Our primary hypothesis was that red-to-near infrared (R-NIR) irradiation would have an effect on the kinetics parameters of the reaction of cytochrome c with isolated cytochrome c oxidase (CCO), and that the magnitude and direction of these changes could be interpreted in the context of the reaction schemes proposed by other authors. New values for the milimolar extinction coefficients of cytochrome c were also determined.Definitive answers to the fundamental processes involved in red-to-near infrared photobiomodulation (R-NIR-PBM) have not been obtained. The consensus is that the electron transport chain enzyme CCO is the target for R-NIR-PBM. This work was undertaken to explore the effect of R-NIR on the activity of isolated CCO.Scans for cytochrome c were obtained in both reduced and oxidized states, and values for the extinction coefficients were calculated. Activity assays were performed by following the oxidation state of cytochrome c at 550 or 415 nm. R-NIR effects on CCO activity were evaluated by pre-irradiating the enzyme at 670 or 830 nm, or by irradiating the reaction mixture with 660 nm light.Milimolar extinction coefficients (L-1 cm-1) were: ɛ550red = 29.1 ± 0.4, ɛ550ox = 8.60 ± 0.15, ɛ415red = 140 ± 2, and ɛ415ox = 89.0 ± 1.1. Reduced-oxidized extinction coefficients were: δɛ550red-ox = 20.5 ± 0.2, and δɛ415red-ox = 51.0 ± 2.0. The second order rate constants k' for irradiated CCO did not show a statistically significant difference from controls.The oxidation of cytochrome c by isolated CCO has not been shown to be affected by R-NIR irradiation, whether applied prior to or concurrently with the enzymatic assays. This lack of effect by R-NIR calls into question the CCO activity model of R-NIR photobiomodulation.