论文的研究目标及其意义
本论文旨在阐明一种名为 FeSII 蛋白(Shethna II 蛋白)的小型铁氧还蛋白如何在氧化应激下保护钼铁固氮酶免受氧化损伤。固氮酶是一类将大气中惰性的 N2 还原为生物可利用的氨的关键酶,对农业生产具有重要意义。然而,固氮酶对 O2 极为敏感,极易受到不可逆的失活。了解固氮酶的保护机制对于开发耐氧固氮系统、减少化肥依赖具有重要意义。论文指出:
The oxygen-sensitive molybdenum-dependent nitrogenase of Azotobacter vinelandii is protected from oxidative damage by a reversible 'switch-off' mechanism.
论文提出的新思路和方法
论文利用冷冻电镜单颗粒分析,解析了 FeSII 蛋白与钼铁固氮酶两个组分(铁蛋白 NifH2 和钼铁蛋白 NifD2K2)形成的三元保护复合物的三维结构。FeSII 蛋白作为一个二聚体,通过其延展的 N 环区与两个 NifH2 和两个 NifD2K2 结合,将它们锁定在一个不活跃但受保护的构象中,同时诱导酶分子聚合成长丝状结构(见图1)。这一结构揭示了一种巧妙的分子开关机制:FeSII 蛋白感知氧化还原状态的改变,构象发生变化,快速响应氧化应激,可逆地抑制固氮酶活性。
相比此前的研究,本论文首次在分子水平上直观展示了完整的固氮酶氧保护机制。FeSII 蛋白仅通过构象改变就能高效地调控固氮酶的活性和聚集状态,无需额外的能量输入,是一种简洁而可逆的调控开关。
Here we report the three-dimensional structure of the protective ternary complex of the catalytic subunit of Mo-nitrogenase, its cognate reductase and the FeSII protein, determined by single-particle cryo-electron microscopy.
贮氮气单胞菌钼铁固氮酶的构象保护
本论文利用冷冻电镜、X射线晶体学和生化分析等方法,系统研究了一种名为FeSII蛋白(Shethna II蛋白)的小分子铁氧还蛋白如何在氧化应激下通过可逆的构象调节保护钼铁固氮酶免受氧化失活。作者的主要发现包括:
FeSII蛋白诱导固氮酶形成高度有序的螺旋状长丝复合物
通过优化体外复合条件,作者发现缺氧还原条件下FeSII、铁蛋白(NifH2)和钼铁蛋白(NifD2K2)三组分不会形成复合物。而短暂通入0.1%的O2后,FeSII与固氮酶两组分快速结合形成稳定的复合物,但在去除O2和加入还原剂后又能解离(图1a)。
Figure 1a: Complex formation monitored by SEC detected by absorption at 280 nm. In a dithionite (DT)-reduced sample (top), the nitrogenase component proteins and FeSII elute as separate peaks without detectable complex formation. Upon removal of reductant and addition of 0.1 vol% O2, complex formation is observed. Reduction and O2 removal lead to complex dissociation (bottom).
利用冷冻电镜单颗粒分析,作者解析了该复合物的高分辨率三维结构。结果表明,二聚体FeSII通过延展的N环区与两个NifH2和两个NifD2K2紧密结合,形成分子量约620 kDa的核心复合物(图1b,e),并进一步聚合成直径约24 nm的螺旋状长丝。长丝呈现明显的周期性,每个螺距含三个基本结构单元,分子量约为1 MDa(图1f,g)。
The dimeric FeSII protein associates with two copies of each component to assemble a 620 kDa core complex that then polymerizes into large, filamentous structures. This structure followed the same architectural principle as the core complex, but reflected the fact that FeSII can bind to both sides of the C2-symmetric MoFe protein. We regularly observed filaments with a length of 150–180 nm (5–6 MDa) that assembled into a right-handed helix with a diameter of 24 nm (Fig. 1f). This assembly includes two Fe protein dimers per single MoFe protein heterotetramer, reflecting the minimal stoichiometric ratio that may exist in the cell, although for in vitro activity assays the more O2-sensitive Fe protein component is typically used in high molar excess. The FeSII–nitrogenase filament had a pitch of 30 nm with almost three basic units—or 1 MDa mass—per turn (Fig. 1g).
