Geobacter's unique ability to perform extracellular electron transfer (EET) to electrodes in Microbial Fuel Cells (MFCs) has sparked the implementation of sustainable production of electrical energy. However, the electrochemical performance of Geobacter's biofilms in MFCs remains challenging to implement industrially. Multiple approaches are being investigated to enhance MFC technologies. Protein engineering of multihaem cytochromes, key components of Geobacter's EET pathways, can, conceivably, be pursued to improve the EET chain. The periplasmic cytochrome PpcA bridges ET from the inner to the outer membrane and its deletion impairs this crucial step. The functional characterisation of PpcA homologs from G. sulfurreducens (Gs) and G. metallireducens (Gm) revealed a significantly different redox behaviour even though they only differ by thirteen amino acids. In a previous study, we found that the single replacement of a tryptophan residue by methionine (W45M) in PpcAGm shifted the reduction potential value 33% towards that of PpcAGs. In this work, we expanded our investigation to include other non-conserved residues by conducting five mutation rounds. We identified the most relevant residues controlling the redox properties of PpcAGm. With just four mutations (K19, G25, N26, W45) the reduction potential value of PpcAGm was shifted 71% toward that of PpcAGs. Additionally, in the quadruple mutant, it was possible to replicate the haem oxidation order and the functional mechanisms of PpcAGs, which differ from those in PpcAGm. Overall, the mutants exhibit diverse redox and functional mechanisms that could be explored as a library for the future design of minimal, synthetic, ET chains in Geobacter.

中文翻译：

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用蛋白质工程调整 Geobacter metallireducens 的 PpcA 的氧化还原特性。


Geobacter 将细胞外电子转移 （EET） 到微生物燃料电池 （MFC） 中的电极的独特能力激发了可持续电能生产的实施。然而，Geobacter 生物膜在 MFC 中的电化学性能在工业上仍然具有挑战性。正在研究多种方法来增强 MFC 技术。可以想象，多血红素细胞色素的蛋白质工程是 Geobacter EET 通路的关键组成部分，可以改进 EET 链。周质细胞色素 PpcA 将 ET 从内膜桥接到外膜，其缺失会损害这一关键步骤。来自硫还原 Gs （Gs） 和金属还原 G. （Gm） 的 PpcA 同源物的功能表征揭示了显着不同的氧化还原行为，即使它们仅相差 13 个氨基酸。在之前的一项研究中，我们发现 PpcAGm 中蛋氨酸 （W45M） 单独取代色氨酸残基后，还原电位值向 PpcAGs 的还原电位值移动了 33%。在这项工作中，我们通过进行五轮突变，将研究范围扩大到包括其他非保守残基。我们确定了控制 PpcAGm 氧化还原特性的最相关残基。仅 4 个突变 （K19 、 G25 、 N26 、 W45） PpcAGm 的还原电位值向 PpcAGs 的还原电位值偏移了 71%。此外，在四重突变体中，可以复制血红素氧化顺序和 PpcAGs 的功能机制，这与 PpcAGm 中的不同。总体而言，突变体表现出多种氧化还原和功能机制，可以作为 Geobacter 中最小合成 ET 链未来设计的库进行探索。