Fluorescent polyene aliphatics as spectroscopic and mechanistic probes for bacterial luciferase: evidence against carbonyl product from aldehyde as the primary excited species.
The fluorescent alpha-parinaric acid (alpha-PAC) and beta-parinaric acid (beta-PAC) were converted to the corresponding aldehydes and alcohols all of which exhibited absorption and fluorescence properties closely resembling those of the parent acids. alpha-PAC and beta-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 alpha-PAC and beta-PAC. These two polyene acids and the beta-parinaryl alcohol were shown to stabilize the luciferase flavin-peroxide intermediate. alpha-Parinaraldehyde (alpha-PAD) and beta-parainaraldehyde (beta-PAD) were active substrates for Vibrio harveyi and Vibrio fischeri luciferases and, for the former enzyme, exhibited Km values similar to and quantum yields about 20-30% as those for decanal and dodecanal. For the V. harveyi luciferase with reduced FMN as a co-substrate, the alpha-PAD- or beta-PAD-initiated luminescence was indistinguishable from the normal emission obtained with octanal (lambda max 495 nm) showing no additional 430-nm component correlatable with emission from excited alpha-PAC or beta-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 beta-PAD again resulted in no additional 430-nm emission. The lack of any emission correlatable with excited alpha-PAC, beta-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Cho
K W
KW
Department of Biochemical and Biophysical Sciences, University of Houston, TX 77204-5934.
Tu
S C
SC
Shao
R
R
D000447:Aldehydes / Q000032:analysis / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D005233:Fatty Alcohols / Q000032:analysis / Q000378:metabolism; D005456:Fluorescent Dyes; D008156:Luciferases / Q000378:metabolism*; D049449:Luminescence; D013050:Spectrometry, Fluorescence / Q000379:methods; D014733:Vibrio / Q000201:enzymology*
D000447:Aldehydes; D005231:Fatty Acids, Unsaturated; D005233:Fatty Alcohols; D005456:Fluorescent Dyes; C080249:parinaraldehyde; D008156:Luciferases; C013641:parinaric acid