High-Repetition-Rate Transient Absorption Spectroscopy of Respiratory Supercomplexes
High-Repetition-Rate Transient Absorption Spectroscopy of Respiratory Supercomplexes
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DOI：
10.1021/acs.jpcb.1c08714
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发表时间：
2022-02-24
影响因子：
3.3
通讯作者：
Wilson, Jesse W.
中科院分区：
文献类型：
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作者：
Wang, Erkang;Specht, Kalyn S.;Wilson, Jesse W.
血红素蛋白是超快瞬态吸收光谱（TAS）的常见对象，因为生物学的重要性，强的紫外-可见吸收，高的光稳定性，以及依赖于氧化还原，构象和配体结合的有趣的瞬态动力学。对血红素蛋白的TAS通常在分离纯化的蛋白质上进行，尽管它们的反应在其天然分子环境中可能不同，这涉及蛋白质复合物和超复合物的形成。最近，我们报道了一个瞬态吸收显微镜（TAM）的实验，引起了一个瞬态的反应，从血红素蛋白在完整的生物组织中使用可见光波长泵（530 nm）和探针（490 nm）。在这里，我们发现，自适应噪声消除加上谐振振镜扫描，使高重复率的光纤激光源，使细胞色素c（Cyt-c）的氧化还原敏感的测量。我们通过凝胶电泳分离的完整线粒体呼吸超复合物的TAS研究生物组织可见波长响应的起源。我们发现，这些高分子量的凝胶带产生的TAS响应特性的细胞色素血红素，这意味着TAS响应的完整细胞和组织来源于不只是Cyt-C，但呼吸细胞色素的混合物。我们还发现了野生型（WT）和他法嗪缺陷（TAZ）线粒体疾病小鼠模型之间的激发态寿命差异。
Hemeproteins are frequent subjects for ultrafast transient absorption spectroscopy (TAS) because of biological importance, strong UV-vis absorption, high photostability, and interesting transient dynamics that depend on redox, conformation, and ligand binding. TAS on hemeproteins is usually performed on isolated, purified proteins, though their response is likely to be different in their native molecular environment, which involves the formation of protein complexes and supercomplexes. Recently, we reported a transient absorption microscopy (TAM) experiment which elicited a transient response from hemeproteins in intact biological tissue using a visible-wavelength pump (530 nm) and probe (490 nm). Here, we find that adaptive noise canceling plus resonant galvanometer scanning enables a high-repetition-rate fiber laser source to make redox-sensitive measurements of cytochrome c (Cyt-c). We investigate the origins of the visible-wavelength response of biological tissue through TAS of intact mitochondrial respiratory supercomplexes, separated via gel electrophoresis. We find that each of these high-molecular-weight gel bands yields a TAS response characteristic of cytochrome hemes, implying that the TAS response of intact cells and tissue originates from not just Cyt-c but a mixture of respiratory cytochromes. We also find differences in excited-state lifetime between wild-type (WT) and a tafazzin-deficient (TAZ) mouse model of mitochondrial disease.