| Autor: |
Natashin PV; Photobiology Laboratory, Institute of Biophysics of Siberian Branch of the Russian Academy of Sciences, Federal Research Center 'Krasnoyarsk Science Center' of Siberian Branch of the Russian Academy of Sciences', Krasnoyarsk 660036, Russia., Burakova LP; Photobiology Laboratory, Institute of Biophysics of Siberian Branch of the Russian Academy of Sciences, Federal Research Center 'Krasnoyarsk Science Center' of Siberian Branch of the Russian Academy of Sciences', Krasnoyarsk 660036, Russia.; Institute of Fundamental Biology and Biotechnology, Siberian Federal University, Krasnoyarsk 660041, Russia., Kovaleva MI; Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia., Shevtsov MB; Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia., Dmitrieva DA; Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia., Eremeeva EV; Photobiology Laboratory, Institute of Biophysics of Siberian Branch of the Russian Academy of Sciences, Federal Research Center 'Krasnoyarsk Science Center' of Siberian Branch of the Russian Academy of Sciences', Krasnoyarsk 660036, Russia.; Institute of Fundamental Biology and Biotechnology, Siberian Federal University, Krasnoyarsk 660041, Russia., Markova SV; Photobiology Laboratory, Institute of Biophysics of Siberian Branch of the Russian Academy of Sciences, Federal Research Center 'Krasnoyarsk Science Center' of Siberian Branch of the Russian Academy of Sciences', Krasnoyarsk 660036, Russia.; Institute of Fundamental Biology and Biotechnology, Siberian Federal University, Krasnoyarsk 660041, Russia., Mishin AV; Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia., Borshchevskiy VI; Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia.; Joint Institute for Nuclear Research, Dubna 141980, Russia., Vysotski ES; Photobiology Laboratory, Institute of Biophysics of Siberian Branch of the Russian Academy of Sciences, Federal Research Center 'Krasnoyarsk Science Center' of Siberian Branch of the Russian Academy of Sciences', Krasnoyarsk 660036, Russia. |
| Abstrakt: |
Hydromedusan photoproteins responsible for the bioluminescence of a variety of marine jellyfish and hydroids are a unique biochemical system recognized as a stable enzyme-substrate complex consisting of apoprotein and preoxygenated coelenterazine, which is tightly bound in the protein inner cavity. The binding of calcium ions to the photoprotein molecule is only required to initiate the light emission reaction. Although numerous experimental and theoretical studies on the bioluminescence of these photoproteins were performed, many features of their functioning are yet unclear. In particular, which ionic state of dioxetanone intermediate decomposes to yield a coelenteramide in an excited state and the role of the water molecule residing in a proximity to the N1 atom of 2-hydroperoxycoelenterazine in the bioluminescence reaction are still under discussion. With the aim to elucidate the function of this water molecule as well as to pinpoint the amino acid residues presumably involved in the protonation of the primarily formed dioxetanone anion, we constructed a set of single and double obelin and aequorin mutants with substitutions of His, Trp, Tyr, and Ser to residues with different properties of side chains and investigated their bioluminescence properties (specific activity, bioluminescence spectra, stopped-flow kinetics, and fluorescence spectra of Ca 2+ -discharged photoproteins). Moreover, we determined the spatial structure of the obelin mutant with a substitution of His64, the key residue of the presumable proton transfer, to Phe. On the ground of the bioluminescence properties of the obelin and aequorin mutants as well as the spatial structures of the obelin mutants with the replacements of His64 and Tyr138, the conclusion was made that, in fact, His residue of the Tyr-His-Trp triad and the water molecule perform the "catalytic function" by transferring the proton from solvent to the dioxetanone anion to generate its neutral ionic state in complex with water, as only the decomposition of this form of dioxetanone can provide the highest light output in the light-emitting reaction of the hydromedusan photoproteins. |
