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

Abstract

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...

Direct Measurement of Charge Regulation in Metalloprotein Electron Transfer

“…Most of the Δ Z values associated with ET in Az (ca. 99 %) can be accounted for by the predicted increase in the p K a  values of both non‐coordinating histidine residues (H35, H83) and α‐NH 3 + ‐A1 (Figures S8 and S9 e, f and Tables S5 and S6); the protonation of H35 upon ET has been observed experimentally 8, 21…”

Electron self-exchange in high-potential iron-sulfur proteins. Characterization of protein I from Ectothiorhodospira vacuolata

“…Electron self-exchange reactions of cytochromes c, C551, and by (Gupta et al, 1972;Gupta, 1973;Concar et al, 1986Concar et al, , 1991Dixon et al, 1989Dixon et al, , 1990Timkovich et al, 1988) and copper proteins azurin (Groeneveld & Canters, 1985;Groeneveld et al, 1988; Ugurbil & Mitra, 1984), plastocyanin (Beattie et al, 1975;Armstrong et al, 1985), and amicyanin (Lommen et al, 1988) have been investigated to clarify the influence of structural and/or external factors (pH, ionic strength) on the rates of electron-transfer reactions. In particular, the dependence of self-exchange rate constants k on ionic strength has been the subject of experimental and theoretical studies (van Leeuwen, 1983; Marcus & Sutin, 1985).…”

Determination of the electron self-exchange rate of azurin from Pseudomonas aeruginosa by a combination of fast-flow/rapid-freeze experiments and EPR

“…This is in contrast to the effect of pH on the kinetics of the redox reaction between azurin and cytochrome c551, on the basis of which the existence of a redox-active and a redox-inactive form of azurin was postulated.15 For a detailed discussion of this pH effect the reader is referred to the literature. 18,[21][22][23]31,32 Regarding the magnitude of the electron self-exchange rate constant there is less agreement between the data in Table I. The room-temperature value of the electron self-exchange rate constant reported in the present study, although less accurate than the data at 4 °C, is clearly much larger (about two orders of magnitude) than the value reported by Ugurbil and Mitra (UM).23 Even the value of the electron self-exchange rate constant at 4 °C, which has a better precision, is still more than one order of magnitude larger than the UM room-temperature value.…”