图1
这一结果首次在分子水平上展示了固氮酶在氧化应激下形成的完整保护结构。有序的聚集态有利于将固氮酶的活性位点与外界隔绝,降低其暴露和失活的风险。同时,聚集态的可逆性确保固氮酶能在恢复厌氧条件后迅速重新活化。
FeSII蛋白氧化还原态变化引发N环构象重排,调控复合物的形成与解离
利用X射线晶体学,研究人员解析了FeSII蛋白在还原态下的高分辨率结构,并与此前获得的氧化态结构进行了对比。发现在还原态下,N环区回折并与蛋白核心区紧密结合,hN1和hN2螺旋分别位于[2Fe:2S]簇的上方(图2a)。K70和D44形成氢键,R92和D93分别与另一亚基的K53和C端A122发生相互作用(Extended Data Fig. 4c),从而稳定了N环的收拢构象。
In the reduced-state structure that represents the inactive state of FeSII (Fig. 2a), the N-loops were folded back onto the core of the dimeric protein, fully ordered and resolved, including the α-helices hN1 (residues 69–78) and hN2 (84–91). In the reduced state the N-loop is fixed by two salt bridges to the body of FeSII (D44-K70 and E71-R99) and interacts with the other protomer via R61 and D93 and a salt bridge (R92-A122) that fixes the C-terminal carboxylate of the other chain.
氧化后N环发生构象变化。在"关闭"构象中,D44-K70和R92-A122相互作用被破坏,N环获得更大的灵活性,但hN1仍位于[2Fe:2S]簇附近(图2b)。而在"开放"构象中,hN1和hN2形成平行的"桨"状二聚体,完全暴露并远离蛋白核心区(图2c,Extended Data Fig. 4d) 。
The predominant conformation of FeSII in the oxidized state, however, had helices hN1 and hN2 in the parallel, paddle-like arrangement described earlier (Fig. 2c). Here, the N-loop was released as the interactions E71:R99 and D44:K70 were severed, and R92 moved away from the carboxylate of the other protomer, releasing its C terminus. The D93:K53′ interaction was retained, so that the N-loop gained flexibility and was released from the core of FeSII, and the formerly separate helices hN1 and hN2 rearranged into the parallel paddle arrangement.
在复合物结构中,N环再次发生重排,形成"锁定"构象(图2d)。桨状结构整体回折,E71和R99重新形成盐桥,但D44和K70未发生相互作用,转而参与了与NifH的界面形成。R92和D93转而背离FeSII核心区,C端A122暴露并指向NifH的[4Fe:4S]簇(Extended Data Fig. 4e)。
In the cryo-EM structure of the complex with the nitrogenase components, the N-loops of FeSII showed yet another conformation that we designate 'locked', as it kept the complex stably in place (Fig. 2d). The N-loop was retracted to the FeSII core, re-establishing the E71:R99 salt bridge, but not the interaction of D44 and K70, as these residues were involved in protein–protein interactions with the enzyme. R92 and D93 rotated away from the other FeSII protomer, so that the C termini at A122 became accessible.
图2
基于以上结构信息,作者提出FeSII蛋白利用N环构象变化感知氧化应激并调控复合物装配的分子机制模型(图2g和图5):还原态FeSII处于休眠状态,N环被锁定在拢状态。氧化诱导N环舒展形成桨状结构,获得与NifH结合的能力。FeSII先结合1个NifH2,再结合第2个NifH2形成FeSII2:2NifH2 "引发复合物",之后再招募2个NifD2K2,最终形成FeSII2:2NifH2:2NifDK2的核心结构单元(图1b),并沿NifDK的C2轴进一步延伸形成长丝(图1f)。在厌氧还原条件下,FeSII构象恢复,复合物解离并释放固氮酶组分。从而实现了固氮酶活性可逆调控和氧保护。
Figure 5: Conformational protection of Mo-nitrogenase is initiated by the oxidative activation of reduced, dimeric FeSII, which leads to the extension of the N-loops in both protomers. This state interacts only with NifH but not with NifDK. The FeSII dimer first binds one NifH dimer, followed by a second one to form an initiator complex that is able to provide significant protection to the more sensitive Fe protein component. The initiator complex then successively recruits two NifD2K2 heterotetramers to assemble the core particle analysed in the present study. As the initiator complex can bind to both sides of the MoFe protein heterotetramer, this interaction leads directly to the polymerization into filaments (Fig. 1g).
