The pH dependence of the electron self‐exchange rate of azurin from Pseudomonas aeruginosa as studied by ¹H‐NMR

Abstract

The electron self-exchange rate of azurin from Pseudomonas aeruginosu has been measured by proton NMR as a function of temperature under various conditions of pH, buffer and ionic strength. The rate does not appear very sensitive to variations in the latter parameters. Qualitative thermodynamic compensation is observed for the entropy and enthalpy of activation, with a compensation temperature of 309 7 K, and an average exchange rate of 1.3 x 106 M -' s K ' . The observed high entropy of activation contributes significantly to the high exchange rate. The data are analyzed by considering an encounter complex in which two azurin molecules associate along their hydrophobic patches. Copper-to-copper electron transfer over a distance of 1.5 nm in the complex is facilitated by the favourable disposition of the His-1 17 ligands.For the study of biological electron transfer, haem proteins like the cytochromes have been used extensively, not least because much structural detail is known about them. A few years ago the three-dimensional structures of a number of blue copper proteins were reported [I -51 and an increasing amount of attention has since been diverted to these proteins, which resemble the cytochromes c in size and function, but harbour a single copper atom, in a distorted tetrahedral environment, as the redox active centre instead of the haem group. Their functional characteristics have been studied by examining electron transfer reactions with coordination complexes of the 3d transition metals [6-91, and with redox proteins, mostly cytochromes [lo-161. By applying Marcus theory, the effect of the driving force on the electron transfer rate could be properly accounted for, and theoretical values of the electron self-exchange rates of various blue copper proteins were obtained [14, 171. Conspicuous is the large spread in rates obtained for a particular protein from the analysis of electron transfer reactions with the transition metal compounds. To rationalize this observation, Gray and coworkers devised a kinetic accessibility scale, with azurin from Psmdomonas aeruginosa a t one end (least accessible), and stellacyanin at the other [8, 181. With the cytochromes as reaction partners, more consistent results were obtained for the self-exchange rates [14, 171, but variations, apparently unaccounted for by Marcus theory, still remained.A logical extension of these studies is the direct experimental determination of the electron self-exchange rates of the blue copper proteins. Not only is it of interest to compare the rates derived from Marcus theory with the experimental values, but self-exchange reactions, in principle, are less complex than electron transfer reactions between different partners (the effect of the driving force, for instance, is absent), and one might hope, therefore, easier to analyse. experiments at 50 T . N M R has also been applied to determine the self-exchange rate of azurin from Ps. aeruginosu. The amount of broadening observed in the NMR spectrum of the reduced protein, when a small qu...

Structure of the azurin mutant Phe114Ala fromPseudomonas aeruginosaat 2.6 Å resolution

“…The first site was identified as the surface close to His35. The second was a hydrophobic patch located around the copper ligand His117, also involved in the self-exhange reaction of azurin (Groeneveld & Canters, 1985. In later investigations the role of the His35 patch has been challenged and it has been concluded further that the His35 patch and His35, in particular, is not directly involved in the electron transfer, Van de Kamp, Floris, Hali & Canters (1990) have tentatively suggested that the hydrophobic patch around His117 in azurin is also involved in the electron-transfer reaction with cytochrome c55~.…”

Electron self-exchange in azurin: calculation of the superexchange electron tunneling rate.

“…The self-exchange ET rate for Pseudomonas azurin has been determined by both 'H NMR and rapid-freeze ESR, and the rate constant at 298 K is in the range 0.4-1.4 x 106 M-ls-1 (14)(15)(16)(17). The rate is fairly insensitive to pH and ionic strength and the activation entropy is relatively favorable (15).…”

“…The rate is fairly insensitive to pH and ionic strength and the activation entropy is relatively favorable (15). In azurin, the copper atoms are buried in the protein interior -7 A below the hydrophobic surface patch around the Cu ligand His-117.…”

“…In this case, the second-order exchange rate constant, kEx is kEx = KA X kET, [4] where KA is the association constant and kET is the rate constant for the intercomplex ET. NMR studies have demonstrated that minimal association takes place in aqueous solutions of 1-2 mM azurin, and from the experimental data a KA value of 1 M-1 was determined (15). We may now calculate the self-exchange rate constant from Eq.…”

“…The three-dimensional structure is now available for a number of both natural (wild type) and single-site mutated azurins (9)(10)(11)(12), which makes this group of proteins highly interesting for studies of the different factors that control the rate of long-range ET (13). The rate of self-exchange reactions in both Pseudomonas aeruginosa azurin (14)(15)(16) and Alcaligenes denitrificans azurin (17) have been determined by 'H NMR line broadening and by rapid-freeze ESR as a function of temperature, pH, and ionic strength. The structure ofthe binary complex in the self-exchange reaction is unknown, but the NMR studies described above as well as mutational studies implicate the hydrophobic surface patch surrounding the Cu ligand His-117 (12).…”