2XV0

Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAHAAM), chemically reduced, pH4.8

Summary for 2XV0

• Entry DOI: 10.2210/pdb2xv0/pdb
• Related: 1AG0 1AZN 1AZR 1AZU 1BEX 1CC3 1E5Y 1E5Z 1E65 1E67 1ETJ 1EZL 1GR7 1I53 1ILS 1ILU 1JVL 1JVO 1JZE 1JZF 1JZG 1JZH 1JZI 1JZJ 1NZR 1R1C 1VLX 1XB3 1XB6 1XB8 2AZU 2FNW 2GHZ 2GI0 2IWE 2TSA 2TSB 2XV2 2XV3 3AZU 4AZU 5AZU
• Descriptor: AZURIN, COPPER (I) ION (3 entities in total)
• Functional Keywords: electron transport, cupredoxin fold
• Biological source: PSEUDOMONAS AERUGINOSA
• Total number of polymer chains: 1
• Total formula weight: 13635.90

Authors

Li, C.,Sato, K.,Monari, S.,Salard, I.,Sola, M.,Banfield, M.J.,Dennison, C. (deposition date: 2010-10-22, release date: 2010-12-29, Last modification date: 2024-10-09)

Primary citation

Li, C.,Sato, K.,Monari, S.,Salard, I.,Sola, M.,Banfield, M.J.,Dennison, C.
Metal-Binding Loop Length is a Determinant of the Pka of a Histidine Ligand at a Type 1 Copper Site
Inorg.Chem., 50:482-, 2011

PubMed Abstract: 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 (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 pKa 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°. 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 C(112)AAAAH(117)AAAM(121) (AZ4A3A) and C(112)AAAAH(117)AAAAM(122) (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 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 pKa of the histidine ligand and other active site properties.

PubMed: 21141901
DOI: 10.1021/IC101413E

Experimental method

X-RAY DIFFRACTION (1.6 Å)