Effects of iodide on the fluorescence and activity of the hydroperoxyflavin intermediate of Vibrio harveyi luciferase.
The 4a-hydroperoxy-4a,5-dihydroFMN intermediate (II or HFOOH) of Vibrio harveyi luciferase is known to transform from a low quantum yield IIx to a high quantum yield (lambdamax 485 nm, uncorrected) IIy fluorescent species on exposure to excitation light. Similar results were observed with II prepared from the alphaH44A luciferase mutant, which is very weak in bioluminescence activity. Because of the rapid decay of the alphaH44A II, its true fluorescence was obscured by the more intense 520 nm fluorescence (uncorrected) from its decay product oxidized flavin mononucleotide (FMN). Potassium iodide (KI) at 0.2 M was effective in quenching the FMN fluorescence, leaving the 485 nm fluorescence of II from both the wild-type (WT) and alphaH44A luciferase readily detectable. For both II species, the luciferase-bound peroxyflavin was well shielded from KI quenching. KI also enhanced the decay rates of both the WT and alphaH44A II. For alphaH44A, the transformation of IIx to IIy can be induced by KI in the dark, and it is proposed to be a consequence of a luciferase conformational change. The WT II formed a bioluminescence-inactive complex with KI, resulting in two distinct decay time courses based on absorption changes and decreases of bioluminescence activity of II.
Huang
Shouqin
S
Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5001, USA.
Tu
Shiao-Chun
SC
D000042:Absorption; D005415:Flavins / Q000737:chemistry* / Q000528:radiation effects; D005453:Fluorescence; D008027:Light; D008156:Luciferases / Q000737:chemistry* / Q000302:isolation & purification / Q000528:radiation effects; D011193:Potassium Iodide / Q000737:chemistry*; D013997:Time Factors; D014733:Vibrio / Q000201:enzymology*
D005415:Flavins; C064157:high fluorescent intermediate II; D011193:Potassium Iodide; D008156:Luciferases