Reconstitution of the Type-1 Active Site of the H145G/A Variants of Nitrite Reductase by Ligand Insertion
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- 作者：Hein J.Wijma ; Martin J. Boulanger ; Annamaria Molon ; Maria Fittipaldi ; Martina Huber ; Michael E. P. Murphy ; Martin Ph. Verbeet ; and Gerard W. Canters
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：April 15, 2003
- 年：2003
- 卷：42
- 期：14
- 页码：4075 - 4083
- 全文大小：294K
- 年卷期：v.42,no.14(April 15, 2003)
- ISSN：1520-4995
文摘
Variants of the copper-containing nitrite reductase (NiR) of Alcaligenes faecalis S6 wereconstructed by site-directed mutagenesis, by which the C-terminal histidine ligand (His145) of the Cu inthe type-1 site was replaced by an alanine or a glycine. The type-1 sites in the NiR variants as isolated,are in the reduced form, but can be oxidized in the presence of external ligands, like (substituted) imidazolesand chloride. The reduction potential of the type-1 site of NiR-H145A reconstituted with imidazole amountsto 505 mV vs NHE (20 
C, pH 7, 10 mM imidazole), while for the native type-1 site it amounts to 260mV. XRD data on crystals of the reduced and oxidized NiR-H145A variant show that in the reducedtype-1 site the metal is 3-coordinated, but in the oxidized form takes up a ligand from the solution. Withthe fourth (exogenous) ligand in place the type-1 site is able to accept electrons at about the same rate asthe wt NiR, but it is unable to pass the electron onto the type-2 site, leading to loss of enzymatic activity.It is argued that the uptake of an electron by the mutated type-1 site is accompanied by a loss of theexogenous ligand and a concomitant rise of the redox potential. This rise effectively traps the electron inthe type-1 site.
C, pH 7, 10 mM imidazole), while for the native type-1 site it amounts to 260mV. XRD data on crystals of the reduced and oxidized NiR-H145A variant show that in the reducedtype-1 site the metal is 3-coordinated, but in the oxidized form takes up a ligand from the solution. Withthe fourth (exogenous) ligand in place the type-1 site is able to accept electrons at about the same rate asthe wt NiR, but it is unable to pass the electron onto the type-2 site, leading to loss of enzymatic activity.It is argued that the uptake of an electron by the mutated type-1 site is accompanied by a loss of theexogenous ligand and a concomitant rise of the redox potential. This rise effectively traps the electron inthe type-1 site.