这一结果阐明了FeSII蛋白如何利用构象动力学感知氧化还原信号,并将其转化为对固氮酶活性和聚集状态的调控。这种设计巧妙且节能的分子开关机制对于理解固氮生物的氧适应机制具有重要启示意义。
FeSII与NifH、NifDK的界面相互作用稳定保护复合物并抑制固氮酶活性
FeSII与NifH的结合是复合物形成的第一步,也是复合物稳定性的关键。结构显示,一个FeSII与NifH2的两个亚基发生不对称结合。主要通过两个区域的相互作用实现(图3d):FeSII的hN1与一个NifH亚基形成多个氢键和盐桥(R72:E69H、K76:E112H和N73-E69H)(图3e);FeSII的核心区紧密包埋NifH的[4Fe:4S]簇,C端A122和E41、E118分别与NifH的R101形成氢键和盐桥(图3f)。
A detailed view of the interface shows that interactions are focused on two areas. One NifH monomer interacts with helix hN1 of the N-loop, and the core of FeSII resides close to the [4Fe:4S] cluster of the Fe protein (Fig. 3d). Key interactions of helix hN1 with NifH are two salt bridges (R72:E69H, K76:E112H) and hydrogen bonds involving N73 and N108H (Fig. 3e). The C terminus of FeSII is placed immediately above the [4Fe:4S] cluster of NifH, and E41 and E118 interact with the Fe protein. The interaction blocks R101H in both NifH monomers (Fig. 3f).
图3
这种结合模式一方面将NifH最敏感的[4Fe:4S]簇与溶剂隔离保护;另一方面阻断了R101与NifDK的盐桥相互作用(Extended Data Fig. 6c),妨碍了固氮酶组分间电子传递和底物还原过程。
FeSII与NifDK主要在NifD亚基表面发生结合(图4a,b),并通过K63和D204D形成盐桥。hN2的负电荷端正对FeMo辅基,可能影响其电子态。结合区覆盖了H196D,这是质子向活性中心转移的入口之一。此外,FeSII的R24侧链延伸至NifD与NifK的C2轴上方(图4e,f),并与NifD的L158D主链羰基形成氢键。总之,FeSII对NifDK结合面的屏蔽和对称性的破坏,很可能阻断了底物分子的进入和还原产物的释放。
FeSII interacts mostly with the surface of NifD (Fig. 4a), covering a patch above the FeMo cofactor that was suggested to serve as one entry point for the protons required for N2 reduction, terminating in residue H196D (Fig. 4b). In addition, K63 of FeSII forms a salt bridge to D204D of MoFe protein, and the negative end of the helix dipole of hN2 points directly at the FeMo cofactor (Fig. 4b). The only interaction with NifK occurs through a short, protruding loop of the subunit, 119TEDAAVFG126, which contains the highly conserved F125K (in bold) that is also found as F125D in NifD (Fig. 4c). FeSII does not cover the surface position above P-cluster, that is, the twofold pseudosymmetry axis of the NifDK heterodimer. However, it extends residue R24 to form a hydrogen bond to the backbone carbonyl of L158D, so that the positively charged guanidinium moiety of the arginine side chain is situated almost precisely on this pseudo-twofold axis (Fig. 4f).
图4
以上结果解释了FeSII复合物中固氮酶组分虽然空间邻近,但酶活性却被完全抑制的原因。同时凸显了FeSII利用界面相互作用精准调控固氮酶构象和功能的能力。
FeSII诱导复合物形成显著提高了钼铁固氮酶的耐氧性但对其他固氮酶无效
作者利用乙炔还原实验评估了FeSII复合物的耐氧保护能力。结果表明,在20%的O2下孵育5分钟后,FeSII能使钼铁固氮酶保留约一半的活性,而无FeSII时酶活性几乎完全丧失(Extended Data Fig. 1c)。
FeSII protein had a strong protective effect on the isolated components of Mo-nitrogenase. The protective effect of FeSII was clearly shown by activity assays (Extended Data Fig. 1c). Acetylene reduction assays for the samples in (a) in the absence (black) and presence (hatched) of FeSII. At incubation times of 1 and 5 min, the stabilizing effect of FeSII is substantial even at 20% of pO2. Error bars represent the standard deviation of three independent measurements.
然而,FeSII不能与钒铁固氮酶(V-nitrogenase)和全铁固氮酶(Fe-nitrogenase)形成保护复合物(Extended Data Fig. 6g)。结构模拟显示,这两种固氮酶的额外δ亚基(VnfG/AnfG)会与FeSII在NifDK表面的结合位
点发生空间位阻(Extended Data Fig. 6d-f),这与实验结果相吻合。这提示FeSII介导的构象保护机制是钼铁固氮酶所特有的。
Figure 6: Hypothetical model for the interaction of FeSII with VndD2K2G2 of V-nitrogenase. The additional subunit VnfG prevents FeSII at the same position as in MoFe protein (d). For Fe-nitrogenase, subunit AnfG causes analogous clashes to (e). While Mo-nitrogenase readily forms a protective complex with FeSII in the presence of oxygen, no such complex formation is observed with either V- or Fe-nitrogenase under the same conditions, highlighting that FeSII acts exclusively in conjunction with Mo-nitrogenase (g).
