The electron-transfer reaction between azurin and the cytochrome c oxidase from Pseudomonas aeruginosa.

A stopped-flow investigation of the electron-transfer reaction between oxidized azurin and reduced Pseudomonas aeruginosa cytochrome c-551 oxidase and between reduced azurin and oxidized Ps. aeruginosa cytochrome c-551 oxidase was performed. Electrons leave and enter the oxidase molecule via its haem c component, with the oxidation and reduction of the haem d1 occurring by internal electron transfer. The reaction mechanism in both directions is complex. In the direction of oxidase oxidation, two phases assigned on the basis of difference spectra to haem c proceed with rate constants of 3.2 X 10(5)M-1-S-1 and 2.0 X 10(4)M-1-S-1, whereas the haem d1 oxidation occurs at 0.35 +/- 0.1S-1. Addition of CO to the reduced enzyme profoundly modifies the rate of haem c oxidation, with the faster process tending towards a rate limit of 200S-1. Reduction of the oxidase was similarly complex, with a fast haem c phase tending to a rate limit of 120S-1, and a slower phase with a second-order rate of 1.5 X 10(4)M-1-S-1; the internal transfer rate in this direction was o.25 +/- 0.1S-1. These results have been applied to a kinetic model originally developed from temperature-jump studies.     

A temperature-jump study of the reaction between azurin and cytochrome c oxidase from Pseudomonas aeruginosa.

The electron-transfer reaction between azurin and the cytochrome oxidase from Pseudomonas aeruginosa was investigated by temperature-jump relaxation in the absence of O2 and in the presence of CO. The results show that: (i) reduced azurin exists in two forms in equilibrium, only one of which is capable of exchanging electrons with the Pseudomonas cytochrome oxidase, in agreement with M. T. Wilson, C. Greenwood, M. Brunori & E. Antonini (1975) (Biochem. J. 145, 449-457); (ii) the electron transfer between azurin and Pseudomonas cytochrome oxidase occurs within a molecular complex of the two proteins; this internal transfer becomes rate-limiting at high reagent concentrations.     

Electron transfer between azurin from Alcaligenes faecalis and cytochrome c551 from Pseudomonas aeruginosa

The electron transfer equilibrium and kinetics between azurin from Alcaligenes faecalis and cytochrome c551 from Pseudomonas aeruginosa have been studied. The equilibrium constant K = ([Cyt(III)] . [Az(I)])/([Cyt(II)] . [Az(II))]) = 0.5 at 25 degrees C is about seven times smaller than that observed between the cytochrome c551 and the titrations confirmed a 43-mV difference between the mid-point potentials of +266 mV and +309 mV for the Alcaligenes and Pseudomonas azurins respectively. The kinetics of the reaction between Alcaligenes azurin and Pseudomonas cytochrome c551 were investigated by the temperature-jump chemical relaxation method. Only a single relaxation mode was observed throughout the range of concentrations and temperatures examined. Thus, the slow relaxation time observed in the reaction between P. aeruginosa azurin and cytochrome c551 is not observed with the Alcaligenes azurin. The simplest mechanism that can therefore be ascribed to the investigated system is: [formula: see text]. This scheme is similar to that proposed earlier for the reaction between P. aeruginosa azurin and cytochrome c551 but does not involve the conformational transition proposed for azurin. The specific rates for the electron transfer are still fast: 1.8 x 10(6) M-1 . s-1 and 3.0 x 10(6) M-1 . s-1 respectively at 25 degrees C.     

Complex formation between the copper protein, azurin and the cytochrome c peroxidase of Pseudomonas aeruginosa.

Reduced azurin reacts with the resting, oxidized cytochrome c peroxidase of Pseudomonas aeruginosa to yield time courses observed at 420 nm, which consist of the sum of two exponential processes. Each process exhibits a hyperbolic dependence of the observed rate constant on the reduced azurin concentration. The fraction of the total optical density change which each process contributes is found to be dependent on the reduced azurin concentration. This pattern of reactivity is maintained at pH values between 5.5 and 8.0. The data has been analyzed in terms of a complex formation between the two proteins followed by an intramolecular electron exchange reaction. This analysis yields values for the binding constants at each pH value. The intramolecular exchange reaction is independent of pH, whilst the pH dependence of the binding reaction suggests the involvement of a histidine residue in this process.     

