Proton nuclear magnetic resonance assignments and the electron self-exchange rate constant for pseudoazurin from Achromobacter cycloclastes

Abstract

A partial assignment of proton resonances has been made for Achromobacter cycloclastes pseudoazurin. These include the imidazole ring resonances of the three histidine residues and also the C^εH₃ resonances of methionines. Some of these assigned resonances have been used to determine the self-exchange rate constant of pseudoazurin by line-broadening measurements on 1 : 1 mixtures of oxidised and reduced protein. The self-exchange rate constant has also been determined using a Marcus analysis of the rate constant for the cross-reaction between pseudoazurin and azurin. Good agreement between the values determined by these two methods is found with a self-exchange rate constants of 2.9 × 10³ M^–1 s^–1 from NMR and 2.7 × 10³ M^–1 s^–1 from the cross-reaction with azurin, both values being at 25 °C, I= 0.100 M. The self-exchange rate constant for pseudoazurin is much smaller than those of most other type 1 blue copper proteins and is quite similar to those found for the higher plant plastocyanins. From the X-ray crystal structure of pseudoazurin it is known that the copper at the active site is co-ordinated by His, Cys, His and Met residues. On the assumption that exchange is via His-81 protruding at the surface of the protein it is likely that the neighbouring basic residues impede the approach of the two proteins in a suitable orientation for this to occur.