Binding of CO to reduced P450 results in a peak at 450 nm in the difference spectrum.

Inhibited, CO-bound P450 can be reactivated by light irradiation at 450 nm. Historically, reversal of CO inhibition was taken as definitive evidence of P450 involvement in a reaction (Estabrook et al., 1963, for the steroid C21 hydroxylation, Cooper et al., 1965, for the p-hydroxylation of acetanilide and the demethylations of codeine and methylaminopyrine). See the reviews by Omura et al., 1965 and Cooper et al., 1979. Similarly, in insects, the Manduca sexta ecdysone 20-monooxygenase shows a photochemical action spectrum with a maximum reversal of inhibition at 450 nm (Smith et al. 1979).

Reversal of CO inhibition by white light is experimentally easier, and was shown for instance for the Locusta migratoria ecdysone 20-monooxygenase (Feyereisen and Durst, 1978) and methyl farnesoate epoxidase (see below, from Feyereisen et al., 1981).

Light (450nm) reversal of CO inhibition remains a defining property of P450-catalyzed reactions, and can therefore be used to distinguish P450 enzymes from other enzymes.

Note that experiments to show CO inhibition should be carried out in CO/O₂ atmosphere, not pure CO, because P450 as monooxygenase needs O₂. Pure CO would be equivalent to anaerobiosis. Note also that CO is a highly poisonous, odorless, colorless gas and should only be handled in a fume hood.