Department of Electrical and Computer Engineering, Colorado State University, Fort Collins, Colorado 80523, United States.
Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523, United States.
J Phys Chem B. 2022 Feb 24;126(7):1404-1412. doi: 10.1021/acs.jpcb.1c08714. Epub 2022 Feb 15.
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 (Cyt-). 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- 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.
血红素蛋白是超快瞬态吸收光谱（TAS）的常见研究对象，这是因为它们具有生物学重要性、较强的紫外可见吸收、高光稳定性以及有趣的瞬态动力学，这些动力学取决于氧化还原、构象和配体结合。TAS 通常在分离的、纯化的蛋白质上进行，但它们在天然分子环境中的反应可能会有所不同，因为天然分子环境涉及蛋白质复合物和超复合物的形成。最近，我们报告了一个瞬态吸收显微镜（TAM）实验，该实验使用可见波长的泵浦（530nm）和探针（490nm）从完整的生物组织中的血红素蛋白中引发瞬态响应。在这里，我们发现自适应噪声消除加上共振旋转镜扫描使高重复率光纤激光源能够对细胞色素（Cyt-）进行氧化还原敏感的测量。我们通过凝胶电泳分离完整的线粒体呼吸超级复合物的 TAS 研究了生物组织中可见波长响应的起源。我们发现这些高分子量凝胶带中的每一个都产生了与细胞色素血红素特征一致的 TAS 响应，这意味着完整细胞和组织的 TAS 响应不仅来自 Cyt-，还来自呼吸细胞色素的混合物。我们还发现野生型（WT）和线粒体疾病的 tafazzin 缺乏（TAZ）小鼠模型之间的激发态寿命存在差异。