Ligand binding to cytochrome c peroxidase from Pseudomonas aeruginosa

The high potential heme site of Pseudomonas cytochrome c peroxidase has His and Met as ligands. On reduction, the Fe-met bond becomes photosensitive. Following photolysis, the bond reforms with a half-time of 35 ps. The low potential heme peroxidatic site of the fully reduced enzyme has been shown to bind to a range of ligands. The compounds with carbon monoxide, methyl, ethyl, n-butyl, and t-butyl isonitriles have been investigated by laser flash photolysis. All are photosensitive and show different degrees of geminate recombination of ligand in the picosecond and nanosecond time ranges. Carbon monoxide shows the least effect. The three straight-chain isonitriles show about 50% geminate recombination with half-times of the order of 10 ns. t-Butyl isonitrile shows more and faster recombination. These results imply considerable freedom of movement within the active site for the smaller ligands.     

Small-angle X-ray scattering studies of oxidized and reduced cytochrome oxidase from Pseudomonas aeruginosa

Small-angle X-rays scattering experiments were performed with oxidized and reduced cytochrome oxidase purified from Pseudomonas aeruginosa. The radii of gyration were calculated to be 40.5 A for the oxidized form and 37.0 A for the reduced. The longest dimension of the oxidized enzyme was 120 A while for the reduced it was 100 A. The volume of the oxidized protein was observed to be slightly greater than that of the reduced. These data indicate that there is a contraction of the structure of the enzyme during reduction of its constituent heme groups.     

A temperature-jump study of the reaction between azurin and cytochrome c-551 from Pseudomonas aeruginosa (Short Communication)

Temperature-jump studies on the electron-transfer reaction between azurin and cytochrome c-551 clearly reveal two chemical relaxations. The amplitudes of these relaxation processes have identical spectral distributions, but the relaxation times show different dependences on the reactant concentrations. These findings are discussed in terms of possible models.     

Oxidation by nitrite of azurin and cytochrome c-551 from Pseudomonas aeruginosa

The nitrite oxidizes reduced azurin and cytochrome c-551 from Pseudomonas aeruginosa. The effects of pH, ionic strength and concentrations of nitrite, EDTA and the protein on the oxidation were investigated. The results obtained indicate that nitrite interacts not only with the terminal electron carrier of the nitrite reducing chain (nitrite reductase, cytochrome cd1) but also with the intermediate electron carrier components of the chain (azurin and cytochrome c-551).     

The reaction of Pseudomonas aeruginosa cytochrome c oxidase with carbon monoxide.

The binding of CO to ascorbate-reduced Pseudomonas cytochrome oxidase was investigated by static-titration, stopped-flow and flash-photolytic techniques. Static-titration data indicated that the binding process was non-stoicheiometric, with a Hill number of 1.44. Stopped-flow kinetics obtained on the binding of CO to reduced Pseudomonas cytochrome oxidase were biphasic in form; the faster rate exhibited a linear dependence on CO concentration with a second-order rate constant of 2 X 10(4) M-1-s-1, whereas the slower reaction rapidly reached a pseudo-first-order rate limit at approx. 1s-1. The relative proportions of the two phases observed in stopped-flow experiments also showed a dependency on CO concentration, the slower phase increasing as the CO concentration decreased. The kinetics of CO recombination after flash-photolytic dissociation of the reduced Pseudomonas cytochrome oxidase-CO complex were also biphasic in character, both phases showing a linear pseudo-first-order rate dependence on CO concentration. The second-order rate constants were determined as 3.6 X 10(4)M-1-s-1 and 1.6 X 10(4)M-1-s-1 respectively. Again the relative proportions of the two phases varied with CO concentration, the slower phase predominating at low CO concentrations. CO dissociation from the enzyme-CO complex measured in the presence of O2 and NO indicated the presence of two rates, of the order of 0.03s-1 and 0.15s-1. When sodium dithionite was used as a reducing agent for the Pseudomonas cytochrome oxidase, the CO-combination kinetics observed by both stopped flow and flash photolysis were extremely complex and not able to be simply analysed.     

The primary structure of Pseudomonas cytochrome c peroxidase

The primary structure of Pseudomonas cytochrome c peroxidase is presented. The intact protein was fragmented with cyanogen bromide into five fragments; partial cleavage was observed at a Met-His bond of the protein. The primary structure was established partly by automatic Edman degradations, partly by manual sequencing of peptides obtained with trypsin, thermolysin, chymotrypsin, pepsin, subtilisin and Staphylococcus aureus V8 endopeptidase. The order of the cyanogen bromide fragments was further confirmed by overlapping peptides obtained by specific cleavage of the whole protein. Pseudomonas cytochrome c peroxidase consists of 302 amino acid residues giving a calculated M_r of 33 690.     

