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

Lookup NU author(s): Dr Chan Li, Dr Katsuko Sato, Dr Isabelle Salard-Arnaud, Dr Mark Banfield, Professor Christopher Dennison


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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 cart 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 (C(112)TFPGH(117)SALM(121), 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 C(112)AAH(115)AAM(118) (AZ2A2A) demonstrate that at pH 4.2 His115 is protonated and no longer coordinated, and the imidazole ring is rotated by 180 degrees. The influence of pH on the reduction potential allows a pK(a) of 5.2 +/- 0.1 for His115 in Cu(I)-AZ2A2A to be determined. In the reduced AZ variants in which the loop sequences C(112)AAAAH(117)AAAM(121) (AZ4A3A) and C(112)AAAAH(117)AAAAM(122) (AZ4A4A) have been introduced, pK(a) 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 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.

Publication metadata

Author(s): Li C, Sato K, Monari S, Salard I, Sola M, Banfield MJ, Dennison C

Publication type: Article

Publication status: Published

Journal: Inorganic Chemistry

Year: 2011

Volume: 50

Issue: 2

Pages: 482-488

Print publication date: 08/12/2010

ISSN (print): 0020-1669

ISSN (electronic): 1520-510X

Publisher: American Chemical Society


DOI: 10.1021/ic101413e


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Funder referenceFunder name
Royal Society (United Kingdom)
BB/C504519/1Biotechnology and Biological Sciences Research Council