Site-directed mutagenesis of pseudoazurin from Alcatigenes faecalis S-6; Pro80Ala mutant exhibits marked increase in reduction potential

Pseudoazurin (a blue copper protein or cupredoxin) of a denitrifyingbacterium Alcaligenes faecalis S-6 is a direct electron carrierfor a Cu-containing nitrite reductase (NIR) of the same organism.Site-directed mutagenesis of the pseudoazurin was carried outusing an Escherichia coli expression system. Replacement ofTyr74 by Phe to remove an internal hydrogen bond in the ß-barrelcaused a slight decrease in heat stability as well as a requirementfor a higher concentration of Cu²⁺ for production in the E.colihost. Exchange of Ala for Pro80 adjacent to His81, one of thefour ligands binding a type I Cu atom, caused a marked increasein reduction potential by 139 mV without change in the opticalabsorption spectrum. The ability of the pseudoazurin to transferelectrons to NIR was markedly diminished but the apparent K_mof NIR for pseudoazurin was not affected by the mutation. X-raydiffraction data collected on the oxidized and reduced formsof the Pro80Ala mutant show that a water molecule occupies thepocket created by the absent side chain. This observation suggeststhat the increase in reduction potential may be caused due tothe increased solvent accessibility to the Cu atom. The electrondensity difference maps on these structures (at 2.0 Å)show that this water moves during the change in oxidation state,and that there are small, but localized, conformational changes>6.5 Å from the copper site, as well as movement ofboth the Cu²⁺ and the cysteinate sulfur.