Oxygen activation of Apo-oberlin-Coelenterazine Complex

Ca2+-regulated photoproteins use a noncovalently bound 2-hydroperoxycoelenterazine ligand to emit light in response to Ca2+ binding. To better understand the mechanism of formation of active photoprotein from apoprotein, coelenterazine and molecular oxygen, we investigated the spectral properties of the anaerobic apo-obelin–coelenterazine complex and the kinetics of its conversion into active photoprotein after exposure to air. Our studies suggest that coelenterazine bound within the anaerobic complex might be a mixture of N7-protonated and C2(-) anionic forms, and that oxygen shifts the equilibrium in favor of the C2(-) anion as a result of peroxy anion formation. Proton removal from N7 and further protonation of peroxy anion and the resulting formation of 2-hydroperoxycoelenterazine in obelin might occur with the assistance of His175. It is proposed that this conserved His residue might play a key role both in formation of active photoprotein and in Ca2+-triggering of the bioluminescence reaction

Ultraviolet fluorescence of coelenteramide and coelenteramide-containing fluorescent proteins. Experimental and theoretical study

Coelenteramide-containing fluorescent proteins are products of bioluminescent reactions of marine coelenterates. They are called ‘discharged photoproteins’. Their light-induced fluorescence spectra are variable, depending considerably on external conditions. Current work studies a dependence of light-induced fluorescence spectra of discharged photoproteins obelin, aequorin, and clytin on excitation energy. It was demonstrated that photoexcitation to the upper electron-excited states (260-300 nm) of the discharged photoproteins initiates a fluorescence peak in the near UV region, in addition to the blue-green emission. To characterize the UV fluorescence, the light-induced fluorescence spectra of coelenteramide (CLM), fluorophore of the discharged photoproteins, were studied in methanol solution. Similar to photoproteins, the CLM spectra depended on photoexcitation energy; the additional peak (330 nm) in the near UV region was observed in CLM fluorescence at higher excitation energy (260-300 nm). Quantum chemical calculations by time depending method with B3LYP / cc-pVDZ showed that the conjugated pyrazine-phenolic fragment and benzene moiety of CLM molecule are responsible for the additional UV fluorescence peak. Quantum yields of CLM fluorescence in methanol were 0.028 ± 0.005 at 270-340 nm photoexcitation. A conclusion was made that the UV emission of CLM might contribute to the UV fluorescence of the discharged photoproteins. The study develops knowledge on internal energy transfer in biological structures – complexes of proteins with low-weight aromatic molecules

The Role of Tyr-His-Trp Triad and Water Molecule Near the N1-Atom of 2-Hydroperoxycoelenterazine in Bioluminescence of Hydromedusan Photoproteins: Structural and Mutagenesis Study

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 Ca2+-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

Bioluminescent and structural features of native folded Gaussia luciferase

The secreted luciferases responsible for light emission of marine copepods have gained popularity for being used in noninvasive imaging of intracellular events. The secreted luciferase of copepod Gaussia princeps is a one-subunit protein catalyzing coelenterazine oxidation to emit blue light. It consists of the N-terminal variable part that bears a signal peptide for secretion and the C-terminal catalytic domain containing ten highly conserved Cys residues supposing the existence of up to five SS bonds. Despite wide application of Gaussia luciferase in biomedical research, its biochemical properties are still insufficiently studied due to the general problem of obtaining the proper folded Cys-rich proteins in bacterial cells. Here we report the properties of the proper folded Gaussia luciferase produced in insect cells using baculovirus expression system. This high purity luciferase reveals the highest activity at 15–20 °C but retains only ~20% activity at 37 °C that may hamper its application for in vivo assays. The maximum of bioluminescent activity of GpLuc is found at NaCl concentrations in the range of 1.0–1.5 M and, furthermore, a high NaCl concentration enhances luciferase stability to thermal denaturation, i.e. Gaussia luciferase displays the features characteristic of halophilic enzymes. The studies on bioluminescence kinetics at different coelenterazine concentrations obviously show a positive cooperativity of Gaussia luciferase with coelenterazine (Hill coefficient – 1.8 ± 0.2; K0.5–2.14 ± 0.17 μM). We suggest this effect to be rather due to the so-called kinetic cooperativity conditioned by conformational changes in response to substrate binding than to the presence of two catalytic sites