Parida Akankshika, Bhattacharyya Gargee, Mallik Swagatika, Behera Rabindra K
Department of Chemistry, National Institute of Technology Rourkela - 769008 Odisha India
Chem Sci. 2025 Jan 20;16(9):3978-3997. doi: 10.1039/d4sc07021f. eCollection 2025 Feb 26.
The self-assembled ferritin protein nanocage plays a pivotal role during oxidative stress, iron metabolism, and host-pathogen interaction by executing rapid iron uptake, oxidation and its safe-storage. Self-assembly creates a nanocompartment and various pores/channels for the uptake of charged substrates (Fe) and develops a concentration gradient across the protein shell. This phenomenon fuels rapid ferroxidase activity by an upsurge in the substrate concentration at the catalytic sites. However, it is difficult to segregate the relative contributions of the catalytic sites and self-assembly towards rapid ferroxidase/mineralization activity owing to the inherent self-assembly propensity of ferritins. In the current work, 3-fold pore electrostatics of bacterioferritin from were rationally altered by site-directed mutagenesis to generate self-assembled (E121A and E121Q) and assembly-defective (E121K and E121F) variants. In comparison to the autoxidation of Fe in buffer, the assembly-defective variants exhibited significantly faster ferroxidase/mineralization activity and O consumption kinetics due to their functional catalytic sites, but failed to level-up with the self-assembled variants even at 100-fold higher Fe concentration. Only the self-assembled variants exhibited cooperativity in iron oxidation, maintained biomineral solubility, and protected DNA against the Fenton reaction. This report highlights the concerted effect of self-assembly and ferroxidase sites that propels the rapid Fe uptake, its oxidation and biomineralization in bacterioferritin. The findings also establish the importance of electrostatic guiding and nanoconfinement offered by ferritin self-assembly towards its enzymatic activity and antioxidative properties. Moreover, this work identifies the key electrostatic interactions ("hot-spots") at the subunit contact points that control the cage/pore formation, impart cage stability and influence ferritin's natural functions. Manipulation of hot-spot residues can be further extended towards the encapsulation of cargo, for various bio-medical applications, by strategically inducing its disassembly and subsequent reassembly through adjustments in ionic strength. This would bypass the need for extreme/harsh reaction conditions and minimize the loss of cargo/protein.
自组装铁蛋白纳米笼通过快速摄取、氧化和安全储存铁，在氧化应激、铁代谢以及宿主-病原体相互作用过程中发挥着关键作用。自组装形成一个纳米隔室以及各种用于摄取带电底物（铁）的孔道/通道，并在蛋白质外壳上形成浓度梯度。这种现象通过催化位点处底物浓度的升高促进了快速的铁氧化酶活性。然而，由于铁蛋白固有的自组装倾向，很难区分催化位点和自组装对快速铁氧化酶/矿化活性的相对贡献。在当前的工作中，通过定点诱变合理改变了来自[具体来源未给出]的细菌铁蛋白的三倍孔静电，以产生自组装变体（E121A和E121Q）和组装缺陷变体（E121K和E121F）。与缓冲液中铁的自氧化相比，组装缺陷变体由于其功能性催化位点而表现出明显更快的铁氧化酶/矿化活性和氧气消耗动力学，但即使在铁浓度高出100倍的情况下也无法与自组装变体相媲美。只有自组装变体在铁氧化中表现出协同性，维持生物矿物的溶解性，并保护DNA免受芬顿反应的影响。本报告强调了自组装和铁氧化酶位点的协同作用，这种作用推动了细菌铁蛋白中铁的快速摄取、氧化和生物矿化。这些发现还确立了铁蛋白自组装提供的静电引导和纳米限域对其酶活性和抗氧化特性的重要性。此外，这项工作确定了亚基接触点处控制笼/孔形成、赋予笼稳定性并影响铁蛋白自然功能的关键静电相互作用（“热点”）。通过在离子强度上进行调整，策略性地诱导其解体并随后重新组装，对热点残基的操纵可以进一步扩展到各种生物医学应用中的货物封装。这将避免对极端/苛刻反应条件的需求，并最大限度地减少货物/蛋白质的损失。
