FLUORESCENT POLYENE ALIPHATICS AS SPECTROSCOPIC AND MECHANISTIC PROBES FOR BACTERIAL LUCIFERASE: EVIDENCE AGAINST CARBONYL PRODUCT FROM ALDEHYDE AS THE PRIMARY EXCITED SPECIES

The fluorescent α‐parinaric acid (α‐PAC) and β‐parinaric acid (β‐PAC) were converted to the corresponding aldehydes and alcohols all of which exhibited absorption and fluorescence properties closely resembling those of the parent acids. α‐PAC and β‐PAC each binds to luciferase in competition with aldehyde. The hydrophobic nature of the aldehyde site was indicated by the enhanced fluorescence quantum yields of the bound α‐PAC and β‐PAC. These two polyene acids and the β‐parinaryl alcohol were shown to stabilize the luciferase flavin‐peroxide intermediate. α‐Parinaraldehyde (α‐PAD) and bt‐parinaraldehyde (β‐PAD) were active substrates for Vibrio harveyi and Vibrio fischeri luciferases and, for the former enzyme, exhibited K m , values similar to and quantum yields about20–30% as those for decanal and dodecanal. For the V. harveyi luciferase with reduced FMN as a co‐substrate, the α‐PAD‐ or β‐PAD‐initiated luminescence was indistinguishable from the normal emission obtained with octanal (γ max 495 nm) showing no additional 430‐nm component correlatable with emission from excited α‐PAC or β‐PAC. In reactions using reduced 2‐thioFMN for V. harveyi luciferase or reduced FMN for V. fischeri luciferase plus yellow fluorescent protein, the replacement of octanal by β‐PAD again resulted in no additional 430‐nm emission. The lack of any emission correlatable with excited α‐PAC, β‐PAC, or equivalent carbonyl product was not due to the quenching of the polyene moiety by chemical transformation, binding to luciferase, or a 100% energy transfer to the flavin 4a‐hydroxide emitter. These results strongly favor singlet state of flavin 4a‐hydroxide rather than singlet or triplet carbonyl product from aldehyde as the primary excited species in the normal luciferase reaction in the absence of any additional fluorescent protein.