A temperature-jump study of the reaction between azurin and cytochrome c-551 from Pseudomonas aeruginosa.
Ontology highlight
ABSTRACT:
SUBMITTER: Brunori M
PROVIDER: S-EPMC1166087 | biostudies-other | 1974 Jan
REPOSITORIES: biostudies-other
Similar Datasets
Project description: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.
| S-EPMC1172342 | biostudies-other
Project description:The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a 'half-hearted' behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady-state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c-551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism.
| S-EPMC1152686 | biostudies-other
Project description: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.
| S-EPMC1165244 | biostudies-other
Project description: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.
| S-EPMC1183676 | biostudies-other
Project description:Hydrogen exchange rates for backbone amide protons of oxidized Pseudomonas aeruginosa cytochrome c-551 (P. aeruginosa cytochrome c) have been measured in the presence of low concentrations of the denaturant guanidine hydrochloride. Analysis of the data has allowed identification of submolecular unfolding units known as foldons. The highest-energy foldon bears similarity to the proposed folding intermediate for P. aeruginosa cytochrome c. Parallels are seen to the foldons of the structurally homologous horse cytochrome c, although the heme axial methionine-bearing loop has greater local stability in P. aeruginosa cytochrome c, in accord with previous folding studies. Regions of low local stability are observed to correspond with regions that interact with redox partners, providing a link between foldon properties and function.
| S-EPMC2654780 | biostudies-literature
Project description:The reaction of ascorbate-reduced Pseudomonas cytochrome oxidase with oxygen was studied by using stopped-flow techniques at pH 7.0 and 25 degrees C. The observed time courses were complex, the reaction consisting of three phases. Of these, only the fastest process, with a second-order rate constant of 3.3 X 10(4) M-1.S-1, was dependent on oxygen concentration. The two slower processes were first-order reactions with rates of 1.0 +/- 0.4s-1 and 0.1 +/- 0.03s-1. A kinetic titration experiment revealed that the enzyme had a relatively low affinity constant for oxygen, approx. 10(4)M-1. Kinetic difference spectra were determined for all three reaction phases, showing each to have different characteristics. The fast-phase difference spectrum showed that changes occurred at both the haem c and haem d1 components of the enzyme during this process. These changes were consistent with the haem c becoming oxidized, but with the haem d1 assuming a form that did not correspond to the normal oxidized state, a situation that was not restored even after the second kinetic phase, which reflected further changes in the haem d1 component. The results are discussed in terms of a kinetic scheme.
| S-EPMC1185743 | biostudies-other
Project description:The redox reaction between cytochrome c-551 and its oxidase from the respiratory chain of pseudomonas aeruginosa was studied by rapid-mixing techniques at both pH7 and 9.1. The electron transfer in the direction of cytochrome c-551 reduction, starting with the oxidase in the reduced and CO-bound form, is monophasic, and the governing bimolecular rate constants are 1.3(+/- 0.2) x 10(7) M-1 . s-1 at pH 9.1 and 4 (+/- 1) x 10(6) M-1 . s-1 at pH 7.0. In the opposite direction, i.e. mixing the oxidized oxidase with the reduced cytochrome c-551 in the absence of O2, both a lower absorbance change and a more complex kinetic pattern were observed. With oxidized azurin instead of oxidized cytochrome c-551 the oxidation of the c haem in the CO-bound oxidase is also monophasic, and the second-order rate constant is 2 (+/- 0.7) x 10(6) M-1 . s-1 at pH 9.1. The redox potential of the c haem in the oxidase, as obtained from kinetic titrations of the completely oxidized enzyme with reduced azurin as the variable substrate, is 288 mV at pH 7.0 and 255 mV at pH 9.1. This is in contrast with the very high affinity observed in similar titrations performed with both oxidized azurin and oxidized cytochrome c-551 starting from the CO derivative of the reduced oxidase. It is concluded that: (i) azurin and cytochrome c-551 are not equally efficient in vitro as reducing substrates of the oxidase in the respiratory chain of Pseudomonas aeruginosa; (ii) CO ligation to the d1 haem in the oxidase induces a large decrease (at least 80 mV) in the redox potential of the c-haem moiety.
