic cytochrome c in algal electron transport and photophosphorylation.

By an improved isolation procedure chloroplasts could be obtained from the alga Bumilleriopsis filiformis (Xanthophyceae) which exhibited high electron transport rates tightly coupled to ATP formation. Uncouplers both stimulate electron transport and inhibit photophosphorylation. These chloroplasts retain almost all soluble cytochrome c-553 besides a membrane-bound cytochrome c-554.5 (=f-554.5). Sonification or iron deficiency removed the soluble cytochrome only with a concurrent decrease of electron transport from water to methyl viologen or to NADP and decreased non-cyclic and cyclic photophosphorylation. However, photosynthetic control and the P/2e ratios remain unaltered. In Bumilleriopsis, which apparently has no plastocyanin, the soluble cytochrome c-553 seemingly links electron transport between the bound cytochrome c and P-700.     

Purification of yeast cytochrome c oxidase with a subunit composition resembling the mammalian enzyme.

Yeast cytochrome c oxidase has been isolated by ion exchange chromatography using lauryl maltoside (n-dodecyl beta-D-maltoside) as the solubilizing detergent. The enzyme prepared in this way has a heme aa3 concentration of 8-9 nmol/mg of protein and a turnover number in the range of 180-210 s-1 at pH 6.2 in 0.01% lauryl maltoside at 20 degrees C. Yeast cytochrome c oxidase prepared by any of several previously published methods which use Triton X-100 contains nine subunits. The enzyme isolated in lauryl maltoside contains these same nine different polypeptides and three others, including homologues of subunits VIa and VIb of the mammalian enzyme.     

Studies on partially reduced mammalian cytochrome oxidase reactions with ferrocytochrome c.

The kinetics of the electron-transfer process which occurs between ferrocytochrome c and partially reduced mammalian cytochrome oxidase were studied by the rapid spectrophotometric techniques of stopped flow and temperature jump. Stopped-flow experiments showed initial very fast extinction changes at 605 nm and at 563 nm, indicating the simultaneous reduction of cytochrome a and oxidation of ferrocytochrome c. During this 'burst' phase, say the first 50 ms after mixing, it was invariably found that more cytochrome c had been oxidized than cytochrome a had been reduced. This discrepancy in electron equivalents may be accounted for by the rapid reduction of another redox site in the enzyme, possibly that associated with the extinction changes observed at 830 nm. During the incubation period in which the partially reduced oxidase was prepared, the rate of reduction of cytochrome a by ferrocytochrome c, at constant reactant concentrations, decreased with time. Temperature-jump experiments showed the presence of two relaxation processes. The faster of the two phases was assigned to the electron-transfer reaction between cytochrome c and cytochrome a. A study of the concentration-dependence of the reciprocal relaxation time for this phase yielded a rate constant of 9 X 10(6)M-1-s-1 for the electron transfer from cytochrome c to cytochrome a, and a value of 8.5 X 10(6)M-1-s-1 for the reverse reaction. The equilibrium constant for the electron-transfer reaction is therefore close to unity. The slower phase has been interpreted as signalling the transfer of electrons between cytochrome a and another redox site within the oxidase molecule.     

Interaction of cytochrome c with cytochrome c oxidase. Photoaffinity labeling of beef heart cytochrome c oxidase with arylazido-cytochrome c.

Cytochrome c derivatives labeled with a 3-nitrophenylazido group at lysine 13, at lysine 22, or at both residues have been prepared. The interaction of the cytochrome c derivatives with beef heart cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) in the presence of ultrviolet light results in formation of a covalent complex between cytochrome c and the oxidase. Using the lysine 22 derivative, the polypeptide composition of the oxidase is not modified, nor is its catalytic activity, whereas with the lysine 13 derivative, the gel electrophoretic pattern is altered and the catalytic activity of the complex diminished. The data are consisten with a specfic covalent interaction of the lysine 13 derivative of cytochrome c with the polypeptide of molecular weight 23,700 (Subunit II) of cytochrome c oxidase.     

Presteady-state and steady-state kinetic properties of human cytochrome c oxidase. Identification of rate-limiting steps in mammalian cytochrome c oxidase.

Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady-state and steady-state kinetic properties of the human cytochrome c oxidase preparations with either human cytochrome c or horse cytochrome c were studied spectrophotometrically and compared with those of bovine heart cytochrome c oxidase. The interaction between human cytochrome c and human cytochrome c oxidase proved to be highly specific. It is proposed that for efficient electron transfer to occur, a conformational change in the complex is required, thereby shifting the initially unfavourable redox equilibrium. The very slow presteady-state reaction between human cytochrome c oxidase and horse cytochrome c suggests that, in this case, the conformational change does not occur. The proposed model was also used to explain the steady-state kinetic parameters under various conditions. At high ionic strength (I = 200 mM, pH 7.4), the kcat was highly dependent on the type of oxidase and it is proposed that the internal electron transfer is the rate-limiting step. The kcat value of the 'high-affinity' phase, observed at low ionic strength (I = 18 mM, pH 7.4), was determined by the cytochrome c/cytochrome c oxidase combination applied, whereas the Km was highly dependent only on the type of cytochrome c used. Our results suggest that, depending on the cytochrome c/cytochrome c oxidase combination, either the dissociation of ferricytochrome c or the internal electron transfer is the rate-limiting step in the 'high-affinity' phase at low ionic strength. The 'low-affinity' kcat value was not only determined by the type of oxidase used, but also by the type of cytochrome c. It is proposed that the internal electron-transfer rate of the 'low-affinity' reaction is enhanced by the binding of a second molecule of cytochrome c.