Influence of some inhibiting and activating substances on the light reaction in vitro of photobacterium phosphoreum

Addition of H₂O₂ to a bacterial luciferase preparation resulted in the formation of a compound with a fluoresence maximum at about 470 mμ. This compond was probably the same as the one that is formed as a result of ultraviolet irradiation of luciferase preparations after addition of FMNH₂. It is assumed to be the luminescent molecule in bacterial luminescence.

The effect of a number of substances (K₃Fe(CN)₆, K₄Fe(CN)₆, ascorbic acid, peroxidase (donor: H₂O₂ oxidoreductase, EC 1.11.1.7), tryptophan and p-chloromecuribenzoate) on both the light reaction and fluorescence increase reaction was investigated. Substances inhibiting the fluorescence increase reaction (K₃Fe(CN)₆, K₄Fe(CN)₆, ascorbic acid) likewise could inhibit the light reaction. Both effects are, at least partly, ascribed to an effect on the fluorescent group of luciferase. Light reaction inhibitors affecting other groups at the enzyme surface (p-chloromercuribenzoate) did not influence the fluorescence increase effect. Activators of the light reaction (cystine, K₄Fe(CN)₆) are suggested to act by their protective action on sulfhydryl groups or on dissociable groups, presumed to be attached to these sulfhydryl groups.

A discussion of the results leads to the conclusion that the light reaction consists of at least two consecutive reactions, involving two different groups of the luciferase molecule.