The catalytic mechanism of Pseudomonas cytochrome c peroxidase

The catalytic mechanism of Pseudomonas cytochrome c peroxidase has been studied using rapid-scan spectrometry and stopped-flow measurements. The reaction of the totally ferric form of the enzyme with H₂O₂ was slow and the complex formed was inactive in the peroxidatic cycle, whereas partially reduced enzyme formed highly reactive intermediates with hydrogen peroxide. Rapid-scan spectrometry revealed two different spectral forms, one assignable to Compound I and the other to Compound II as found in the reaction cycle of other peroxidases. The formation of Compound I was rapid approaching that of diffusion control. The stoichiometry of the peroxidation reaction, deduced from the formation of oxidized electron donor, indicates that both the reduction of Compound I to Compound II and the conversion of Compound II to resting (partially reduced) enzyme are one-electron steps. It is concluded that the reaction mechanism generally accepted for peroxidases is applicable also to Pseudomonas cytochrome c peroxidase, the intramolecular source of one electron in Compound I formation, however, being reduced heme c.     

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 . 10(7) M-1 . s-1 at low ionic strength (I = 223 mM, 10 degrees C). The value of this rate constant decreases to 1.8 . 10(5) 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 . 10(5) M-1 . s-1 and k-1 = 3.3 . 10(5) M-1 . s-1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10 degrees 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+ in equilibrium or formed from cytochrome c3+1 + cytochrome c2+.     

Electron transfer between azurin and cytochrone c-551 from Pseudomonas aeruginosa.

The electron-transfer reaction between azurin and cytochrome c1 isolated from Pseudomonas aeruginosa was investigated by rapid-reaction techniques. Temperture-jump studies clearly reveal two chemical relaxations, the amplitudes of which have ikentical spectral distributions, but relaxation times show different dependencies on reactant concentrations. Stopped experiments also showed complex kinetics. A model is proposed which is consistent with the kinetic and equilibrium data obtained. The central feature of this model is the proposal that two intercenvertible forms of reduced azurin exist in solution, only one of which si able to participate directly in the electron-transfer reaction with cytochrome c-551. Support for the hypothesis that two forms of reduced azurin exist is derived from studies on the electron-transfer reaction between azurin and Pseudomonas cytochrome oxidase. The possible physiological significance of such a situation is discussed.     

Chromatography of oxidized and reduced cytochrome c on carboxymethylcellulose

1. Cytochrome c was isolated from horse heart by a chromatographic method. 2. Oxidized and reduced cytochrome c were chromatographed on CM-cellulose that was in equilibrium with several buffer systems of constant composition at pH values of 8.4, 6.75 and 4.9. 3. Separation was better at the higher pH values; the oxidized form was retarded more than twice as much as the reduced form, though they differed by only a single charge. 4. Self-competition between cytochrome molecules is suggested to account for the peak distortion observed at high loads (above 20mum protein concentration).     

Isolation and properties of cytochrome c peroxidase from Pseudomonas denitrificans.

The isolation of cytochrome c peroxidase, cytochrome c4, cytochrome c-551 and azurin from Pseudomonas dentrificans is described. The peroxidase has a molecular weight of 63,000 and an isoelectric point of 5.6. Its absorption spectrum suggests that it contains two haem c groups/molecule. Preliminary steady-state kinetic data are reported with cytochromes c-551 and c4 and azurin as the second substrate.     

Small substrates and cytochrome c are oxidized at different sites of cytochrome c peroxidase.

Modeling studies suggest that electrons are transferred from cytochrome c to cytochrome c peroxidase (CcP) with cytochrome c predominantly bound at a site facing the gamma-meso edge of the CcP prosthetic heme group (Poulos, T.L., and Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330). As shown here, guaiacol and ferrocyanide are oxidized at a different site of CcP. Thus, the oxidations of cytochrome c and guaiacol are differentially inactivated by phenylhydrazine and sodium azide. The loss of guaiacol oxidation activity correlates with covalent binding of 1 equivalent of [14C]phenylhydrazine to the protein, whereas the slower loss of cytochrome c activity correlates with the appearance of a 428-nm absorbance maximum attributed to the formation of a sigma-phenyl-iron heme complex. The delta-meso-phenyl and 8-hydroxymethyl derivatives of heme are formed as minor products. Catalytic oxidation of azide to the azidyl radical results in inactivation of CcP and formation of delta-meso-azidoheme. Reconstitution of apo-CcP with delta-meso-azido-, -ethyl-, and -(2-phenylethyl)heme yields holoproteins that give compound I species with H2O2 and exhibit 80, 59, and 31%, respectively, of the control kcat value for cytochrome c oxidation but little or no guaiacol or ferrocyanide oxidizing activity. Conversely, CcP reconstituted with gamma-meso-ethylheme is fully active in the oxidation of guaiacol and ferrocyanide but only retains 27% of the cytochrome c oxidizing activity. These results indicate that guaiacol and ferrocyanide are primarily oxidized near the delta-meso-heme edge rather than, like cytochrome c, at a surface site facing the gamma-meso edge.