Alternate Substrate Binding Modes to Two Mutant (D98N and H255N) Forms of Nitrite Reductase from Alcaligenes faecalis S-6: Structural Model of a Transient Catalytic Intermediate
详细信息 查看全文
- 作者：Martin J. Boulanger and Michael E. P. Murphy
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：August 7, 2001
- 年：2001
- 卷：40
- 期：31
- 页码：9132 - 9141
- 全文大小：503K
- 年卷期：v.40,no.31(August 7, 2001)
- ISSN：1520-4995
文摘
High-resolution nitrite soaked oxidized and reduced crystal structures of two active site mutants,D98N and H255N, of nitrite reductase (NIR) from Alcaligenes faecalis S-6 were determined to betterthan 2.0 Å resolution. In the oxidized D98N nitrite-soaked structures, nitrite is coordinated to the type IIcopper via its oxygen atoms in an asymmetric bidentate manner; however, elevated B-factors and weakelectron density indicate that both nitrite and Asn98 are less ordered than in the native enzyme. Thisdisorder likely results from the inability of the N
2 atom of Asn98 to form a hydrogen bond with thebound protonated nitrite, indicating that the hydrogen bond between Asp98 and nitrite in the native NIRstructure is essential in anchoring nitrite in the active site for catalysis. In the oxidized nitrite soakedH255N crystal structure, nitrite does not displace the ligand water and is instead coordinated in an alternativemode via a single oxygen to the type II copper. His255 is clearly essential in defining the nitrite bindingsite despite the lack of direct interaction with the substrate in the native enzyme. The resultingpentacoordinate copper site in the H255N structure also serves as a model for a proposed transientintermediate in the catalytic mechanism consisting of a hydroxyl and nitric oxide molecule coordinatedto the copper. The formation of an unusual dinuclear type I copper site in the reduced nitrite soakedD98N and H255N crystal structures may represent an evolutionary link between the mononuclear type Icopper centers and dinuclear CuA sites.
2 atom of Asn98 to form a hydrogen bond with thebound protonated nitrite, indicating that the hydrogen bond between Asp98 and nitrite in the native NIRstructure is essential in anchoring nitrite in the active site for catalysis. In the oxidized nitrite soakedH255N crystal structure, nitrite does not displace the ligand water and is instead coordinated in an alternativemode via a single oxygen to the type II copper. His255 is clearly essential in defining the nitrite bindingsite despite the lack of direct interaction with the substrate in the native enzyme. The resultingpentacoordinate copper site in the H255N structure also serves as a model for a proposed transientintermediate in the catalytic mechanism consisting of a hydroxyl and nitric oxide molecule coordinatedto the copper. The formation of an unusual dinuclear type I copper site in the reduced nitrite soakedD98N and H255N crystal structures may represent an evolutionary link between the mononuclear type Icopper centers and dinuclear CuA sites.