Metal-Binding Loop Length Is a Determinant of the pKa of a Histidine Ligand at a Type 1 Copper Site

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• 作者：Chan Li ; Katsuko Sato ; Stefano Monari ; Isabelle Salard ; Marco Sola ; Mark J. Banfield ; Christopher Dennison
• 刊名：Inorganic Chemistry
• 出版年：2011
• 出版时间：January 17, 2011
• 年：2011
• 卷：50
• 期：2
• 页码：482-488
• 全文大小：814K
• 年卷期：v.50,no.2(January 17, 2011)
• ISSN：1520-510X

文摘

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 pK_a 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 pK_a 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掳. The influence of pH on the reduction potential allows a pK_a of 5.2 卤 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, pK_a values of 4.5 卤 0.1 and 4.4 卤 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 pK_a of the coordinating histidine by approximately 1 pH unit. The pK_a for this histidine ligand varies in native cupredoxins by >5 pH units. Other structural and electronic features, governed primarily by the second-coordination 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 pK_a of the histidine ligand and other active site properties.