Metal-binding Loop Length is a Determinant of the pKa of Histidine Ligand at a Type 1 Copper Site.

The type 1 copper site of a cupredoxin involves coordination by cysteine, histidine, and methionine residues from a single loop. Dissociation and protonation of the histidine ligand on this loop is observed in only certain reduced cupredoxins and can regulate electron-transfer reactivity. This effect is introduced in azurin (AZ) (the wild-type protein has an estimated pKa of <2) by mutating the native copper-binding loop (C112TFPGH117SALM121, ligands numbered). In this work, we have investigated the influence of loop length alone on histidine ligand protonation by determining the pKa value in AZ variants with ligand-containing polyalanine loops of different length. Crystal structures of the Cu(I)- variant with the loop sequence C112AAH115AAM118 (AZ2A2A) demonstrate that at pH 4.2 His115 is protonated and no longer coordinated, and the imidazole ring is rotated by 180. The influence of pH on the reduction potential allows a pKa of 5.2 ( 0.1 for His115 in Cu(I)-AZ2A2A to be determined. In the reduced AZ variants in which the loop sequences C112AAAAH117AAAM121 (AZ4A3A) and C112AAAAH117AAAAM122 (AZ4A4A) have been introduced, pKa values of 4.5 ( 0.1 and 4.4 ( 0.1, respectively, are obtained for the His117 ligand. Consistent with these data, the crystal structure of Cu(I)-AZ4A4A at pH 5.3 shows no sign of His117 protonation (crystals were unstable at lower pH values). The loop length range studied matches that which occurs naturally and these investigations indicate that length alone can alter the pKa of the coordinating histidine by approximately 1 pH unit. The pKa for this histidine ligand varies in native cupredoxins by >5 pH units. Other structural and electronic features, governed primarily by the secondcoordination sphere, to which the ligand-binding loop is a major contributor, also alter this important feature. A longer ligand-containing loop made of residues whose side chains are larger and more complex than a methyl group increases the second coordination sphere providing additional scope for tuning the pKa of the histidine ligand and other active site properties.