Effect of Red-to-Near Infrared Light on the Reaction of Isolated Cytochrome c Oxidase with Cytochrome c.

OBJECTIVE

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.

BACKGROUND DATA

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.

METHODS

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.

RESULTS

Milimolar extinction coefficients (L cm) were: ɛ = 29.1 ± 0.4, ɛ = 8.60 ± 0.15, ɛ = 140 ± 2, and ɛ = 89.0 ± 1.1. Reduced-oxidized extinction coefficients were: δɛ = 20.5 ± 0.2, and δɛ = 51.0 ± 2.0. The second order rate constants k' for irradiated CCO did not show a statistically significant difference from controls.

CONCLUSIONS

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.