The electron transfer reaction between the "blue" single copper protein azurin (from Pseudomonas aeruginosa) and the hexacyanoiron (II/III) couple has been studied. Equilibrium constants for the reduction of azurin were measured spectrophotometrically in the temperature range 5-33 degrees C (K = 1.1 X 10(-2) at 25 degrees C, deltaH degrees = 10.9 kcal/mol, 0.1 M potassium phosphate, pH 7.0, I = 0.22). The enthalpy change was also determined by microcalorimetry and from the analysis of chemical relaxation amplitudes. Following a temperature-jump perturbation of this equilibrium, only a single relaxation was observed. The reciprocal of the relaxation time increased linearly as oxidized azurin was reacted with increasing amounts of ferrocyanide, yet reached saturation when reduced azurin was titrated with ferricyanide. This behavior as well as the analysis of the relaxation amplitudes led to the following scheme for this system: see article. At 25 degrees C the rate constants for the electron transfer were k+3=6.4s-1 and k-3=45s-1, the association constants K1=54 M-1 and K2-1=610 M-1. The activation and overall thermodynamic parameters as well as the individual thermodynamic values for the different steps were combined to construct a self-consistent energy profile for the reaction.
The interaction of Rhus laccase with 0 2 and its reduction intermediates leads to characteristic changes in the circular dichroism (CD) spectrum. Those occurring in the 300-400-nm range can be resolved in terms of three distinct parameters: the intensity and exact position of a negative band at 310-330 nm and the intensity of the positive 370-nm band. The former C D transition is absent in oxidized laccase (native as well as reoxidized), but appears in all cases which are assumed to involve enzyme-bound 0 2 reduction intermediates. Peroxy-laccase, prepared from the nativeoxidized enzyme and H202, displays the most intense band (at 330 nm), with the same anisotropy factor as oxy-tyrosinases and oxy-hemocyanins. A similar, though somewhat blue-shifted, negative band is obtained from the presumed peroxy derivative produced by reacting partially reduced laccase with 0 2 . Aerobic reduction of laccase with a limited amount of ascorbate leads to a transient spectrum which, characterized by a negative band at 330 nm and a strong enhancement of the (+) 370-nm band intensity, is possibly related to a bound oxygen intermediate. After the first-order decay of this transient (k = 5.1 x s-', 25 "C, pH 7.0) a stable spectrum is established which clearly differs from that of native laccase and is attributed to a different, metastable form of the oxidized enzyme (C state). The CD spectrum of laccase formed during turnover at low ascorbate concentrations suggests that under these conditions peroxy intermediates and enzyme molecules in the C state are the major species present in the steady-state phase. We propose a catalytic scheme for laccase which allows for the reactions of 0 2 with laccase molecules reduced to different extents and which suggests cooperation between the type 2 and type 3 sites in the bond-breaking reduction of the peroxide intermediate.The nature of oxygen reduction intermediates in the catalytic conversion of dioxygen to water by Rhus vernicifera laccase and related blue copper oxidases is a topic of considerable interest [l]. Since the process involves the transfer of four electrons and four protons, many different intermediates can be expected. We have recently obtained evidence that 0 2 interacts with laccase molecules at all stages of reduction, i.e. whether containing 1, 2, 3 or 4 redox electrons [2]. The formation of a peroxy-laccase complex by the reaction of hydrogen peroxide with the type 3 site of oxidized native laccase, has previously been demonstrated [2-61. Its formation is accompanied by a relatively small change in the absorption spectrum of the enzyme, but pronounced effects are observed in its circular dichroic (CD) spectrum. The Abbreviation. CD, circular dichroism. Enzyme. Rhus vernicifera laccase (EC 1.10.3.2). most conspicuous one is a large negative Cotton effect appearing in the near-ultraviolet region [ 5 ] . CD measurements therefore seem to be particularly useful for monitoring chemical and conformational changes in the enzyme resulting from the reaction with 0 2 and its reductio...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.