Structural study of [NiFe]hydrogenase

[NiFe]氢化酶的结构研究

• 批准号: 12680654
• 负责人: HIGUCHI Yoshiki
• 金额: $ 2.3万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680654/
• 关键词: [Ni-Fe]hydrogenase; CO-bound hydrogenase; Hydrogen metabolism; Cryogenic diffraction study; quasi-dynamic X-ray analysis; High resolution X-ray analysis; 準動的X線結晶解析; ヒドロゲナーゼ; Ni-Fe活性部位; CO配位子; 超高分解能結晶解析; タンパク質間相互作用; タンパク質-配位子相互作用; 極低温結晶解析

Hydrogenases catalyze the reversible oxidation of molecular hydrogen and play a key role in hydrogen metabolism in various bacteria. Hydrogenases are classified into [non-metal], [Fe], [NiFe], and [NiFeSe] hydrogenases according to the metal composition of their active sites. The [NiFe]hydrogenase from D. vulgaris Miyazaki F is composed of a heterodimer with a total molecular mass of 91 kDa. The active site of the [NiFe] hydrogenases in the oxidized form is composed of Ni and Fe atoms with four cysteinyl sulfur and four non-protein ligands (S/O, CO, CN or SO). Carbon monoxide (CO) was known as a reversible inhibitor of hydrogenases. The carbon monoxide complex of [NiFe]hydrogenase from D. v. Miyazaki has been prepared and characterized by X-ray crystallography, and absorption and Raman spectroscopy. The exogenously added CO was found to be bound to the Ni atom at the Ni-Fe active site. Distinct changes were observed in the electron density distribution of the Ni and sγ(Cys546) atoms between the CO-bound and CO-liberated structures for all the crystals tested. One of the CO-bound structures showed an additional electron density peak between the CO and Sγ(Cys546), suggesting that an intermediate of the catalytic reaction was trapped at the active site. The novel structural features found near the Ni and Sγ(Cys546) atoms suggest that these two atoms at the Ni-Fe active site play a key role during the initial process to bind H_2.

氢化酶催化分子氢的可逆氧化，在各种细菌的氢代谢中发挥关键作用。氢化酶根据其金属组成分为[非金属]、[Fe]、[NiFe]和[NiFeSe]氢化酶。来自 D. vulgaris Miyazaki F 的 [NiFe] 氢化酶由总分子量为 91 kDa 的异二聚体组成。氧化形式的[NiFe]氢化酶的位点由Ni和Fe原子以及四个半胱氨酰硫和四个非蛋白质配体（S/O、CO、CN或SO）组成，被称为可逆的一氧化碳（CO）。 D. v. Miyazaki 制备了 [NiFe] 氢化酶的一氧化碳复合物，并通过 X 射线晶体学、吸收和拉曼进行了表征。发现外源添加的 CO 与 Ni-Fe 活性位点的 Ni 原子结合，在 CO 结合和 CO 释放之间的 Ni 和 sγ(Cys546) 原子的电子密度分布观察到明显的变化。所有测试晶体的结构中，其中一个 CO 结合结构在 CO 和 Sγ(Cys546) 之间显示出额外的电子密度峰，表明催化反应的中间体被捕获在活性位点处。在 Ni 和 Sγ(Cys546) 原子附近发现的新结构特征表明，Ni-Fe 活性位点上的这两个原子在结合 H_2 的初始过程中发挥着关键作用。

项目成果

樋口芳樹: "水素の物性・反応の機能性化と応用"生体反応・ヒドロゲナーゼ. 249-253 (2001)

Yoshiki Higuchi：“氢的物理性质、反应的功能化和应用”《生物反应和氢化酶》249-253 (2001)。

八木達彦,樋口芳樹: "廣川タンパク質化学"廣川書店. 340 (2000)

八木龙彦、樋口义树：《广川蛋白质化学》广川书店 340 (2000)。

樋口芳樹: "ヒドロゲナーゼ活性中心の反応機構"現代化学. 8. 51-56 (2000)

樋口义树：“氢化酶活性中心的反应机制”现代化学8. 51-56 (2000)。

樋口芳樹: "ヒドロゲナーゼの立体構造と機能発現"日本生物物理学会誌. 40・4. 249-253 (2000)

樋口义树：“氢化酶的三级结构和功能表达”日本生物物理学会杂志40・4（2000）。

O.Trofanchuk 等: "Single Crystal EPR Studies of the Oxidized Active Site of [NiFe] Hydrogenase from Desulfovibrio vulgaris Miyazaki F"J.Bioinorganic Chemistry. 5. 36-44 (2000)

O. Trofanchuk 等人：“来自 Desulfovibrio vulgaris Miyazaki F 的 [NiFe] 氢化酶氧化活性位点的单晶 EPR 研究”J. Bioinorganic Chemistry 5. 36-44 (2000)。