Physicochemical Analysis of Bacterial and Firefly Bioluminescence

Abstract

The light emitting reaction of bioluminescence is one of the most visible processes in nature. The present work provides new significant information about the bioluminescent reaction in bacteria. A radical intermediate during the catalytic process was detected by means of spin trapping in combination with EPR spectroscopy. In the presence of luciferase, the relative amplitude of the EPR signal of this spin adduct correlates with the protein concentration. As to the identity of the observed radical species, the EPR signals contain no information at present. The kinetic assays in the present work provide direct information about the activity of the luciferase and the effect of different substrates. The phosphate moiety of the flavin-cofactor was shown to be obligatory for the process of bacterial bioluminescence. With regard to the aldehyde cosubstrate, kinetic measurements provide evidence for the important role of the aldehyde moiety. Moreover, the light emission increased with extending chain length of the aliphatic aldehyde. The commonly used assay including a chemically reduced FMN by sodium dithionite was found to have a high impact on the bioluminescence efficiency. Sodium dithionite is involved in additional reaction pathways and induces the formation of aldehyde radicals, making interpretation of rate constants difficult.<br /> The electronic structure of FMN has been investigated at different pH values and in the presence of AgNO₃ by UV/VIS spectroscopy, EPR spectroscopy of the triplet state, DFT and TDDFT calculations. Upon lowering the pH the bands at 355 and 450 nm in the UV/VIS spectrum of FMN fall together and form one intense band at 397 nm. These changes have been attributed to a protonation of nitrogen atom N1. ZFS parameters change only slightly upon pH change, since the HOMO and LUMO remain virtually the same. The first six doubly occupied orbitals that are in energy below the HOMO react stronger to the pH change. Upon addition of AgNO₃, all UV/VIS bands display a bathochromic shift, which originates from the coordination of Ag⁺ to nitrogen atom N5, stabilizing the LUMO. In the presence of AgNO₃, the ZFS parameters slightly increase and the polarization pattern changes, owing to a contribution by SOC of Ag⁺. The addition of AgNO₃ did not lead to an increase of signal, which would be large enough to perform pulsed ENDOR experiments of the triplet state for direct examination of the hyperfine coupling constants of the nitrogen atoms and the protons of FMN. Nevertheless, the decrease of the lifetimes of the triplet sublevels by up to several hundred microseconds by the increased SOC is an advance, since it allows excitation and detection with a larger rate than previously possible. Additionally, the interpretation of the electronic structure of free FMN may be useful when compared with the same investigations of enzyme-bound FMN. <br /> The results in this study contribute to a better understanding of the catalytic cycle of bacterial bioluminescence and its high efficiency.