支撑图6
总结
本文通过冷冻电镜揭示了FeSII诱导钼铁固氮酶形成螺旋状长丝复合物,并阐明了FeSII感知氧化还原信号、调控构象动力学以实现固氮酶可逆失活和聚集的分子机制。同时,文章系统表征了复合物的界面相互作用,阐释了其抑制固氮酶活性和提高酶耐氧性的结构基础。此外,研究还发现FeSII介导的构象保护机制具有固氮酶种属特异性。这些发现极大地深化了我们对固氮生物氧适应机制的认知,为开发新型固氮体系提供了重要的结构和机制见解。
研究成果的影响与潜在应用
本研究揭示了一种新颖的固氮酶氧保护机制,对于理解固氮生物如何在有氧条件下维持固氮具有重要意义。论文指出,FeSII 蛋白对钼铁固氮酶的保护机制在异源固氮系统中可能也很重要:
The action of this small ferredoxin represents a straightforward means of protection from O2 that may be crucial for the maintenance of recombinant nitrogenase in food crops.
将固氮酶导入粮食作物,实现"自养"固氮,是减少化肥依赖的理想途径。然而异源固氮系统面临的最大瓶颈之一就是固氮酶极易受氧失活。FeSII 蛋白提供了一种内源的保护机制,使植物线粒体中的异源固氮酶在昼夜周期的氧浓度波动下得以维持活性。针对这一机制开发更高效的分子开关,有望进一步提高异源固氮系统的稳定性。
本研究还为开发新型固氮催化剂和工程菌提供了新思路。模仿 FeSII 蛋白独特的 N 环结构和构象变化特点,有望设计出对氧不敏感的固氮酶变体。利用 FeSII 调控异源固氮基因的表达,有望构建更高效的固氮工程菌株。
未来研究方向与机遇
固氮酶的氧敏感性仍然是限制其应用的关键瓶颈。虽然 FeSII 蛋白能在一定程度上缓解氧化失活,但对氧的耐受能力仍然很有限。未来需要在以下方面开展更深入的研究:
FeSII 蛋白的分子感氧和传导机制。虽然构象变化与氧化还原状态有关,但具体的分子机制尚不清楚。阐明 FeSII 蛋白的氧感受机制,有望设计出更灵敏的分子开关。 FeSII-固氮酶复合物的解离机制。FeSII 诱导形成的聚集体中所有金属中心均不可及,目前对其解离机制知之甚少。了解复合物的解离诱因,有助于动态调控异源固氮系统以适应内环境。 其他辅助蛋白在固氮酶保护中的作用。一些固氮生物中存在多种保护机制协同作用,如何整合不同因素以构建更稳健的固氮体系值得深入探索。
Biosyn导师：Oliver Einsle
https://www.frias.uni-freiburg.de/en/people/fellows/current-fellows/einsle
Oliver Einsle教授是一位成就卓著的结构生物学家,目前担任德国弗莱堡大学生物化学研究所所长。他的研究兴趣主要集中在金属蛋白和膜蛋白的结构与功能研究。
Einsle教授的学术生涯始于德国康斯坦茨大学,在那里他主修生物学。随后,他前往位于马尔蒂斯里德的马普生物化学研究所,师从Robert Huber和Peter Kroneck,研究细胞色素c亚硝酸盐还原酶。2001年,他加入美国加州理工学院Douglas Rees实验室,开始了对固氮酶的研究。
2003年,Einsle教授回到德国,在哥廷根大学担任蛋白质晶体学青年教授。2008年,他受聘为弗莱堡大学生物化学教授,并兼任化学和药学院生物化学研究所所长。在他的领导下,研究组在多种金属蛋白(如固氮酶、一氧化二氮还原酶和多血红素细胞色素c)的结构和功能表征方面取得了开创性的成果。此外,他们还致力于膜蛋白,特别是细菌转运蛋白和通道蛋白的研究。
Einsle教授发表了一系列高影响力的研究论文,其中多篇发表在Nature和Science等顶级期刊上。这些工作极大地推动了金属蛋白和膜蛋白领域的发展。例如,他们首次揭示了固氮酶FeMo辅基中存在插入碳原子(Science, 2011),阐明了一氧化二氮还原酶铜硫簇结合底物的独特方式(Nature, 2011),发现了FocA甲酸盐通道的pH门控机制(Science, 2011)等。
除了科研工作,Einsle教授还积极推动学术交流与合作。他主持的"化学表观遗传学"项目旨在整合有机合成化学、药物化学、结构生物学和生物信息学的力量,探索组蛋白赖氨酸乙酰化和甲基化识别蛋白的结构基础及其药物设计潜力,并致力于建立一个国际化的博士生培养项目。
