High-Repetition-Rate Transient Absorption Spectroscopy of Respiratory Supercomplexes

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.