13C ENDOR Characterization of the Central Carbon within the Nitrogenase Catalytic Cofactor Indicates That the CFe6 Core Is a Stabilizing "Heart of Steel".
13C ENDOR Characterization of the Central Carbon within the Nitrogenase Catalytic Cofactor Indicates That the CFe6 Core Is a Stabilizing "Heart of Steel".
复制标题
固氮酶催化辅因子内中心碳的 13C ENDOR 表征表明 CFe6 核心是稳定的“钢之心”。
DOI：
10.1021/jacs.2c06149
复制
发表时间：
2022
影响因子：
15
通讯作者：
Hoffman,BrianM
中科院分区：
文献类型：
--
作者：
Lukoyanov,DmitriyA;Yang,Zhi-Yong;Pérez-González,Ana;Raugei,Simone;Dean,DennisR;Seefeldt,LanceC;Hoffman,BrianM
钼依赖性固氮酶的底物和抑制剂与活性位点FeMo-辅因子[7 Fe-9 S-C-Mo-高柠檬酸盐]的铁离子结合并反应，该辅因子包含在MoFe蛋白α-亚基中。辅因子包含一个CFe 6核心，一个位于六个Fe的三棱柱内的碳，其在催化中的作用未知。碳的靶向13 C标记使得电子-核双共振（ENDOR）光谱能够灵敏地监测Fe-C键的电子性质和FeMo-辅因子金属离子所采用的自旋耦合方案。本报告比较了13 CFe 6 ENDOR测量值（i）野生型蛋白静息状态（ii）α-Ile 70，（iii）α-Ala 70-取代蛋白质;（iv）晶体学表征的CO抑制的“hi-CO”状态;（v）E4（4 H）Janus中间体，通过积累4[e-/H+]而活化N2结合/还原;（vi）E4（2 H）* 态，其含有双重还原的FeMo-辅因子而没有Fe结合的底物;和（vii）炔丙醇还原中间体，其具有作为铁结合剂结合到FeMo-辅因子Fe 6的烯丙醇。所有的状态检查，bothS= 1/2和3/2表现出近零的13 C各向同性超精细耦合常数，Ca= [−1.3 Participate +2.7] MHz。密度泛函理论计算和自然键轨道分析表明，这是因为CFe 6 Fe离子自旋的（3自旋向上/3自旋向下）自旋交换配置在每个Fe-C键中产生了对碳的大自旋转移的抵消。先前的X射线衍射和DFT都表明，在所有这些状态下，碳周围的三角棱柱几何形状都保持着高精度。CFe 6核心的持久结构和Fe-C键合表明，它不提供功能动态（半不稳定）的“跳动的心脏”，而是作为“钢心”，在固氮酶催化过程中稳定FeMo-辅因子-活性位点的结构。
Substrates and inhibitors of Mo-dependent nitrogenase bind and react at Fe ions of the active-site FeMo-cofactor [7Fe–9S–C–Mo–homocitrate] contained within the MoFe protein α-subunit. The cofactor contains a CFe6core, a carbon centered within a trigonal prism of six Fe, whose role in catalysis is unknown. Targeted13C labeling of the carbon enables electron-nuclear double resonance (ENDOR) spectroscopy to sensitively monitor the electronic properties of the Fe–C bonds and the spin-coupling scheme adopted by the FeMo-cofactor metal ions. This report compares13CFe6ENDOR measurements for (i) the wild-type protein resting state (E0; α-Val70) to those of (ii) α-Ile70, (iii) α-Ala70-substituted proteins; (iv) crystallographically characterized CO-inhibited “hi-CO” state; (v)E4(4H) Janus intermediate, activated for N2binding/reduction by accumulation of 4[e–/H+]; (vi)E4(2H)* state containing a doubly reduced FeMo-cofactor without Fe-bound substrates; and (vii) propargyl alcohol reduction intermediate having allyl alcohol bound as a ferracycle to FeMo-cofactor Fe6. All states examined, bothS= 1/2 and 3/2 exhibited near-zero13C isotropic hyperfine coupling constants,Ca= [−1.3 ↔ +2.7] MHz. Density functional theory computations and natural bond orbital analysis of the Fe−C bonds show that this occurs because a (3 spin-up/3 spin-down) spin-exchange configuration of CFe6Fe-ion spins produces cancellation of large spin-transfers to carbon in each Fe–C bond. Previous X-ray diffraction and DFT both indicate that trigonal-prismatic geometry around carbon is maintained with high precision in all these states. The persistent structure and Fe–C bonding of the CFe6core indicate that it does not provide a functionally dynamic (hemilabile) “beating heart”─instead it acts as “a heart of steel”, stabilizing the structure of the FeMo-cofactor-active site during nitrogenase catalysis.