The mutation Met121-> His creates a type-1.5 copper site in Alcaligenes denitrificans azurin

被引:49
作者
Kroes, SJ
Hoitink, CWG
Andrew, CR
Ai, JY
SandersLoehr, J
Messerschmidt, A
Hagen, WR
Canters, GW
机构
[1] LEIDEN UNIV, LEIDEN INST CHEM, GORLAEUS LABS, NL-2300 RA LEIDEN, NETHERLANDS
[2] NV ORGANON, DEPT BIOTECHNOL & BIOCHEM, NL-5340 BH OSS, NETHERLANDS
[3] OREGON GRAD INST SCI & TECHNOL, DEPT CHEM BIOCHEM & MOL BIOL, PORTLAND, OR USA
[4] MAX PLANCK INST BIOCHEM, D-82152 MARTINSRIED, GERMANY
[5] AGR UNIV WAGENINGEN, DEPT BIOCHEM, WAGENINGEN, NETHERLANDS
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1996年 / 240卷 / 02期
关键词
site-directed mutagenesis; blue-copper protein; type-1 copper site; electron transfer;
D O I
10.1111/j.1432-1033.1996.0342h.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The Cu ligand Met121 in azurin of Alcaligenes denitrificans was mutated to His. The spectroscopic and mechanistic properties of [M121H]azurin appear to be pH dependent with a pK(a) of 3.8 due to the ionization of His121. The [M121H]azurin mutant exhibits two major distinct metal-site-coordination geometries which coexist in solution according to a pH-dependent equilibrium. Both species have been spectroscopically characterized by ultraviolet-visible, EPR and resonance Raman spectroscopies. At neutral DH. His121 is deprotonated and acts as the fourth ligand of the Cu; the spectroscopic characteristics of the Cu site at this pH are halfway between those of a type-1 and a type-2 Cu site, and the site is referred to as a type-1.5 or intermediate Cu site. The spectral data are compatible with a tetrahedral geometry of this site. At low pH, the spectroscopic data indicate that [M121H]azurin has a trigonal type-1 rhombic Cu site.
引用
收藏
页码:342 / 351
页数:10
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