Kinetics and equilibriums of the electron transfer between azurin and the hexacyanoiron (II/III) couple

Goldberg, M; Pecht, Israel

doi:10.1021/bi00664a011

Cited by 66 publications

(32 citation statements)

References 36 publications

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“…[6]. The concentration was determined spectrophotometrically at 628 nm, using an absorbance coefficient of 5700 M -l .cm 1 [7]. The Hll7G mutant of azurin was obtained in the apo-form according to ref.…”

Section: Methodsmentioning

confidence: 99%

The role of His117 in the redox reactions of azurin from Pseudomonas aeruginosa

Gorren¹,

Blaauwen²,

Canters³

et al. 1996

FEBS Letters

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The electron-transfer properties of HI 17G-and wildtype azurin were compared by applying both as electron acceptors in the conversion of 4-ethylphenol by 4-ethylphenol methylenehydroxylase (4-EPMH). The reactivity of Hll7G-azurin was determined in the absence and presence of imidazoles, which can substitute the missing fourth ligand. In the absence of imidazoles, Hll7G-azurin reacted directly with 4-ethylphenol, this reaction was abolished in the presence of imidazoles. The enzymatic reduction of H117G-azurin by 4-EPMH was 40 times slower than that of wild-type azurin. The rate of this reaction was enhanced by some imidazoles, diminished by others. In all cases the reduction of Hll7G-azurin was irreversible. These results demonstrate that Hisll7 is vital for electron transfer and effectively protects the copper site against aspecific reactions.

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Section: Methodsmentioning

confidence: 99%

The role of His117 in the redox reactions of azurin from Pseudomonas aeruginosa

Gorren¹,

Blaauwen²,

Canters³

et al. 1996

FEBS Letters

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“…As can be seen, the slopes of the lines are close to unity (0.95 and 1.02 for the Alcaligenes and Pseudomonas azurins, respectively), as expected for a single electron transfer. There is a difference of [9].…”

Section: Potentiometric Titrutionsmentioning

confidence: 99%

Electron Transfer between Azurin from Alcaligenes faecalis and Cytochrome c551 from Pseudomonas aeruginosa

1981

Self Cite

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The electron transfer equilibrium and kinetics between azurin from Alruli~enr.s,fueru/i.s and cytochrome r~s I from Pseudomonas aeruginosa have been studied. The equilibrium constant. [Az(IIj]) = 0.5 at 25°C is about seven times smaller than that observed between the cytochrome c5s1 and the azurin both from P. ae~ugizzosa [Rosen, P. and Pecht,1. ( 1 976 The kinetics of the reaction between Akaligenc~s azurin and Pseudomonas r~torlz~oznc cSs1 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 betwecn P. acwginosu azurin and cytochrome cSs1 is not observed with the AIc~ligezz~.~ azurin. The simplest mechanism that can therefore be ascribed to the investigated system is:This scheme is similar to that proposed earlier for the reaction between P. ucmginosa azurin and cytochrome ~' 5 5~ but does not involve the conformational transition proposed for azurin. The specific rates for the elcctron transfcr are still fast: 1

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“…Because of their convenient size and the unusual characteristics of their redox center, the structure and the electron exchange properties of this protein have been examined in detail (e.g., refs. [1][2][3][4][5][6][7][8][9][10][11][12]. In several of these studies, the electron self-exchange rate for azurin has been calculated based on Marcus theory and electron transfer rates between azurin and a variety of redox partners (7)(8)(9)(10).…”

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confidence: 99%

1H NMR studies of electron exchange rate of Pseudomonas aeruginosa azurin.

Uğurbil

Mitra

1985

Proc. Natl. Acad. Sci. U.S.A.

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T1 values of the His-35 C-2 proton resonance of reduced Pseudomonas aeruginosa azurin were determined at 25C and pH values 4.5, 7.3, and 9.0 in the presence of different fractional amounts of oxidized azurin. The C-2 proton of His-35 undergoes very rapid spin relaxation in oxidized azurin because of its close proximity to the paramagnetic copper. In the presence of oxidized protein, the T1 values of this proton in reduced azurin depend on the lifetime of the reduced protein. From the T1 data, the electron self-exchange rate constant for azurin was calculated to be 1.4 x 104 M-s-, 4.3 x 10 M-l s'l, and 6.0 x 103 M-l s'1 at pH values 4.5, 7.3, and 9, respectively. At pH 7.3, the C-2 proton of His-35 is in slow exchange between the imidazole and imidazolium forms and gives rise to two separate resonances at 9.39 and 8.00 ppm. By using these two resonances, the electron self-exchange rate constants were determined separately for the two species of azurin for which the His-35 residue is in the imidazole or the imidazolium forms; results showed that both species participate in self-exchange of electrons with equal efficiency.Azurins are low molecular weight bacterial proteins that contain one "blue" copper redox center per molecule of protein. Because of their convenient size and the unusual characteristics of their redox center, the structure and the electron exchange properties of this protein have been examined in detail (e.g., refs. 1-12). In several of these studies, the electron self-exchange rate for azurin has been calculated based on Marcus theory and electron transfer rates between azurin and a variety of redox partners (7-10). Given the important role played by this parameter in theoretical considerations of the overall electron exchange process, it would be highly desirable to determine its value experimentally.In this paper we report direct measurements of the selfexchange rate constant for Pseudomonas aeruginosa azurin using 1H nuclear magnetic resonance spectroscopy. The measurements rely on determinations of the spin-lattice relaxation time (T,) gen by the Grain Processing Corp., Muscatine, IA. Azurin was purified as described (12). The final product had a purity ratio of (Ag25/A280) > 0.52 and also was checked by the appearance of a single band corresponding to azurin in a polyacrylamide gel.Azurin samples were prepared for NMR by taking lyophilized protein and incubating in excess 2H20 at pH 3.5 and 50'C for 1 hr. The pH was then adjusted to 7.5, and the samples were lyophilized and subsequently redissolved in 0.1 M NaCl in 2H20. Reduced azurin was prepared by addition of dithionite and subsequent dialysis against 0.1 M NaCl in 2H20 under an argon atmosphere. Oxidized azurin concentrations were measured at 625 nm with an extinction coefficient of 5.7 x 103 M-cm-(2). The pH values of the solutions were adjusted with NaO2H or 2HCl in 2H2Q. The pH readings reported represent direct electrode readings and were not corrected for the deuterium isotope effect.The 1H NMR spectra were recorded at 25...

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Kinetics and equilibriums of the electron transfer between azurin and the hexacyanoiron (II/III) couple

Cited by 66 publications

References 36 publications

The role of His117 in the redox reactions of azurin from Pseudomonas aeruginosa

The role of His117 in the redox reactions of azurin from Pseudomonas aeruginosa

Electron Transfer between Azurin from Alcaligenes faecalis and Cytochrome c551 from Pseudomonas aeruginosa

1H NMR studies of electron exchange rate of Pseudomonas aeruginosa azurin.

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