Data Sheet 1_Cytochrome “nanowires” are physically limited to sub-picoamp currents that suffice for cellular respiration.docx

Mineral-respiring microorganisms from hydrothermal vents to terrestrial soils express filaments that electrically connect intracellular respiration to extracellular geochemistry. Filaments dubbed “cytochrome nanowires” (CNs) have been resolved by CryoEM, but whether they are the two-decades-long sought-after physiological “nanowires” remains unproven. To assess their functional competence, we analyzed biological redox conduction in all CNs by computing driving forces in the presence of redox anti-cooperativities, reorganization energies with electronic polarizability, and Marcus rates for diffusive and protein-limited flux models. The chain of heme cofactors in any CN must be densely packed to realize weak (≤0.01 eV) electronic coupling for electron transfer, as evidenced by a single Soret band produced from coincidental absorptions on multiple hemes. Dense packing, in turn, has three consequences: (1) limited driving forces (≤|0.3| eV) due to shared electrostatic microenvironments, (2) strong (≤0.12 eV) redox anti-cooperativities that would accentuate the free energy landscape if the linear heme arrangement did not dictate a contra-thermodynamic oxidation order, and (3) an entropic penalty that is offset by thioether ‘tethers’ of the hemes to the protein backbone. These linkages physically necessitate the rate-throttling T-stacked motif (10-fold slower than the other highly conserved slip-stacked motif). If the sequence of slip- and T-stacked hemes in the CNs had the fastest known nanosecond rates at every step, a micron-long filament would carry a diffusive 0.02 pA current at a physiological 0.1 V, or a protein-limited current of 0.2 pA. Actual CNs have sub-optimal (≤102-fold lower), but sufficient conductivities for cellular respiration, with at most thousands of filaments needed for total cellular metabolic flux. Reported conductivities once used to argue for metallic-like pili against the cytochrome hypothesis and now attributed to CNs remain inconsistent by 102–105-fold with the physical constraints on biological redox conduction through multiheme architectures.

从热液喷口到陆地土壤的矿物呼吸微生物（Mineral-respiring microorganisms），可表达出能够将胞内呼吸作用与胞外地球化学过程电学连接的丝状结构。被称为‘细胞色素纳米线（cytochrome nanowires，CNs）’的细丝已通过冷冻电镜（CryoEM）解析，但它们是否是学界追寻二十年的生理学意义上的‘纳米线’，仍未得到证实。为评估其功能活性，我们通过计算氧化还原反协同效应存在时的驱动力、结合电子极化率的重组能，以及针对扩散受限与蛋白受限通量模型的马库斯电子转移速率（Marcus rates），分析了所有细胞色素纳米线的生物氧化还原传导特性。任一细胞色素纳米线中的血红素辅因子（heme cofactors）链都必须紧密排布，以实现弱（≤0.01 eV）的电子转移耦合，这一点可由多个血红素的巧合吸收产生单一索雷特带（Soret band）所佐证。而紧密排布又会带来三项后果：（1）由于共享静电微环境，驱动力受限（≤|0.3| eV）；（2）存在强（≤0.12 eV）的氧化还原反协同效应，若线性排布的血红素未规定逆热力学的氧化顺序，该效应会加剧自由能景观；（3）存在熵罚，而该熵罚可由血红素与蛋白骨架之间的硫醚连接臂抵消。这些连接结构从物理上要求形成限速的T堆叠基序（T-stacked motif），其速率比另一高度保守的滑移堆叠基序（slip-stacked motif）慢10倍。若细胞色素纳米线中的滑移堆叠与T堆叠血红素序列在每一步都拥有已知最快的纳秒级速率，那么一根微米级长度的细丝在生理电位0.1 V下，可承载0.02皮安的扩散受限电流，或0.2皮安的蛋白受限电流。实际的细胞色素纳米线的传导性未达最优（低10²倍以内），但仍足以支撑细胞呼吸——仅需数千根此类细丝即可满足细胞整体代谢通量的需求。此前曾被用于佐证类菌毛金属导电、反驳细胞色素假说的已报道传导率，如今虽被归因为细胞色素纳米线的作用，但与多血红素结构的生物氧化还原传导的物理约束仍存在10²~10⁵倍的偏差。