Photoexcited bacterial luminescence. Spectral properties and mechanistic implication of a reduced flavine-like prosthetic group associated with photoexcitable luciferase.

Abstract

A prosthetic group, designated B, has been isolated from bacterial photoexcitable luciferase and found to possess spectral and photochemical properties characteristic of substituted reduced flavines. Its fluorescence when bound to luciferase has an excitation maximum at 375 nm, correlating well with the absorption spectrum, and an emission peaking at 495 nm. However, free B is nonfluorescent in aqueous solution at ambient temperature. Both free and luciferase-bound B show similar negative circular dichroism in the region 330-475 nm with troughs at 375 and 380 nm, respectively. In the luciferase reaction initiated by FMNH2, B is an inhibitor competitive with FMNH2. Irradiation of photoexcitable luciferase converts B to FMN, the latter identified spectrally, enzymatically, and chromatographically. These findings lead to the suggestion that B is a substituted FMNH2. The luciferase-bound B resembles but is not identical with the normal flavine intermediate obtainable by reacting luciferase with reduced flavine mononucleotide and oxygen. It is hypothesized that B is a false intermediate of the bacterial bioluminescence reaction, and a mechanism for the photoexcited bioluminescence reaction is suggested.