One Protein, Two Chromophores: Comparative Spectroscopic Characterization of 6,7-Dimethyl-8-ribityllumazine and Riboflavin Bound to Lumazine Protein

Abstract

We investigated the lumazine protein from Photobacterium leiognathi in complex with its biologically active cofactor, 6,7-dimethyl-8-ribityllumazine, at different redox states and compared the results with samples containing a riboflavin cofactor. Using anaerobic photoreduction, we were able to record optical absorption kinetics from both cofactors in similar protein environments. It could be demonstrated that the protein is able to stabilize a neutral ribolumazine radical with ∼35% yield. The ribolumazine radical state was further investigated by W-band continuous-wave EPR and X-band pulsed ENDOR spectroscopy. Here, both the principal values of the g-tensor and an almost complete mapping of the proton hyperfine couplings (hfcs) could be obtained. Remarkably, the g-tensor's principal components are similar to those of the respective riboflavin-containing protein; however, the proton hfcs show noticeable differences. Comparing time-resolved optical absorption and fluorescence data from ribolumazine-containing samples, solely fluorescence but no signs of any intermediate radical or a triplet state could be identified. This is in contrast to lumazine protein samples containing the riboflavin cofactor, for which a high yield of the photogenerated triplet state and some excited flavin radical could be detected using time-resolved spectroscopy. These results clearly demonstrate that ribolumazine is a redox-active molecule and could, in principle, be employed as a cofactor in other enzymatic reactions.

Spectroscopic characterization of radicals and radical pairs in fruit fly cryptochrome – protonated and nonprotonated flavin radical‐states

“…Crys have also been suggested to play a major role in the magnetic orientation system of migratory birds, fruit flies and other animals . The most thoroughly investigated animal Cry is that of Drosophila melanogaster ( Dm Cry), which has been shown to be responsible for the entrainment of the circadian cycle and involved in magnetic orientation .…”

Generation of Fluorescent Bacteria with the Genes Coding for Lumazine Protein and Riboflavin Biosynthesis

“…Based on a previous study that reported that riboflavin competes with lumazine to bind with the lumazine protein [ 13 ], we fixed the excitation wavelength of riboflavin at 450 nm and examined the emission spectrum. As you can see in Figure 5 a, we found that the emission spectrum of riboflavin excited at 450 nm was similar to that of lumazine shown in Figure 5 b.…”

“…They show differences in the quaternary structure of protein, the lumazine protein has a monomeric structure whereas the riboflavin synthase forms trimeric structure [ 6 , 13 ]. The peculiar characteristics of lumazine protein is the intramolecular sequence similarity between N-terminal and C-terminal domain half ( Figure 3 a), and it was reported that the protein binds to one molecule of lumazine at N-terminal domain half [ 14 , 15 ] ( Figure 3 b).…”

Perspectives on Bioluminescence Mechanisms

“…Although the spatial structures of the lumazine protein variants are quite homologous overall, there are some small variations around the binding site. They have identical spectroscopic properties to the native proteins and the variations among the ligands can be rationalized by examination of interactions in the binding site . The lumazine derivative is buried in a hydrophobic pocket held in place by an extensive hydrogen bond network .…”