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

Rosen, Philip; Segal, Michael G.; Pecht, Israel

doi:10.1111/j.1432-1033.1981.tb05709.x

Cited by 24 publications

(27 citation statements)

References 28 publications

(20 reference statements)

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“…It was suggested that through structural perturbations induced near the copper center, "the electron exchange efficiency between azurin and the Cyt c551 may be affected by the ionization of this histidine and/or that the PKa of this residue is different in the oxidized and reduced azurins, resulting in complex equilibria and decay kinetics in the reported temperature-jump experiments, which were carried out at neutral pH" (3). All subsequent experiments concerned with this question (6,12,(22)(23)(24)(25) indicate that the protonation reaction of the His-35 residue is indeed the source of the complex decay kinetics observed in T-jump studies of electron exchange between azurin and Cyt c551. However, any model in which the electron exchange rate between azurin and Cyt c is proposed to be significantly altered by the ionization state of His-35 is inconsistent with the fact that the electron transfer rate between Cyt c551 and azurin is independent of pH in the range 5.0-9.0 (6, 23, 26) and with our current results, which show that the self-exchange rate constant is not affected by His-35 ionization.…”

Section: Rate Measurementsmentioning

confidence: 99%

“…It was inferred from this unusual kinetic behavior that reduced azurin in solution exists in two conformations, one of which is absolutely inactive in the electron transfer reaction with Cyt c551 (1,21). In subsequent reports (22)(23)(24) the "active" and the "inactive" conformers were proposed to be the azurin molecules that differ in the state of protonation of His-35. The possibility that the electron exchange kinetics of azurin may be affected by His-35 ionization, although not in the all-or-nothing fashion later envisioned (22)(23)(24), was first suggested on the basis of natural abundance 13C NMR studies on P. aeruginosa azurin (3).…”

Section: Rate Measurementsmentioning

confidence: 99%

“…In subsequent reports (22)(23)(24) the "active" and the "inactive" conformers were proposed to be the azurin molecules that differ in the state of protonation of His-35. The possibility that the electron exchange kinetics of azurin may be affected by His-35 ionization, although not in the all-or-nothing fashion later envisioned (22)(23)(24), was first suggested on the basis of natural abundance 13C NMR studies on P. aeruginosa azurin (3). The 13C NMR data was found to be inconsistent with the presence of fully "active" and fully "inactive" conformers of azurin.…”

Section: Rate Measurementsmentioning

confidence: 99%

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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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Section: Rate Measurementsmentioning

confidence: 99%

Section: Rate Measurementsmentioning

confidence: 99%

Section: Rate Measurementsmentioning

confidence: 99%

See 1 more Smart Citation

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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“…The long exchange time of 0O. 1 sec actually found shows that the protonation must be accompanied by a local conformation change that alters the effectiveness of the protein in electron donation.Our interest in the azurin from Alcaligenes faecalis was aroused by the fact that this azurin in its reaction with cytochrome c551 shows only a single fast relaxation time (12). Therefore, we undertook a NMR study to see how the imidazole side chains behave as a function of pH.…”

mentioning

confidence: 99%

“…Our interest in the azurin from Alcaligenes faecalis was aroused by the fact that this azurin in its reaction with cytochrome c551 shows only a single fast relaxation time (12). There-fore, we undertook a NMR study to see how the imidazole side chains behave as a function of pH.…”

mentioning

confidence: 99%

Proton NMR of the histidines of azurin from Alcaligenes faecalis: linkage of histidine-35 with redox kinetics.

Mitra¹,

Bersohn²

1982

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

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On the basis of redox kinetic studies, Rosen and Pecht [Rosen, P. & Pecht, I. (1976) Biochem. 120,[339][340][341][342][343][344] observed that the azurin from the bacterium Alcaligenesfaecalis shows no such slowly attained equilibrium between two forms. Therefore, a 1H NMR study was carried out on this azurin with emphasis on the downfield region. A resonance was found at 7.95 ppm downfield that does not move with pH, is not seen in the oxidized protein, has the same pseudocontact shift in the Co(II) derivative as the C-2 proton of histidine-35 has in the Co(E) derivative of P. aeruginosa azurin, and, in the apoprotein, exhibits a typical protonation shift downfield at pH <5. Therefore, this resonance is assigned to the C-2 proton of histidine-35. The crystal structure ofP. aeruginosa azurin shows that at pH 7 the imidazole side chain ofhistidine-35 is in a crevice within the protein, where its ring is adjacent and parallel to that ofhistidine-47, a copper ligand. The preceding observations combined with others show that the kinetics of some redox reactions involving azurin depend on the position of histidine-35. The implication is that there is a pathway for electron transport to the copper atom involving passage through histidine-35.An azurin is a deep blue, low molecular weight (Mr 14,000), bacterial protein that contains one Cu(II) per molecule. The crystal structure of one of them, the azurin from Pseudomonas aeruginosa has been solved (1,2), and the copper ligands are nitrogen atoms from His-46 and His-117 and sulfur atoms from Cys-112 and Met-121. These four amino acids (and many others) are invariant in all of the azurins with sequences determined so far (3). Indeed, the deep blue color has been convincingly explained as being due to a charge-transfer transition from the cysteinate sulfur to Cu(II) (4, 5).The function of azurins in bacteria has not yet been elucidated, but a generous hint is furnished by the fast rate (6) of electron transfer between P. aeruginosa azurin and cytochrome c551, another small protein found in the same bacterium. When the temperature is suddenly raised, the redox equilibrium shifts toward reduced azurin with a fast time constant and then shifts back again with a slow time constant (7,8). Thus, there are two interesting aspects to the reaction. One is the rapid electron transfer (k 106 M -1sec 1) between two proteins, both ofwhose metal ions are well shielded from the solvent. The other is the slow interconversion of two conformers of the same protein, reduced azurin, one ofwhich is much more reactive in electron transfer than the other. Previous NMR (9-11) and temperaturejump studies (unpublished data) have shown that the slowly attained protonation-deprotonation equilibrium of the imidazole side chains of His-35 may be the slow step in the redox process. Normally the exchange time near neutral pH is short (<1 psec). The long exchange time of 0O. 1 sec actually found shows that the protonation must be accompanied by a local conformation change that alters the ef...

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Electron transfer in biological systems

Larsson

2009

Int. J. Quantum Chem.

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The conditions for bridged resonance transfer are derived from a time-dependent quantum mechanical treatment of a model system. It is suggested that biological transfer is characterized by small reaction barriers and that uncontrolled diffusion of electrons is prevented by nonadiabatic behavior in the electron transfer process. Adiabatic transfer pathways should have well overlapping orbitals without gaps. Some examples are given. It is finally suggested that sulfur atoms also may act as exchange centers and thereby divide up the transfer distance into smaller parts.

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

Cited by 24 publications

References 28 publications

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

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

Proton NMR of the histidines of azurin from Alcaligenes faecalis: linkage of histidine-35 with redox kinetics.

Electron transfer in biological systems

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