Wavelength- and Oxygen-Dependent Single-Molecule Kinetics of Cytochrome c Interactions with Cytochrome c Oxidase Offer Insights into the Molecular Basis of Photobiomodulation

Cytochrome c oxidase (CCO) is widely regarded as the primary photoacceptor underlying photobiomodulation (PBM), in which light absorption stimulates mitochondrial respiration by promoting electron transfer through CCO. Single-molecule plasmonic nanopore measurements were used to examine cytochrome c (CC)–CCO interactions across three excitation wavelengths spanning the near-infrared range, which is preferred for PBM studies in vivo due to increased tissue penetration. Wavelength-dependent measurements of CC–CCO interactions were performed under normoxic, hypoxic, and allosterically inhibited conditions, revealing distinct interaction populations that are obscured in ensemble measurements. CC–CCO interactions were resolved into short-duration, long-duration, and heterogeneous populations for all wavelengths and oxygenation conditions. The short-duration population was selectively and substantially depleted by both hypoxia and ADDA5-mediated allosteric inhibition, supporting its assignment as a distinct, putative electron-transfer-associated interaction state. Allosteric inhibition strongly favored the long-duration population, whereas hypoxia predominantly increased heterogeneous duration interactions, consistent with non-specific binding. Under normoxia, 820 nm and 905 nm produced statistically comparable population fractions of the putative electron-transfer-active population, whereas the corresponding population fraction at 1064 nm was significantly lower but remained substantial. The short-duration binding population kinetics remained largely unchanged across wavelength and oxygenation states, indicating that these conditions primarily altered access to the CCO binding site rather than affecting its binding lifetime. These findings reveal the previously unresolved heterogeneity in CC–CCO interactions and provide a single-molecule framework for understanding how the excitation wavelength and oxygenation state may influence molecular interactions relevant to PBM efficiency.