Toggle Main Menu Toggle Search

Open Access padlockePrints

Loop-directed mutagenesis of the blue copper protein amicyanin from Paracoccus versutus and its effect on the structure and the activity of the type-1 copper site

Lookup NU author(s): Professor Christopher Dennison

Downloads

Full text for this publication is not currently held within this repository. Alternative links are provided below where available.


Abstract

Four loop-mutants of the blue copper protein amicyanin from Paracoccus versutus have been constructed and characterized. The mutations replaced the loop containing three (Cys93, His96, Met99) of the four copper ligands in amicyanin by the 'ligand loops' of P. aeruginosa azurin (AmiAzu), A. faecalis pseudoazurin (AmiPaz), P. nigra plastocyanin (AmiPcy), and P. aureofaciens nitrite reductase (AmiNiR). The copper centers of all variants appear to be perfect type-1 Cu sites although the AmiNiR variant exhibits diminished stability. The optical spectra of the AmiAzu and AmiPaz variants display a significant dependence on temperature. Excitation at 457 nm as well as 647 nm results in similar resonance Raman spectra. The reduction potentials of the three stable variants are all higher than that of wt amicyanin. The reduced forms of the loop-mutants protonate at the C-terminal histidine, with pK(a) values of 5.6 (AmiAzu), 5.4 (AmiPaz), and 5.7 (AmiPcy) (6.8 for wt amicyanin). AmiAzu is the first known cupredoxin with more than two amino acids between the cysteine and histidine ligands that undergoes this protonation. The electron self-exchange rate constants at 25 °C are 5.7-7.5 times lower for the loop mutants than for wt amicyanin (1.2 x 105 M-1 s-1). The results are interpreted by taking into consideration that three metal ligands are intimately connected with the stable β-sandwich structure of the cupredoxin. This leaves considerable freedom in positioning the fourth one, the C-terminal His, on the 'ligand loop'. This explains the ease by which the C-terminal histidine ligand can be excised and replaced by external ligands without losing the metal binding property of the protein. The results also help understand the remarkable evolutionary success of the combination of cupredoxin fold and Cu site for mediating biological electron transfer.


Publication metadata

Author(s): Buning C, Canters GW, Comba P, Dennison C, Jeuken L, Melter M, Sanders-Loehr J

Publication type: Article

Publication status: Published

Journal: Journal of the American Chemical Society

Year: 2000

Volume: 122

Issue: 2

Pages: 204-211

Print publication date: 31/12/1999

ISSN (print): 0002-7863

ISSN (electronic): 1520-5126

Publisher: American Chemical Society

URL: http://dx.doi.org/10.1021/ja992796b

DOI: 10.1021/ja992796b


Altmetrics

Altmetrics provided by Altmetric


Actions

Find at Newcastle University icon    Link to this publication


Share