ИСТИНА

Firefly luciferases catalyse the bioluminescent reaction of firefly luciferin oxidation. The mechanism of color modulation in this reaction remains unclear. The studies concerning the correlation between the structure of luciferase and the resulting bioluminescent spectra are mostly focused on the N-domain of the enzyme, in which many strong color-shifting mutations were discovered. The role of the C-domain in color modulation is rarely discussed, and very few mutations in this region are known to affect the color of bioluminescence. Recently we reported a mutation of the absolutely conserved C-domain residue E457K (L. mingrelica luciferase) [1]; the mutation reduced the enzymatic activity to ~40% and resulted in a strongly red-shifted bioluminescent spectrum. The possible mechanism behind this effect is of interest because it can reveal the role of the C-domain in maintaining the bioluminescence color. By means of site-directed mutagenesis, we obtained the following set of mutants: E457K, E457V, E457Q, E457D, A534R, and E457V/A534R. The set was design to vary the properties of the side-chain at the position 457 and to introduce an additional hydrogen bond between the residues 457 and 534 (A534R mutant). The TS form of L. mingrelica luciferase with a thermostabilized N-domain was used as a parent enzyme in order to increase the enzyme tolerance for mutagenesis [2]. The enzymes were purified to homogeneity by Ni2+-affinity chromatography. Their catalytic properties, thermal stability and bioluminescence spectra at three different temperatures were studied. The properties of the mutants are summarized in the Table. In most cases, the effect of the mutations on the catalytic parameters of the TS enzyme was rather mild. However, the bioluminescent spectra were significantly altered (see Figure). The bioluminescent spectra of TS enzyme were bimodal in the temperature range 10-42°C: at 10°C, the emission maximum (λmax) is in the green region; at 42°C, in the red. The spectra obtained for the E457D and A534R mutants were similar to that of TS, yet the shoulder in the green region is less pronounced. The spectra obtained for the E457Q, E457V and E457V/A534R mutants were strongly red-shifted even at low temperatures; the spectra of the E457K mutant were red-shifted, monomodal and temperature independent. At 42°C the bioluminescent spectra are monomodal for all the mutants; their λmax values were equal within the error limits. We managed to represent the experimental spectra as the superposition of two theoretical spectra, where the λmax(1) of the red spectra was equal to that of the monomodal experimental spectra of the mutants at 42°C; the λmax(2) of the green spectra was also equal for all the mutants within the error limits. The difference between the values of λmax for these spectra was λmax(2)- λmax(1) ~ 30 nm. It implies that there are two forms of the emitter in the system. Our data correspond to the previously obtained fluorescence parameters of keto- and enol/enolate forms of oxyluciferin [3] supporting the theory of keto-enol tautomerism as a possible mechanism for the color modulation in luciferases. The structures of the emitters should be stabilized by two conformations of the enzyme active center that differ by rigidity and polarity. Our data shows that the relative stability of these conformations is controlled by the mutations in the C-domain. The analysis of the computer models of mutated enzymes demonstrate that most of the mutation cause steric hindrances in the enzyme structure, weakening the interactions between the elements of the C-domain. Thus, the intact structure of the C-domain is essential for maintaining the green color of bioluminescence. The results of this study reveal certain aspects of the role of the C-domain in luciferase structure and supports the theory of the oxyluciferin keto-enol tautomerism as one of the color-tuning mechanisms in luciferase reaction. This work was supported by the Russian Foundation for Basic Research (grants 08-04-00624 and 11-04-00698).