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
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-1cm-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.


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.
Department of Neurology, The Medical College of Wisconsin , Milwaukee, Wisconsin.
Brendan J
BJ
Quirk
Department of Neurology, The Medical College of Wisconsin , Milwaukee, Wisconsin.
Harry T
HT
Whelan
D045304:Cytochromes c / Q000737:chemistry / Q000528:radiation effects*; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000528:radiation effects*; D007259:Infrared Rays; D010084:Oxidation-Reduction
D045304:Cytochromes c; D003576:Electron Transport Complex IV
cytochrome c oxidase kinetics; light therapy; light-emitting diode (LED); near infrared radiation; photobiology; photobiomodulation