| S-EPMC1158249 | biostudies-other
Project description:Cytochrome c-551 was prepared from nine different strains of Pseudomonas aeruginosa and six of Pseudomonas fluorescens biotype C, and their amino acid sequences were compared with the sequences previously determined for the cytochromes of type strains of each species. The standard of sequence examination was such that all single amino acid substitutions, delections or insertions ought to have been detected. Balanced double changes in sites in the same part of the sequence might have escaped detection. The standard of some of the quantitative amino acid analyses was not as high as would be required for the investigation of completely unknown sequences. Eight of the Ps. aeruginosa sequences could not be distinguished from the type sequence, whereas the ninth had a single amino acid substitution. The sequences from Ps. fluorescens biotype C were more varied, differing in from zero to four substitutions from the type sequence, with the most diverse sequences differing in seven positions. The results for Ps. aeruginosa are interpreted as evidence that neutral mutations are not responsible for much molecular evolution. The superficially paradoxical differences in the results for the two species are discussed.
| S-EPMC1166074 | biostudies-other
Project description:We have designed ruthenium-modified Pseudomonas aeruginosa azurins that incorporate 3-nitrotyrosine (NO2YOH) between Ru(2,2'-bipyridine)2(imidazole)(histidine) and Cu redox centers in electron transfer (ET) pathways. We investigated the structures and reactivities of three different systems: RuH107NO2YOH109, RuH124NO2YOH122, and RuH126NO2YOH122. RuH107NO2YOH109, unlabeled H124NO2YOH122, and unlabeled H126NO2YOH122 were structurally characterized. The pKa's of NO2YOH at positions 122 and 109 are 7.2 and 6.0, respectively. Reduction potentials of 3-nitrotyrosinate (NO2YO(-))-modified azurins were estimated from cyclic and differential pulse voltammetry data: oxidation of NO2YO(-)122 occurs near 1.1 versus NHE; oxidation of NO2YO(-)109 is near 1.2 V. Our analysis of transient optical spectroscopic experiments indicates that hopping via NO2YO(-) enhances Cu(I) oxidation rates over single-step ET by factors of 32 (RuH107NO2YO(-)109), 46 (RuH126NO2YO(-)122), and 13 (RuH124NO2YO(-)122).
| S-EPMC3839300 | biostudies-literature
Project description:The electron-transfer reactions of cellobiose oxidase (CBO) have been investigated by conventional and by rapid-scan stopped-flow spectroscopy at pH 6.0. Analysis of the absorbance/time/wavelength matrix by Singular Value Decomposition (SVD) confirms earlier studies showing that cellobiose rapidly reduces the flavin group (7.7 s-1; cellobiose, 100 microM) which in turn slowly (0.2 s-1) reduces the cytochrome b moiety. In the presence of CBO, cellobiose reduces cytochromes c in a reaction that does not depend on oxygen or superoxide. The rate limit for this process is independent of the source of the cytochromes c and is identical with the rate of cytochrome b reduction. Rapid-mixing experiments show that cytochrome b may donate electrons very rapidly to either mammalian cytochrome c or bacterial cytochrome c-551. The reactions were second-order (kc = 1.75 x 10(7) M-1 x s-1; kc-551 = 4.3 x 10(6) M-1 x s-1; pH 6.0, 21 degrees C and I0.064) and strongly ionic-strength (I)-dependent: kc decreasing with I and kc-551 increasing with I. These results suggest the electron-transfer site near cytochrome b bears a significant negative charge. Equilibrium gel chromatography confirms that CBO oxidase and positively charged mammalian cytochrome c make stable complexes. These results are discussed in terms of a model suggesting an electron-transfer role for cytochrome b in vivo, possibly connected with radical-mediated cellulose breakdown.
| S-EPMC1137943 | biostudies-other