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 CεH3 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.