PROTON NUCLEAR-MAGNETIC-RESONANCE ASSIGNMENTS AND THE ELECTRON SELF-EXCHANGE RATE-CONSTANT FOR PSEUDOAZURIN FROM ACHROMOBACTER-CYCLOCLASTES

A partial assignment of proton resonances has been made for Achromobac ter cycloclastes pseudoazurin. These include the imidazole ring resona nces of the three histidine residues and also the C(epsilon)H3 resonan ces of methionines. Some of these assigned resonances have been used t o determine the self-exchange rate constant of pseudoazurin by line-br oadening measurements on 1:1 mixtures of oxidised and reduced protein. The self-exchange rate constant has also been determined using a Marc us analysis of the rate constant for the cross-reaction between pseudo azurin and azurin. Good agreement between the values determined by the se two methods is found with a self-exchange rate constants of 2.9 x 1 0(3) M-1 s-1 from NMR and 2.7 x 10(3) M-1 s-1 from the cross-reaction with azurin, both values being at 25-degrees-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 f ound for the higher plant plastocyanins. From the X-ray crystal struct ure 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 th e two proteins in a suitable orientation for this to occur.