Luminescence Activity Decreases When v‐coelenterazine Replaces Coelenterazine in Calcium‐Regulated Photoprotein—A Theoretical and Experimental Study

Abstract

Calcium‐regulated photoproteins are found in at least five phyla of organisms. The light emitted by those photoproteins can be tuned by mutating the photoprotein and/or by modifying the substrate coelenterazine (CTZ). Thirty years ago, Shimomura observed that the luminescence activity of aequorin was dramatically reduced when the substrate CTZ was replaced by its analog v‐CTZ. The latter is formed by adding a phenyl ring to the π‐conjugated moiety of CTZ. The decrease in luminescence activity has not been understood until now. In this paper, through combined quantum mechanics and molecular mechanics calculations as well as molecular dynamics simulations, we discovered the reason for this observation. Modification of the substrate changes the conformation of nearby aromatic residues and enhances the π‐π stacking interactions between the conjugated moiety of v‐CTZ and the residues, which weakens the charge transfer to form light emitter and leads to a lower luminescence activity. The microenvironments of CTZ in obelin and in aequorin are very similar, so we predicted that the luminescence activity of obelin will also dramatically decrease when CTZ is replaced by v‐CTZ. This prediction has received strong evidence from currently theoretical calculations and has been verified by experiments.

Crystal structure of semisynthetic obelin‐v

“…There are only a few spatial differences to be mentioned, namely in obelin-v the Trp92 and Trp179 and to a lesser extent His22 and Phe88 are slightly shifted as compared to their positions in obelin. It is worth noting that the small shifts in the region of His22-Phe88-Trp92 triad and 2-hydroperoxyCTZ-v observed in the obelin-v crystal structure are overall consistent with the results of optimization of S 1 -state structures in both obelin and obelin-v, 23 where changes in spatial arrangement of the key residues and hydrogen bond distances due to the substrate modification are clearly visible suggesting good prognostication ability of the theoretical calculations.…”

“…47 Of particular note is that the experimentally revealed long wavelength shifts of the emission maxima of obelin-v were successfully predicted through combined QM/MM calculations as well as molecular dynamics simulations and were calculated as 25 and 84 nm for the emission at shorter and longer wavelengths, respectively. 23 The quantum yield (Φ BL ) of bioluminescent reaction is an important characteristic that, in fact, shows the efficiency of conversion of substrate molecules into photons during reaction. The quantum yields of reactions catalyzed by even cognate bioluminescent proteins utilizing the same substrate can vary significantly.…”

“…22 A similar result is obtained for the recombinant obelin activated with CTZ-v (obelin-v) which retains only 1.7% of bioluminescence light output of obelin activated with unmodified CTZ. 23 The most remarkable feature of CTZ-v as a bioluminescent substrate is its ability to shift light emission of several CTZ-dependent bioluminescence proteins toward longer wavelengths when switching substrate from CTZ to the CTZ-v analog. For instance, bioluminescence spectrum maxima of native Renilla and Oplophorus luciferases with CTZ-v are shifted from 475 to 512 nm and from 454 to 480 nm, respectively.…”

Specific Activities of Hydromedusan Ca2+‐Regulated Photoproteins

“…It is worth mentioning here that according to theoretical studies, in aequorin, the His‐Tyr‐Trp triads residing close to the 6‐( p ‐hydroxy)‐phenyl substituent and peroxide group can severely affect the pathways of dioxetanone decomposition and, consequently, the efficiency of generation of singlet excited state (45). A recent theoretical study on the properties of coelenterazine‐ v analogue showed that in both aequorin and obelin, the increased coplanarity of the conjugated region of coelenterazine‐ v due to the additional vinylene bridge causes the aromatic residues Trp173 and Phe66 to move toward coelenteramide‐ v and consequently form π–π stacking interactions between rings, which weaken the charge transfer between phenol and pyrazine moiety of coelenteramide‐ v and strongly decrease the bioluminescence activity of photoproteins activated with coelenterazine‐ v (46). It is possible that in different photoproteins the different distances between aromatic amino acid residues of the coelenterazine‐binding pocket and coelenteramide atoms affect the π–π stacking interactions as well and consequently influence the charge transfer process.…”

Computational Bioluminescence

“…The combined QM and molecular mechanics (MM) scheme is used to calculate the real system and consider all kinds of effects. In the past 14 years, we have investigated the mechanism or chemistry of BL of firefly, [ 24‐37 ] Cypridina , [ 38‐40 ] Aequorin, [ 41‐42 ] Obelia, [ 43‐44 ] bacteria, [ 45‐47 ] firefly squid, [ 48 ] dinoflagellate, [ 49‐50 ] Latia, [ 51 ] and earthworm. [ 52 ] Some of our theoretical studies were involved in the latest book “ Bioluminescence, the nature of light ” [ 53 ] written by John Lee, one of the pioneers in BL field.…”