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Abstract— The reaction of luciferase‐bound flavin hydroperoxide with both I‐1H and 1–2H decanal has been examined at 2°C in both low (0.01 M) and high (0.35 M) phosphate buffer, pH 7, where the kinetics and deuterium isotope effects are quite different. Upon reaction in both buffers there are rapid (<2 ms) increases in absorption at both 380 and W nm, followed by decay over the subsequent seconds and minutes. The changes at 380 nm exhibit a primary isotope effect and are rapid compared to bioluminescence, indicating that the scission of the aldehyde C — H bond occurs prior to the step responsible for populating the electronically excited state. However, the final absorbance change at 600 nm decays in parallel to bioluminescence under the several different conditions studied, suggesting the involvement of a long‐wavelength absorbing flavin species in the production of, the excited state. Evidence is also presented indicating that under certain conditions there may be two (sequential) steps, each of which exhibits a primary isotope effect involving the same H atom.

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A luciferase-bound neutral flavin semiquinone radical can be formed upon the oxidation of the luciferase-FMNH2 complex by molecular oxygen. This species can also be formed anaerobically by comproportionation of FMN and FMNHz in 1.he presence of luciferase. The radical is kinetically stable ( t 1 , 2 z 20 h at 0°C in air; the Arrhenius AHie,,,, being about 170 kJ/mol) and can be prepared in pure form by Sephadex G-25 chromatography at 0 -4 "C. The pure enzyme-bound radical is inactive for light emission either with or without aldehyde, and is not in (relevantly rapid) equilibrium with the luciferase 4a-peroxyflavin, the active intermediate in the bioluminescent reaction.In the steps have bacterial bioluminescence reaction two distinct been distinguished, both involving enzyme(1uci-ferase)-bound intermediates; the reaction of reduced flavin with molecular oxygen to form an oxygen-containing (4a-peroxyflavin) intermediate (step a), and the reaction of this with a long-chain aliphatic aldehyde to give oxidized FMN, the corresponding fatty acid and light emission (photon yield, about 0.1 -0.2) centered at about 490 nm (step b) When originally isolated and chamcterized, the preparation of the active intermediate was shown to exhibit absorption with a peak at 373 nm, a shoulder at about 460 nm, tailing off around 500 nm with no absorption above 520 nm [3]. Spectral comparison with an authentic 4a-hydroperoxyflavin [5] (see also [4]), as well as I3C NMR studies [6], provided direct evidence that the compound is a 4a-hydroperoxyflavin.In later work, however, appreciable absorption in the 550 -650-nm region was found to accompany preparations made either in the presence or absence of aldehyde [7,8]. Although the absorption was reminiscent of that of neutral flavin semiquinone, no EPR signatl was detected. Kinetic data raised the possibility that this absorbing species was associated with (possibly in rapid equilibration with) luciferase intermediates in the pathway leading to light emission.One of the basic current questions to be addressed in the flavin-catalyzed bioluminescent reaction is the mechanism of the oxidation of aldehyde by luciferas,e peroxyflavin to produce an excited state. The possible role of this red-absorbing blue species thus prompted interest, speculation and conflicting proposals concerning this reaction mechanism [9-121. The present work was undertaken in order to elucidate the structure of this blue species and to study in some detail its properties and possible role in the catalytic light emission reaction.Abbreviations. FMN, FMNHz and FMNH; oxidized, reduced and neutral semiquinone forms of flavin mononucleotide, respectively: EPR, electron paramagnetic resonance.Enzyme. Luciferase (EC 1.14.14.3). MATERIALS AND METHODSLuciferase was isolated from the luminous bacterium Beneckea harveyi, mutant strain M-17 [I31 and purified according to Hastings et al. [14]. The enzyme concentration of luciferase preparations, measured by absorption using an ~2 8 0 value of 74 mM-' cm-' for the heterodimer [15], ...

Abstract— The bioluminescent oxidation of reduced flavin mononucleotide by bacterial luciferase involves a long‐lived flavoenzyme intermediate whose chromophore has been postulated to be the 4a‐sub‐stituted peroxy anion of reduced flavin. Reaction of long chain aldehyde with this intermediate results in light emission and formation of the corresponding acid. These experiments show that the typical aldehyde‐dependent, luciferase‐catalyzed bioluminescence can also be obtained starting with FMN and H2O2 instead of FMNH2 and O2. We postulate that the 4a‐peroxy anion intermediate is formed directly by attack of H2O2 on FMN. The latter may be bound to luciferase. An enzyme bound intermediate is formed which by kinetic analysis, flavin specificity for luminescence, aldehyde dependence, and bioluminescent emission spectrum appears to be identical with the species generated by reaction of FMNH, and O2 with luciferase. The quantum yield of the H2O2‐‐ and FMN‐initiated biolumlnescence is low but can be enhanced by certain metal ions, which also stimulate a chemiluminescent reaction of oxidized flavin with H2O2. The peak of this chemiluminescence. however, appears to be at a shorter wavelength than that (490 nm) of the bioluminescence.